WorldWideScience

Sample records for live staphylococcal cells

  1. Staphylococcal enterotoxins in the Etiopathogenesis of Mucosal Autoimmunity within the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    MaryAnn Principato

    2014-04-01

    Full Text Available The staphylococcal enterotoxins (SEs are the products of Staphylococcus aureus and are recognized as the causative agents of classical food poisoning in humans following the consumption of contaminated food. While illness evoked by ingestion of the SE or its producer organism in tainted food are often self-limited, our current understanding regarding the evolution of S. aureus provokes the utmost concern. The organism and its associated toxins, has been implicated in a wide variety of disease states including infections of the skin, heart, sinuses, inflammatory gastrointestinal disease, toxic shock, and Sudden Infant Death Syndrome. The intricate relationship between the various subsets of immunocompetent T cells and accessory cells and the ingested material found within the gastrointestinal tract present daunting challenges to the maintenance of immunologic homeostasis. Dysregulation of the intricate balances within this environment has the potential for extreme consequences within the host, some of which are long-lived. The focus of this review is to evaluate the relevance of staphylococcal enterotoxin in the context of mucosal immunity, and the underlying mechanisms that contribute to the pathogenesis of gastrointestinal autoimmune disease.

  2. FDA approved Immunosuppressants Targeting Staphylococcal Superantigens: Mechanisms and Insights

    Science.gov (United States)

    2016-12-02

    cells , resulting in polyclonal T- cell activation [4-6]. Staphylococcal superantigens hyperactivate cells of the innate immune system and adaptive T... innate host defense responses, antiviral genes, apoptotic programs, immunoproteasomes, and has many immunomodulatory functions. The cell death...1692. 104. Mendis C, Das R, Hammamieh R, Royaee A, Yang D, Peel S, et al. Transcriptional response signature of human lymphoid cells to

  3. Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts.

    Science.gov (United States)

    Hussain, Muzaffar; Haggar, Axana; Heilmann, Christine; Peters, Georg; Flock, Jan-Ingmar; Herrmann, Mathias

    2002-06-01

    To initiate invasive infection, Staphylococcus aureus must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of S. aureus strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable eap::ermB deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.

  4. Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

    Directory of Open Access Journals (Sweden)

    Bao G Vu

    Full Text Available BACKGROUND: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin. METHODOLOGY/PRINCIPAL FINDINGS: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8. CONCLUSIONS/SIGNIFICANCE: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to

  5. Staphylococcal enterotoxin-like X (SElX is a unique superantigen with functional features of two major families of staphylococcal virulence factors.

    Directory of Open Access Journals (Sweden)

    Ries J Langley

    2017-09-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs and the Staphylococcal Superantigen-Like (SSL exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX reveals that SElX is a unique single-domain SAg. In summary, SElX is an 'SSL-like' SAg.

  6. Therapeutic Down-Modulators of Staphylococcal Superantigen-Induced Inflammation and Toxic Shock

    Directory of Open Access Journals (Sweden)

    Teresa Krakauer

    2010-07-01

    Full Text Available Staphylococcal enterotoxin B (SEB and related superantigenic toxins are potent stimulators of the immune system and cause a variety of diseases in humans, ranging from food poisoning to toxic shock. These toxins bind directly to major histocompatibility complex (MHC class II molecules on antigen-presenting cells and specific Vb regions of T-cell receptors (TCR, resulting in hyperactivation of both monocytes/macrophages and T lymphocytes. Activated host cells produce massive amounts of proinflammatory cytokines and chemokines, activating inflammation and coagulation, causing clinical symptoms that include fever, hypotension, and shock. This review summarizes the in vitro and in vivo effects of staphylococcal superantigens, the role of pivotal mediators induced by these toxins in the pathogenic mechanisms of tissue injury, and the therapeutic agents to mitigate the toxic effects of superantigens.

  7. Evaluation of in-situ fatty acid extraction protocols for the analysis of staphylococcal cell membrane associated fatty acids by gas chromatography.

    Science.gov (United States)

    Crompton, Marcus J; Dunstan, R Hugh

    2018-05-01

    The composition and integrity of the bacterial cytoplasmic membrane is critical to the survival of staphylococci in dynamic environments and it is important to investigate how the cell membrane responds to changes in the environmental conditions. The staphylococcal membrane differs from eukaryotic and many other bacterial cell membranes by having a high abundance of branch fatty acids and relatively few unsaturated fatty acids. The range of available methods for extraction and efficient analyses of staphylococcal fatty acids was initially appraised to identify the best potential procedures for appraisal. Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 29213) was grown under optimal conditions to generate a cell biomass to compare the efficiencies of three approaches to extract and prepare methyl esters of the membrane fatty acids: (1) acidic direct transesterification of lipids, (2) modified basic direct transesterification of membrane lipids with adjusted reaction times and temperatures, and (3) base catalysed hydrolysis followed by acid catalysed esterification in two separate chemical reactions (MIDI process). All methods were able to extract fatty acids from the cell mass effectively where these lipids represented approximately 5% of the cellular dry mass. The acidic transesterification method had the least number of steps, the lowest coefficient of variation at 6.7% and good resistance to tolerating water. Basic transesterification was the least accurate method showing the highest coefficient of variation (26%). The MIDI method showed good recoveries, but had twice the number of steps and a coefficient of variation of 16%. It was also found that there was no need to use an anti-oxidant such as BHT for the protection of polyunsaturated fatty acids when the GC-MS injection liner was clean. It was concluded that the acidic transesterification procedures formed the most efficient and reproducible method for the analyses of staphylococcal membrane fatty acids

  8. Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

    Directory of Open Access Journals (Sweden)

    William D. Cornwell

    2010-06-01

    Full Text Available Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy. We characterized the components of the T cell receptor (TCR during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  9. Manipulation of Innate and Adaptive Immunity by Staphylococcal Superantigens

    Directory of Open Access Journals (Sweden)

    Stephen W. Tuffs

    2018-05-01

    Full Text Available Staphylococcal superantigens (SAgs constitute a family of potent exotoxins secreted by Staphylococcus aureus and other select staphylococcal species. SAgs function to cross-link major histocompatibility complex (MHC class II molecules with T cell receptors (TCRs to stimulate the uncontrolled activation of T lymphocytes, potentially leading to severe human illnesses such as toxic shock syndrome. The ubiquity of SAgs in clinical S. aureus isolates suggests that they likely make an important contribution to the evolutionary fitness of S. aureus. Although the apparent redundancy of SAgs in S. aureus has not been explained, the high level of sequence diversity within this toxin family may allow for SAgs to recognize an assorted range of TCR and MHC class II molecules, as well as aid in the avoidance of humoral immunity. Herein, we outline the major diseases associated with the staphylococcal SAgs and how a dysregulated immune system may contribute to pathology. We then highlight recent research that considers the importance of SAgs in the pathogenesis of S. aureus infections, demonstrating that SAgs are more than simply an immunological diversion. We suggest that SAgs can act as targeted modulators that drive the immune response away from an effective response, and thus aid in S. aureus persistence.

  10. c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin

    Directory of Open Access Journals (Sweden)

    Alejandro J. Moyano

    2018-05-01

    Full Text Available c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant

  11. Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

    Science.gov (United States)

    Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

    2013-02-01

    The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  12. Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis.

    Science.gov (United States)

    Mordmuang, Auemphon; Voravuthikunchai, Supayang Piyawan

    2015-10-01

    Antibiotic residues in dairy products as well as emergence of antimicrobial resistance in foodborne pathogens have been recognized as global public health concerns. The present work was aimed to study a potent antibacterial extract from natural product as an alternative treatment for staphylococcal bovine mastitis. Staphylococcal isolates (n=44) were isolated from milk samples freshly squeezed from individual cows. All staphylococcal isolates were resistant to ampicillin, ciprofloxacin, erythromycin, gentamicin, penicillin, except vancomycin. Rhodomyrtus tomentosa leaf ethanolic extract was accessed for its antibacterial activity and anti-inflammatory potential. The extract exhibited profound antibacterial activity against all of staphylococcal isolates with MIC and MBC values ranged from 16-64 μg/ml and 64->128 μg/ml, respectively. Moreover, the extract also exerted anti-protein denaturation and human red blood cell membrane stabilizing activity. The results support the use of R. tomentosa extract that could be applied to cure bovine mastitis and to reduce inflammatory injury caused by the bacterial infections. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Nematode Infections Are Risk Factors for Staphylococcal Infection in Children

    Directory of Open Access Journals (Sweden)

    Sandra F Moreira-Silva

    2002-04-01

    Full Text Available Nematode infection may be a risk factor for pyogenic liver abscess in children and we hypothesized that the immunomodulation induced by those parasites would be a risk factor for any staphylococcal infection in children. The present study was designed to compare, within the same hospital, the frequency of intestinal nematodes and Toxocara infection in children with and without staphylococcal infections. From October 1997 to February 1998, 80 children with staphylococcal infection and 110 children with other diseases were submitted to fecal examination, serology for Toxocara sp., evaluation of plasma immunoglobulin levels, and eosinophil counts. Mean age, gender distribution, birthplace, and socioeconomic conditions did not differ significantly between the two groups. Frequency of intestinal nematodes and positive serology for Toxocara, were remarkably higher in children with staphylococcal infections than in the non-staphylococcal group. There was a significant correlation between intestinal nematodes or Toxocara infection and staphylococcal infection in children, reinforced by higher eosinophil counts and higher IgE levels in these children than in the control group. One possible explanation for this association would be the enhancement of bacterial infection by the immunomodulation induced by helminth infections, due to strong activation of the Th2 subset of lymphocytes by antigens from larvae and adult worms.

  14. Lactobacilli require physical contact to reduce staphylococcal TSST-1 secretion and vaginal epithelial inflammatory response.

    Science.gov (United States)

    Younes, Jessica A; Reid, Gregor; van der Mei, Henny C; Busscher, Henk J

    2016-06-01

    ITALIC! Staphylococcus aureusbiofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species ITALIC! Lactobacilluscan alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is hypothesized that these effects of lactobacilli require direct physical contact between lactobacilli, staphylococci and the epithelium. Indeed, lactobacilli only reduced ITALIC! S. aureus-induced inflammatory cytokine expression when allowed physical contact with vaginal epithelial cells. Furthermore, a reduction in toxic shock syndrome toxin-1 secretion only occurred when a probiotic ITALIC! Lactobacillusstrain was allowed contact, but not when being physically separated from ITALIC! S. aureus Bacterial-probe atomic force microscopy demonstrated that lactobacilli and staphylococci strongly adhere to epithelial cells, while lactobacilli adhere stronger to staphylococci than staphylococci to each other, giving lactobacilli opportunity to penetrate and reside in staphylococcal biofilms, as visualized using confocal laser scanning microscopy with fluorescence ITALIC! in situhybridization probes. These results identify that physical contact and biochemical signaling by lactobacilli are intrinsically linked mechanisms that reduce virulence of ITALIC! S. aureusbiofilm. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Probabilistic risk model for staphylococcal intoxication from pork-based food dishes prepared in food service establishments in Korea.

    Science.gov (United States)

    Kim, Hyun Jung; Griffiths, Mansel W; Fazil, Aamir M; Lammerding, Anna M

    2009-09-01

    Foodborne illness contracted at food service operations is an important public health issue in Korea. In this study, the probabilities for growth of, and enterotoxin production by, Staphylococcus aureus in pork meat-based foods prepared in food service operations were estimated by the Monte Carlo simulation. Data on the prevalence and concentration of S. aureus as well as compliance to guidelines for time and temperature controls during food service operations were collected. The growth of S. aureus was initially estimated by using the U.S. Department of Agriculture's Pathogen Modeling Program. A second model based on raw pork meat was derived to compare cell number predictions. The correlation between toxin level and cell number as well as minimum toxin dose obtained from published data was adopted to quantify the probability of staphylococcal intoxication. When data gaps were found, assumptions were made based on guidelines for food service practices. Baseline risk model and scenario analyses were performed to indicate possible outcomes of staphylococcal intoxication under the scenarios generated based on these data gaps. Staphylococcal growth was predicted during holding before and after cooking, and the highest estimated concentration (4.59 log CFU/g for the 99.9th percentile value) of S. aureus was observed in raw pork initially contaminated with S. aureus and held before cooking. The estimated probability for staphylococcal intoxication was very low, using currently available data. However, scenario analyses revealed an increased possibility of staphylococcal intoxication when increased levels of initial contamination in the raw meat, andlonger holding time both before and after cooking the meat occurred.

  16. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    Directory of Open Access Journals (Sweden)

    Leśniewicz Krzysztof

    2012-10-01

    Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

  17. Production of staphylococcal enterotoxin A in cream-filled cake.

    Science.gov (United States)

    Anunciaçao, L L; Linardi, W R; do Carmo, L S; Bergdoll, M S

    1995-07-01

    Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.

  18. Rauvolfia grandiflora (Apocynaceae extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Lanamar de Almeida Carlos

    2010-10-01

    Full Text Available Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction (RGE on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 mg.mL-1 RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections.

  19. Rauvolfia grandiflora (apocynaceae) extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity.

    Science.gov (United States)

    de Almeida Carlos, Lanamar; da Silva Amaral, Kenas Aguiar; Curcino Vieira, Ivo José; Mathias, Leda; Braz-Filho, Raimundo; Silva Samarão, Solange; Vieira-da-Motta, Olney

    2010-07-01

    Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction) (RGE) on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 μg.mL(-1) RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections.

  20. Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly

    Science.gov (United States)

    Stewart, Elizabeth J.; Ganesan, Mahesh; Younger, John G.; Solomon, Michael J.

    2015-01-01

    We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism. PMID:26272750

  1. Sulfasalazine Attenuates Staphylococcal Enterotoxin B-Induced Immune Responses

    Directory of Open Access Journals (Sweden)

    Teresa Krakauer

    2015-02-01

    Full Text Available Staphylococcal enterotoxin B (SEB and related exotoxins are important virulence factors produced by Staphylococcus aureus as they cause human diseases such as food poisoning and toxic shock. These toxins bind directly to cells of the immune system resulting in hyperactivation of both T lymphocytes and monocytes/macrophages. The excessive release of proinflammatory cytokines from these cells mediates the toxic effects of SEB. This study examined the inhibitory activities of an anti-inflammatory drug, sulfasalazine, on SEB-stimulated human peripheral blood mononuclear cells (PBMC. Sulfasalazine dose-dependently inhibited tumor necrosis factor α, interleukin 1 (IL-1 β, IL-2, IL-6, interferon γ (IFNγ, and various chemotactic cytokines from SEB-stimulated human PBMC. Sulfasalazine also potently blocked SEB-induced T cell proliferation and NFκB activation. These results suggest that sulfasalazine might be useful in mitigating the toxic effects of SEB by blocking SEB-induced host inflammatory cascade and signaling pathways.

  2. METHICILLIN RESISTANCE IN STAPHYLOCOCCAL ISOLATES ...

    African Journals Online (AJOL)

    The study assessed the importance of Staphylococcus aureus as a urinary pathogen and the incidence of multidrug resistant (MDR), methicillin-resistant Staphylococcus aureus (MRSA). A total of 86 staphylococcal isolates made up of 50 clinical isolates from urine samples submitted to the Medical Microbiology Laboratory ...

  3. Adhesion of staphylococcal and Caco-2 cells on diamond-like carbon polymer hybrid coating.

    Science.gov (United States)

    Kinnari, Teemu J; Soininen, Antti; Esteban, Jaime; Zamora, Nieves; Alakoski, Esa; Kouri, Vesa-Petteri; Lappalainen, Reijo; Konttinen, Yrjö T; Gomez-Barrena, Enrique; Tiainen, Veli-Matti

    2008-09-01

    Staphylococci cause the majority of the nosocomial implant-related infections initiated by adhesion of planktonic bacteria to the implant surface. It was hypothesized that plasma accelerating filtered pulsed arc discharge method enables combination of the advantageous properties of diamond with the antisoiling properties of polymers. Diamond-like carbon polytetrafluoroethylene hybrid (DLC-PTFE-h) coating was produced. The adhesion of S. aureus ATCC 25923 (10(8) colony-forming units/mL) to surfaces diminished from 2.32%, 2.35%, and 2.57% of high quality DLC, titanium, and oxidized silicon, respectively, to 1.93% of DLC-PTFE-h. For S. epidermidis ATCC 35984 the corresponding figures were 3.90%, 3.32%, 3.47%, and 2.57%. Differences in bacterial adhesion between recombinant DLC-PTFE-h and other materials were statistically significant (p DLC-PTFE-h as to DLC, titanium, or silicon, which were all in the MTT test found to be cytocompatible. DLC-PTFE-h coating can be used to modify the surface properties of any surgical implants and is an unfavorable substrate for staphylococcal cells, but compatible with human Caco-2 cells. DLC-PTFE-h coating may help in the combat against Staphylococcus-related implant infections which usually require both antibiotics and surgical removal of the implant for cure.

  4. Staphylococcal scalded skin syndrome in a 4-year-old child: a case report.

    Science.gov (United States)

    Haasnoot, P J; De Vries, A

    2018-01-29

    Staphylococcal scalded skin syndrome is an exfoliating skin disease which primarily affects children. Differential diagnosis includes toxic epidermal necrolysis, staphylococcal scalded skin syndrome, epidermolysis bullosa, and Stevens-Johnson syndrome. Staphylococcal scalded skin syndrome primarily affects children and can cause serious morbidity. In this case report we highlight the case of a 4-year-old Caucasian boy. Diagnostic and therapeutic challenges are discussed. Differential diagnoses are considered and therapy is described and discussed. The latest treatment options are used and described. Successful results are achieved in this case due to timely and correct management. Some therapeutic options are widely used without thorough research bases. This case report highlights staphylococcal scalded skin syndrome and its treatment, and future challenges. Further research is warranted and this case report aims to further research in exfoliating skin disorders.

  5. Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation

    Directory of Open Access Journals (Sweden)

    Nikoleta Zeaki

    2015-09-01

    Full Text Available The prophage-encoded staphylococcal enterotoxin A (SEA is recognized as the main cause of staphylococcal food poisoning (SFP, a common foodborne intoxication disease, caused by Staphylococcus aureus. Studies on the production of SEA suggest that activation of the SOS response and subsequent prophage induction affect the regulation of the sea gene and the SEA produced, increasing the risk for SFP. The present study aims to evaluate the effect of NaCl and sorbic acid, in concentrations relevant to food production, on SOS response activation, prophage induction and SEA production. The impact of stress was initially evaluated on steady state cells for a homogenous cell response. NaCl 2% was found to activate the SOS response, i.e., recA expression, and trigger prophage induction, in a similar way as the phage-inducer mitomycin C. In contrast, sorbic acid decreased the pH of the culture to a level where prophage induction was probably suppressed, even when combined with NaCl stress. The impact of previous physiological state of the bacteria was also addressed on cells pre-exposed to NaCl, and was found to potentially affect cell response upon exposure to further stress. The results obtained highlight the possible SFP-related risks arising from the use of preservatives during food processing.

  6. Panton-Valentine Leukocidin-Producing Staphylococcal aureus: Report of Four Siblings.

    LENUS (Irish Health Repository)

    2012-01-31

    Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus results in leukocyte destruction and tissue necrosis (Pediatric Dermatology 2007;24:401). It can be associated with a spectrum of clinical manifestations that range from localized staphylococcal skin infections to sometimes severe necrotizing pneumonia (Clin Infect Dis 1999;29:1128). We report a case of four siblings, three brothers whose atopic dermatitis was complicated by cutaneous lesions and furunculosis, while their 21-month-old sister had a fatal PVL positive staphylococcal pneumonia.

  7. Evolutionary paths of streptococcal and staphylococcal superantigens

    Directory of Open Access Journals (Sweden)

    Okumura Kayo

    2012-08-01

    Full Text Available Abstract Background Streptococcus pyogenes (GAS harbors several superantigens (SAgs in the prophage region of its genome, although speG and smez are not located in this region. The diversity of SAgs is thought to arise during horizontal transfer, but their evolutionary pathways have not yet been determined. We recently completed sequencing the entire genome of S. dysgalactiae subsp. equisimilis (SDSE, the closest relative of GAS. Although speG is the only SAg gene of SDSE, speG was present in only 50% of clinical SDSE strains and smez in none. In this study, we analyzed the evolutionary paths of streptococcal and staphylococcal SAgs. Results We compared the sequences of the 12–60 kb speG regions of nine SDSE strains, five speG+ and four speG–. We found that the synteny of this region was highly conserved, whether or not the speG gene was present. Synteny analyses based on genome-wide comparisons of GAS and SDSE indicated that speG is the direct descendant of a common ancestor of streptococcal SAgs, whereas smez was deleted from SDSE after SDSE and GAS split from a common ancestor. Cumulative nucleotide skew analysis of SDSE genomes suggested that speG was located outside segments of steeper slopes than the stable region in the genome, whereas the region flanking smez was unstable, as expected from the results of GAS. We also detected a previously undescribed staphylococcal SAg gene, selW, and a staphylococcal SAg -like gene, ssl, in the core genomes of all Staphylococcus aureus strains sequenced. Amino acid substitution analyses, based on dN/dS window analysis of the products encoded by speG, selW and ssl suggested that all three genes have been subjected to strong positive selection. Evolutionary analysis based on the Bayesian Markov chain Monte Carlo method showed that each clade included at least one direct descendant. Conclusions Our findings reveal a plausible model for the comprehensive evolutionary pathway of streptococcal and

  8. The staphylococcal enterotoxin (SE) family: SEB and siblings.

    Science.gov (United States)

    Krakauer, Teresa; Stiles, Bradley G

    2013-11-15

    Staphylococcus aureus plays an important role in numerous human cases of food poisoning, soft tissue, and bone infections, as well as potentially lethal toxic shock. This common bacterium synthesizes various virulence factors that include staphylococcal enterotoxins (SEs). These protein toxins bind directly to major histocompatibility complex class II on antigen-presenting cells and specific Vβ regions of T-cell receptors, resulting in potentially life-threatening stimulation of the immune system. Picomolar concentrations of SEs ultimately elicit proinflammatory cytokines that can induce fever, hypotension, multi-organ failure, and lethal shock. Various in vitro and in vivo models have provided important tools for studying the biological effects of, as well as potential vaccines/therapeutics against, the SEs. This review succinctly presents known physical and biological properties of the SEs, including various intervention strategies. In particular, SEB will often be portrayed as per biodefense concerns dating back to the 1960s.

  9. Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin

    Science.gov (United States)

    Hayworth, J L; Kasper, K J; Leon-Ponte, M; Herfst, C A; Yue, D; Brintnell, W C; Mazzuca, D M; Heinrichs, D E; Cairns, E; Madrenas, J; Hoskin, D W; McCormick, J K; Haeryfar, S M M

    2009-01-01

    Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome. PMID:19659771

  10. Live-cell imaging.

    Science.gov (United States)

    Cole, Richard

    2014-01-01

    It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions.

  11. Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins.

    Directory of Open Access Journals (Sweden)

    Wouter L W Hazenbos

    Full Text Available Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR. The prototype member of this family is clumping factor A (ClfA, a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

  12. Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins

    NARCIS (Netherlands)

    Hazenbos, Wouter L. W.; Kajihara, Kimberly K.; Vandlen, Richard; Morisaki, J. Hiroshi; Lehar, Sophie M.; Kwakkenbos, Mark J.; Beaumont, Tim; Bakker, Arjen Q.; Phung, Qui; Swem, Lee R.; Ramakrishnan, Satish; Kim, Janice; Xu, Min; Shah, Ishita M.; Diep, Binh An; Sai, Tao; Sebrell, Andrew; Khalfin, Yana; Oh, Angela; Koth, Chris; Lin, S. Jack; Lee, Byoung-Chul; Strandh, Magnus; Koefoed, Klaus; Andersen, Peter S.; Spits, Hergen; Brown, Eric J.; Tan, Man-Wah; Mariathasan, Sanjeev

    2013-01-01

    Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that

  13. Lactobacilli require physical contact to reduce staphylococcal TSST-1 secretion and vaginal epithelial inflammatory response

    NARCIS (Netherlands)

    Younes, Jessica A.; Reid, Gregor; van der Mei, Henny C.; Busscher, Henk J.

    Staphylococcus aureus biofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species Lactobacillus can alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is

  14. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections.

    Science.gov (United States)

    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N

    2015-06-26

    Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections.

  15. Modified wound dressing with phyto-nanostructured coating to prevent staphylococcal and pseudomonal biofilm development

    Science.gov (United States)

    Anghel, Ion; Holban, Alina Maria; Grumezescu, Alexandru Mihai; Andronescu, Ecaterina; Ficai, Anton; Anghel, Alina Georgiana; Maganu, Maria; Lazǎr, Veronica; Chifiriuc, Mariana Carmen

    2012-12-01

    This paper reports a newly fabricated nanophyto-modified wound dressing with microbicidal and anti-adherence properties. Nanofluid-based magnetite doped with eugenol or limonene was used to fabricate modified wound dressings. Nanostructure coated materials were characterized by TEM, XRD, and FT-IR. For the quantitative measurement of biofilm-embedded microbial cells, a culture-based method for viable cell count was used. The optimized textile dressing samples proved to be more resistant to staphylococcal and pseudomonal colonization and biofilm formation compared to the uncoated controls. The functionalized surfaces for wound dressing seems to be a very useful tool for the prevention of wound microbial contamination on viable tissues.

  16. Meta-analysis of staphylococcal diarrhea in some developing African countries

    Directory of Open Access Journals (Sweden)

    George P. Einstein

    2017-06-01

    Full Text Available Staphylococcal diarrhea is a common gastro-intestinal illness caused by poor water supply and unhygienic food preparation. Although, about 70% of diarrheal cases per year have been attributed to the consumption of contaminated foods, in Africa, few studies have reported the incidence of Staphylococcus-related diarrhea and the implicated virulence factors. We investigated the implications of common factors such as age of children, age of mothers, maternal level of education, gender of children, overall bacteria isolated, sanitary condition status and feeding type of mothers on the burden of staphylococcal diarrhea. A literature search was performed using Pubmed, Googlescholar, American society for microbiology (ASM journals and other sources. The quality of studies was assessed. Adjusted odds ratios with 95% confidence intervals (CI for the occurrence of Staphylococcus aureus amongst other causes were extracted. For each study, a multivariate logistic regression analysis of the adjusted odds ratio was performed to identify the risk factors on the burden of staphylococcal diarrhea. Six case-control studies were included in the meta–analysis. Quality of individual studies rages from 0.57–0.84 (median, 0.69. meta-analysis gave pooled odds ratios 3.27 (95% CI; 2.88–4.17. The results identified inadequate sanitary conditions and type of feeding as major risk factors.

  17. Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

    Directory of Open Access Journals (Sweden)

    Mark Fenton

    2013-01-01

    Full Text Available New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

  18. Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ib alpha and alpha(IIb)beta(3)

    NARCIS (Netherlands)

    De Haas, C. J. C.; Weeterings, C.; Vughs, M. M.; De Groot, P. G.; Van Strijp, J. A.; Lisman, T.

    2009-01-01

    Objectives: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results: When purified

  19. How we live and why we die the secret lives of cells

    CERN Document Server

    Wolpert, Lewis

    2009-01-01

    Cells are the basis of all life in the universe. Our bodies are made up of billions of them: an incredibly complex society that governs everything, from movement to memory and imagination. When we age, it is because our cells slow down; when we get ill, it is because our cells mutate or stop working. In "How We Live and Why we Die", Wolpert provides a clear explanation of the science that underpins our lives. He explains how our bodies function and how we derived from a single cell - the embryo. He examines the science behind the topics that are much discussed but rarely understood - stem-cell research, cloning, DNA - and explains how all life evolved from just one cell. Lively and passionate, "How We Live and Why we Die" is an accessible guide to understanding the human body and, essentially, life itself.

  20. Fast automatic quantitative cell replication with fluorescent live cell imaging

    Directory of Open Access Journals (Sweden)

    Wang Ching-Wei

    2012-01-01

    Full Text Available Abstract Background live cell imaging is a useful tool to monitor cellular activities in living systems. It is often necessary in cancer research or experimental research to quantify the dividing capabilities of cells or the cell proliferation level when investigating manipulations of the cells or their environment. Manual quantification of fluorescence microscopic image is difficult because human is neither sensitive to fine differences in color intensity nor effective to count and average fluorescence level among cells. However, auto-quantification is not a straightforward problem to solve. As the sampling location of the microscopy changes, the amount of cells in individual microscopic images varies, which makes simple measurement methods such as the sum of stain intensity values or the total number of positive stain within each image inapplicable. Thus, automated quantification with robust cell segmentation techniques is required. Results An automated quantification system with robust cell segmentation technique are presented. The experimental results in application to monitor cellular replication activities show that the quantitative score is promising to represent the cell replication level, and scores for images from different cell replication groups are demonstrated to be statistically significantly different using ANOVA, LSD and Tukey HSD tests (p-value Conclusion A robust automated quantification method of live cell imaging is built to measure the cell replication level, providing a robust quantitative analysis system in fluorescent live cell imaging. In addition, the presented unsupervised entropy based cell segmentation for live cell images is demonstrated to be also applicable for nuclear segmentation of IHC tissue images.

  1. Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCCmec)

    DEFF Research Database (Denmark)

    Rolo, Joana; Worning, Peder; Nielsen, Jesper Boye

    2017-01-01

    , many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several...

  2. Congenital staphylococcal scalded skin syndrome in a premature infant

    NARCIS (Netherlands)

    Haveman, LM; Fleer, A; de Vries, LS; Gerards, LJ

    2004-01-01

    A case of congenital staphylococcal scalded skin syndrome (SSSS) with fatal outcome in a premature infant is reported. An intrauterine infection with Staphylococcus aureus was probably the cause for the fulminant course of the disease. Despite adequate antibiotic treatment, the infant died within 24

  3. Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci

    Directory of Open Access Journals (Sweden)

    Somayyeh Hosseinzadeh

    2014-06-01

    Full Text Available Staphylococcal mastitis is a major and costly problem of dairy cattle all over the world. The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 7 dairy herds located in 4 different areas of East and West Azerbaijan provinces, Iran. Of the 158 mastitic milk samples collected, 113 staphylococcal isolates were identified (71.5% on the basis of cultural and biochemical features as well as by genus specific PCR. Then, species level identification of staphylococcal isolates was carried out using restriction fragment length polymorphism (RFLP analysis of the gap gene (933 bp. On the basis of polymerase chain reaction-RFLP, 10 different patterns were identified. Of 113 isolates, 5 (4.4% were Staphylococcus aureus and 108 (95.6% were coagulase-negative staphylococci (CoNS. Overall, nine different species of CoNS were identified as: 44 Staphylococcus haemolyticus (40.7%, 17 Staphylococcus chromogenes (15.7%, 11 Staphylococcus epidermidis, Staphylococcus warneri and Staphylococcus cohnii each (10.2%, 6 Staphylococcus simulans (5.5%, 4 Staphylococcus hominis (3.7%, 3 Staphylococcus capitis (2.7% and 1 Staphylococcus xylosus (0.9%. S. haemolyticus, S. chromogenes and S. warneri were the only species identified from clinical mastitis. No significant difference in staphylococcal IMI was found among the studied herds and regions. This study demonstrated that CoNS, especially S. haemolyticus and S. chromogenes, were predominant and thus be considered as emerging pathogens causing mastitis in the North West of Iran. Our results also revealed that the gap PCR-RFLP was useful for identifying staphylococcal isolates derived from bovine mastitis at species level.

  4. Persistent production of TH2-type cytokines and polyclonal B cell activation after chronic administration of staphylococcal enterotoxin B in mice

    NARCIS (Netherlands)

    Florquin, S.; Amraoui, Z.; Goldman, M.

    1996-01-01

    In order to study the immunopathological consequences of repeated exposure to bacterial superantigens, we evaluated the production of cytokines, the profile of serum immunoglobulins and the tissue damage in BALB/c mice injected twice a week for 3 weeks with 50 micrograms of staphylococcal

  5. Staphylococcal superantigens; toxic shock syndrome toxin-1 and enterotoxins in pediatric otitis media effusion: a brief report

    Directory of Open Access Journals (Sweden)

    Noorbakhsh S

    2013-03-01

    Full Text Available Background: Staphylococcal superantigens (SAg's may have some role in otitis media with effusion (OME. The aim of this study was the search of staphylococcal SAg's in middle ear effusion of children with OME.  Methods: This cross sectional-analytic study was done in ENT & pediatric wards upon 64 children with otitis media with effusion (OME between 1-15 years, (mean age=7.42+4 years of Rasoul Akram University Hospital, Tehran, Iran in 2009-2011. Fifty six percent (36 of cases were male, 43.8% (28 were female. Staphylococcal SAg's; Toxic Shock Syndrome Toxin-1 (TSST-1, Staphylococcal enterotoxin A, B, C, D (Enzyme immune assay, AB Cam, USA were detected in middle ear effusion samples after conventional culture.Results: None type of SAg's found in 39% of OME cases, enterotoxin B found in: 22%; enterotoxin A: 17%, enterotoxin C: 15.6%, enterotoxin D: 12.5%, Toxic Shock Syndrome Toxin-1 (TSST-1: 7.8% Mean age of cases with positive TSST-1, enterotoxin A, B, C, and D was: 1, 5, 8.6, 9.6 and 9.6 years respectively. Positive TSST had no agreement with positive enterotoxin A and C but had weak agreement with type B and D. Mean age of cases with positive TSST was one years which had significant difference with (7.9 years in cases with negative TSST test (P<0.0001.Conclusion: At least one or more type of staphylococcal toxins had found in middle ear effusion of 70% of OME cases with negative culture for Staphylococcus aureus. Even in culture negative cases, staphylococcal toxins might have some immunologic role in middle ear effusion forming. Finding the SAg's (at least one type are important for treatment of immunosuppressive or corticosteroid in cases with resistant OME.

  6. Staphylococcal phenotypes induced by naturally occurring and synthetic membrane-interactive polyphenolic β-lactam resistance modifiers.

    Directory of Open Access Journals (Sweden)

    Lucia Palacios

    Full Text Available Galloyl catechins, in particular (--epicatechin gallate (ECg, have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA; they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (--epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.

  7. Polyvalent Display of Biomolecules on Live Cells.

    Science.gov (United States)

    Shi, Peng; Zhao, Nan; Lai, Jinping; Coyne, James; Gaddes, Erin R; Wang, Yong

    2018-06-04

    Surface display of biomolecules on live cells offers new opportunities to treat human diseases and perform basic studies. Existing methods are primarily focused on monovalent functionalization, that is, the display of single biomolecules across the cell surface. Here we show that the surface of live cells can be functionalized to display polyvalent biomolecular structures through two-step reactions under physiological conditions. This polyvalent functionalization enables the cell surface to recognize the microenvironment one order of magnitude more effectively than with monovalent functionalization. Thus, polyvalent display of biomolecules on live cells holds great potential for various biological and biomedical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

    Science.gov (United States)

    Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  9. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

    Science.gov (United States)

    Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  10. Staphylococcal Superantigens Spark Host-Mediated Danger Signals

    Directory of Open Access Journals (Sweden)

    Terry eKrakauer

    2016-02-01

    Full Text Available Staphylococcal enterotoxin B (SEB of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific V regions of T-cell receptors (TCR and major histocompatibility complex (MHC class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes / macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1, IL-2, interferon γ (IFNγ, and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed towards TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR / MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase / mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an SEB-only model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA / RNA sensors, apoptosis / DNA damage-related molecules, immunoproteasome components, as well as anti-viral and IFN-stimulated genes. The host-wide induction of these, and other, anti-microbial defense genes provide evidence that SEB elicits danger signals resulting in multi

  11. Staphylococcus aureus and staphylococcal enterotoxin A in breaded chicken products: detection and behavior during the cooking process.

    Science.gov (United States)

    Pepe, Olimpia; Blaiotta, Giuseppe; Bucci, Francesca; Anastasio, Marilena; Aponte, Maria; Villani, Francesco

    2006-11-01

    In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a(w)) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4 degrees C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a(w) values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.

  12. Pharmacokinetic/Toxicity Properties of the New Anti-Staphylococcal Lead Compound SK-03-92

    Directory of Open Access Journals (Sweden)

    William R. Schwan

    2015-11-01

    Full Text Available Because of the potential of a new anti-staphylococcal lead compound SK-03-92 as a topical antibiotic, a patch, or an orally active drug, we sought to determine its safety profile and oral bioavailability. SK-03-92 had a high IC50 (125 μg/mL in vitro against several mammalian cell lines, and mice injected intraperiteonally at the highest dose did not exhibit gross toxicity (e.g., altered gait, ungroomed, significant weight loss. Single dose (100 μg/g pharmacokinetic (PK analysis with formulated SK-03-92 showed that peak plasma concentration (1.64 μg/mL was achieved at 20–30 min. Oral relative bioavailability was 8%, and the drug half-life was 20–30 min, demonstrating that SK-03-92 is likely not a candidate for oral delivery. Five-day and two-week PK analyses demonstrated that SK-03-92 plasma levels were low. Multi-dose analysis showed no gross adverse effects to the mice and a SK-03-92 peak plasma concentration of 2.12 μg/mL with the presence of significant concentrations of breakdown products 15 min after dosing. SK-03-92 appeared to be very safe based on tissue culture and mouse gross toxicity determinations, but the peak plasma concentration suggests that a pro-drug of SK-03-92 or preparation of analogs of SK-03-92 with greater bioavailability and longer half-lives are warranted.

  13. Interactions between semiconductor nanowires and living cells.

    Science.gov (United States)

    Prinz, Christelle N

    2015-06-17

    Semiconductor nanowires are increasingly used for biological applications and their small dimensions make them a promising tool for sensing and manipulating cells with minimal perturbation. In order to interface cells with nanowires in a controlled fashion, it is essential to understand the interactions between nanowires and living cells. The present paper reviews current progress in the understanding of these interactions, with knowledge gathered from studies where living cells were interfaced with vertical nanowire arrays. The effect of nanowires on cells is reported in terms of viability, cell-nanowire interface morphology, cell behavior, changes in gene expression as well as cellular stress markers. Unexplored issues and unanswered questions are discussed.

  14. 4Pi-confocal microscopy of live cells

    Science.gov (United States)

    Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

    2002-06-01

    By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

  15. Staphylococcal scalded skin syndrome in a premature newborn caused by methicillin-resistant Staphylococcus aureus: case report

    Directory of Open Access Journals (Sweden)

    Andreas Hörner

    Full Text Available CONTEXT: Staphylococcal scalded skin syndrome is an exfoliative skin disease. Reports of this syndrome in newborns caused by methicillin-resistant Staphylococcus aureus are rare but, when present, rapid diagnosis and treatment is required in order to decrease morbidity and mortality. CASE REPORT: A premature newly born girl weighing 1,520 g, born with a gestational age of 29 weeks and 4 days, developed staphylococcal scalded skin syndrome on the fifth day of life. Cultures on blood samples collected on the first and fourth days were negative, but Pseudomonas aeruginosa and Enterococcus sp. (vancomycin-sensitive developed in blood cultures performed on the day of death (seventh day, and Pseudomonas aeruginosa and Serratia marcescens were identified in cultures on nasopharyngeal, buttock and abdominal secretions. In addition to these two Gram-negative bacilli, methicillin-resistant Staphylococcus aureus was isolated in a culture on the umbilical stump (seventh day. The diagnosis of staphylococcal scalded skin syndrome was based on clinical criteria.

  16. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-05-01

    Nanostructures fabricated by different methods have become increasingly important for various applications at the cellular level. In order to understand how these nanostructures “behave” and for studying their internalization kinetics, several attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic pH-dependent dye pHrodo™ Red, covalently bound to the aminosilane surface. Time-lapse live imaging of human colon carcinoma HCT 116 cells interacting with the labeled iron nanowires is performed for 24 hours. As the pHrodo™ Red conjugated nanowires are non-fluorescent outside the cells but fluoresce brightly inside, internalized nanowires are distinguished from non-internalized ones and their behavior inside the cells can be tracked for the respective time length. A machine learning-based computational framework dedicated to automatic analysis of live cell imaging data, Cell Cognition, is adapted and used to classify cells with internalized and non-internalized nanowires and subsequently determine the uptake percentage by cells at different time points. An uptake of 85 % by HCT 116 cells is observed after 24 hours incubation at NW-to-cell ratios of 200. While the approach of using pHrodo™ Red for internalization studies is not novel in the literature, this study reports for the first time the utilization of a machine-learning based time-resolved automatic analysis pipeline for quantification of nanowire uptake by cells. This pipeline has also been used for comparison studies with nickel nanowires coated with APTES and labeled with pHrodo™ Red, and another cell line derived from the cervix carcinoma, HeLa. It has thus the potential to be used for studying the interaction of different types of nanostructures with potentially any live cell types.

  17. Knowledge and Perceptions About Community-acquired Staphylococcal Infections Among Health Care Workers in Hawai‘i

    Science.gov (United States)

    Tice, Alan D; Hurwitz, Eric L; Katz, Alan R

    2013-01-01

    Since the early 1990s, national rates of methicillin-resistant Staphylococcus aureus (MRSA) infections have increased dramatically.1,2 Initially identified in health care settings, community-acquired MRSA is now a major public health concern. With Hawai‘i's expanding S. aureus and MRSA epidemic closely approximating the national trend in inpatient and outpatient settings,7,8 a high level of knowledge and awareness among health care workers is essential to successfully control this evolving epidemic. Health care and related workers were surveyed to assess their knowledge and perceptions about staphylococcal and MRSA infections. Knowledge was estimated by demonstrated ability to correctly identify risk factors including diabetes and obesity, as well as to demonstrate awareness of a growing staphylococcal and MRSA epidemic.9,10 Perceptions were estimated by level of concern of antibiotic resistance as well as of the severity of the staphylococcal and MRSA epidemic. Variations in knowledge and perception concerning basic principles associated with S. aureus infections as well as characteristics of the evolving S. aureus and MRSA epidemic were observed among various occupations (advance clinical practitioners, nurses, public health professionals, athletic trainers, and non-medical workers) as well as work locations (hospital, community, and non-clinical community). Overall, health care and related workers in community settings demonstrated disparities in knowledge regarding S. aureus and MRSA infections. They were also more likely to misperceive this growing threat. These findings provide support for focused educational interventions targeting community health care and related workers to improve awareness of staphylococcal infections in order to successfully address and combat this evolving epidemic. PMID:24069572

  18. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

    Directory of Open Access Journals (Sweden)

    Bettina Stieber

    Full Text Available S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

  19. Knowledge and perceptions about community-acquired staphylococcal infections among health care workers in Hawai'i.

    Science.gov (United States)

    Dunn, Brandyn S; Tice, Alan D; Hurwitz, Eric L; Katz, Alan R

    2013-09-01

    Since the early 1990s, national rates of methicillin-resistant Staphylococcus aureus (MRSA) infections have increased dramatically.1,2 Initially identified in health care settings, community-acquired MRSA is now a major public health concern. With Hawai'i's expanding S. aureus and MRSA epidemic closely approximating the national trend in inpatient and outpatient settings,7,8 a high level of knowledge and awareness among health care workers is essential to successfully control this evolving epidemic. Health care and related workers were surveyed to assess their knowledge and perceptions about staphylococcal and MRSA infections. Knowledge was estimated by demonstrated ability to correctly identify risk factors including diabetes and obesity, as well as to demonstrate awareness of a growing staphylococcal and MRSA epidemic.9,10 Perceptions were estimated by level of concern of antibiotic resistance as well as of the severity of the staphylococcal and MRSA epidemic. Variations in knowledge and perception concerning basic principles associated with S. aureus infections as well as characteristics of the evolving S. aureus and MRSA epidemic were observed among various occupations (advance clinical practitioners, nurses, public health professionals, athletic trainers, and non-medical workers) as well as work locations (hospital, community, and non-clinical community). Overall, health care and related workers in community settings demonstrated disparities in knowledge regarding S. aureus and MRSA infections. They were also more likely to misperceive this growing threat. These findings provide support for focused educational interventions targeting community health care and related workers to improve awareness of staphylococcal infections in order to successfully address and combat this evolving epidemic.

  20. Microencapsulation Of Living Cells

    Science.gov (United States)

    Chang, Manchium; Kendall, James M.; Wang, Taylor G.

    1989-01-01

    In experimental technique, living cells and other biological materials encapsulated within submillimeter-diameter liquid-filled spheres. Sphere material biocompatible, tough, and compliant. Semipermeable, permitting relatively small molecules to move into and out of sphere core but preventing passage of large molecules. New technique promises to make such spherical capsules at high rates and in uniform, controllable sizes. Capsules injected into patient through ordinary hypodermic needle. Promising application for technique in treatment of diabetes. Also used to encapsulate pituitary cells and thyroid hormone adrenocortical cells for treatment of other hormonal disorders, to encapsulate other secreting cells for transplantation, and to package variety of pharmaceutical products and agricultural chemicals for controlled release.

  1. Live cell refractometry using microfluidic devices.

    Science.gov (United States)

    Lue, Niyom; Popescu, Gabriel; Ikeda, Takahiro; Dasari, Ramachandra R; Badizadegan, Kamran; Feld, Michael S

    2006-09-15

    Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

  2. Staphylococcal enterotoxin C2 promotes osteogenesis and suppresses osteoclastogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Fu, Wei-ming; Zhu, Xiao; Wang, Hua; Wei-Mao Wang; Chen, Ju-yu; Liang, Yan; Zhang, Jin-fang; Kung, Hsiang-fu

    2014-03-10

    As a super-antigen, staphylococcal enterotoxin C2 (SEC2) stimulates the release of massive inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and interleukin-2 (IL-2) which are documented to implicate osteoblast differentiation. In the present study, SEC2 was found to significantly improve the osteoblast differentiation by up-regulating BMP2 and Runx2/Cbfa1 expression. Interferon (IFN)-inducible gene IFI16, a co-activator of Runx2/Cbfa1, was also activated by SEC2 in the osteoblast differentiation. In addition, exogenous introduction of SEC2 stimulated OPG expression and suppressed RANKL, suggesting suppression of osteoclastogenesis in hMSCs. Therefore, our results displayed that SEC2 plays an important role in the commitment of MSC to the osteoblast and it might be a potential new therapeutic candidate for bone regeneration. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Staphylococcal Entertotoxins of the Enterotoxin Gene Cluster (egcSEs Induce Nitrous Oxide- and Cytokine Dependent Tumor Cell Apoptosis in a Broad Panel of Human Tumor Cells

    Directory of Open Access Journals (Sweden)

    David eTerman

    2013-08-01

    Full Text Available The egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing theses proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25% in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 µM, a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA (0.3 mM, an NO synthase antagonist. Cell free supernatants (CSFs of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies respectively and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma>osteogenic sarcoma>melanoma>breast carcinoma>neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower respectively than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells.

  4. Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

    Science.gov (United States)

    Macori, Guerrino; Bellio, Alberto; Bianchi, Daniela Manila; Gallina, Silvia; Adriano, Daniela; Zuccon, Fabio; Chiesa, Francesco; Acutis, Pier Luigi; Casalinuovo, Francesco; Decastelli, Lucia

    2016-04-19

    In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus , and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa -type. Showing a high heterogeneity of isolates, spa -type t 13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  5. Molecular typing of Staphylococcus aureus isolate responsible for staphylococcal poisoning incident in homemade food

    Directory of Open Access Journals (Sweden)

    Guerrino Macori

    2016-06-01

    Full Text Available In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA and D (SED, respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  6. Recent advances in live cell imaging of hepatoma cells

    Science.gov (United States)

    2014-01-01

    Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

  7. Functional living biointerfaces to direct cell-material interaction

    OpenAIRE

    Rodrigo Navarro, Aleixandre

    2016-01-01

    [EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engine...

  8. Prevalence of Staphylococcal Enterotoxins in Ready-to-Eat Foods Sold in Istanbul.

    Science.gov (United States)

    Ulusoy, Beyza H; Çakmak Sancar, Burcu; Öztürk, Muhsin

    2017-10-01

    The aim of this study was to investigate the prevalence of staphylococcal enterotoxins (SEs) in ready-to-eat (RTE) foods sold in Istanbul, Turkey. A total of 5,241 samples were randomly collected from various caterers, hotels, and restaurants from 2014 to 2016. The samples were classified into four groups: (i) various cooked RTE meat and vegetable meals, (ii) various RTE salads, charcuterie, and cold appetizers, (iii) various cooked RTE bakery products (pasta, pastries, pizza, pita, ravioli, etc.), and (iv) any cooked RTE sweets and desserts (pudding, custard, cream, ashura, etc.). The samples were examined for the presence of SEs by 3M Tecra Staph Enterotoxin Visual Immunoassay method, which is a manual enzyme-linked immunosorbent assay method. Among all samples, only 1 (0.019%) RTE meal (vegetable meal with meat) was found to be contaminated with SEs, a good result in terms of staphylococcal food poisoning risk and public health.

  9. Staphylococcal endogenous endophthalmitis in association with pyogenic vertebral osteomyelitis.

    Science.gov (United States)

    Steeples, L R; Jones, N P

    2016-01-01

    PURPOSE To describe pyogenic vertebral osteomyelitis as a rare infection associated with endogenous endophthalmitis.METHODS A retrospective review of three patients with endogenous endophthalmitis and sepsis due to underlying Staphylococcal vertebral osteomyelitis presenting during a 21-month time period. The ophthalmic and systemic features and management and outcomes are presented.RESULTS One patient developed unilateral endophthalmitis with cervical spine osteomyelitis, Staphylococcus aureus being isolated from blood cultures. The second presented with bilateral endophthalmitis with disseminated Methicillin-resistant S. aureus (MRSA) infection, with thoracic and lumbar discitis and para-spinal abscesses. MRSA was cultured from vitreous, blood, and synovial fluid. Both patients received prolonged courses of intravenous antibiotics. Intravitreal antibiotic therapy was used in the second patient. Excellent visual and systemic outcomes were achieved in both cases with no ocular complications. The third patient developed lumbar osteomyelitis following spinal surgery and presented with disseminated S. aureus sepsis including unilateral endogenous endophthalmitis. Despite systemic antibiotics and intensive care the patient died.CONCLUSIONS Endogenous endophthalmitis should be suspected in septic patients developing eye symptoms. Endogenous endophthalmitis with staphylococcal bone infection is a rare but serious condition. Osteomyelitis should be considered as an infective source in any such patient reporting bone pain or reduced spinal mobility. Prompt investigation and treatment can achieve favourable visual and systemic outcomes.

  10. Live cell imaging of in vitro human trophoblast syncytialization.

    Science.gov (United States)

    Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

    2014-06-01

    Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

  11. Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guojun [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Xu, Mingkai, E-mail: mkxu@iae.ac.cn [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Zhang, Huiwen [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Song, Yubo [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Wang, Jian; Zhang, Chenggang [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China)

    2016-12-15

    Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis

  12. Detecting and Tracking Nonfluorescent Nanoparticles Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Gufeng; Fang, Ning

    2012-01-17

    Precisely imaging and tracking dynamic biological processes in live cells are crucial for both fundamental research in life sciences and biomedical applications. Nonfluorescent nanoparticles are emerging as important optical probes in live-cell imaging because of their excellent photostability, large optical cross sections, and low cytotoxicity. Here, we provide a review of recent development in optical imaging of nonfluorescent nanoparticle probes and their applications in dynamic tracking and biosensing in live cells. A brief discussion on cytotoxicity of nanoparticle probes is also provided.

  13. Long term imaging of living brain cancer cells

    Science.gov (United States)

    Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

    2018-02-01

    QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

  14. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  15. Assessing resolution in live cell structured illumination microscopy

    Science.gov (United States)

    Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

    2017-12-01

    Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

  16. Paracentrin 1, a synthetic antimicrobial peptide from the sea-urchin Paracentrotus lividus, interferes with staphylococcal and Pseudomonas aeruginosa biofilm formation.

    Science.gov (United States)

    Schillaci, Domenico; Cusimano, Maria Grazia; Spinello, Angelo; Barone, Giampaolo; Russo, Debora; Vitale, Maria; Parrinello, Daniela; Arizza, Vincenzo

    2014-01-01

    The rise of antibiotic-resistance as well as the reduction of investments by pharmaceutical companies in the development of new antibiotics have stimulated the investigation for alternative strategies to conventional antibiotics. Many antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells and, due to their mode of action the development of resistance is considered unlikely. We recently characterized an antimicrobial peptide that was called Paracentrin 1 from the 5-kDa peptide fraction from the coelomocyte cytosol of the Paracentrotus lividus. In this study, the chemically synthesized Paracentrin 1, was tested for its antimicrobial and antibiofilm properties against reference strains of Gram positive and Gram negative. The Paracentrin 1 was active against planktonic form of staphylococcal strains (reference and isolates) and Pseudomonas aeruginosa ATCC 15442 at concentrations ranging from 12.5 to 6.2 mg/ml. The Paracentrin 1 was able to inhibit biofilm formation of staphylococcal and Pseudomonas aeruginosa strains at concentrations ranging from 3.1 to 0.75 mg/ml. We consider the tested peptide as a good starting molecule for novel synthetic derivatives with improved pharmaceutical potential.

  17. Hiding in plain sight: immune evasion by the staphylococcal protein SdrE.

    Science.gov (United States)

    Herr, Andrew B; Thorman, Alexander W

    2017-05-10

    The human immune system is responsible for identification and destruction of invader cells, such as the bacterial pathogen Staphylococcus aureus In response, S. aureus brings to the fight a large number of virulence factors, including several that allow it to evade the host immune response. The staphylococcal surface protein SdrE was recently reported to bind to complement Factor H, an important regulator of complement activation. Factor H attaches to the surface of host cells to inhibit complement activation and amplification, preventing the destruction of the host cell. SdrE binding to Factor H allows S. aureus to mimic a host cell and reduces bacterial killing by granulocytes. In a new study published in Biochemical Journal , Zhang et al. describe crystal structures of SdrE and its complex with the C-terminal portion of Factor H. The structure of SdrE and its interaction with the Factor H peptide closely resemble a family of surface proteins that recognize extracellular matrix components such as fibrinogen. However, unbound SdrE forms a novel 'Closed' conformation with an occluded peptide-binding groove. These structures reveal a fascinating mechanism for immune evasion and provide a potential avenue for the development of novel antimicrobial agents to target SdrE. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  18. Analysis of live cell images: Methods, tools and opportunities.

    Science.gov (United States)

    Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

    2017-02-15

    Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

  19. Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

    Science.gov (United States)

    Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

    2016-04-01

    The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM

  20. Nosocomial coagulase-negative staphylococcal infections in bone marrow transplantation recipients with central vein catheter. A 5-year prospective study.

    Science.gov (United States)

    Engelhard, D; Elishoov, H; Strauss, N; Naparstek, E; Nagler, A; Simhon, A; Raveh, D; Slavin, S; Or, R

    1996-02-15

    The purpose of this study was to examine coagulase-negative staphylococcal infections in bone marrow transplantation (BMT) patients with central vein catheters by investigating incidence, clinical relevance, risk factors, methicillin resistance, clinical impact of initial empiric antimicrobial therapy without vancomycin, and management of documented catheter-related infections. A 5-year prospective study was conducted with daily evaluation of 242 BMT patients during hospitalization, including clinical assessment and blood culture via the Hickman/Broviac catheter. If fever or infected appearance occurred, peripheral blood cultures or exit site cultures, respectively, were done. Results showed a septicemia incidence of 7.0%, including in 6 patients following colonization, in 1 patient with tunnel infection, in 1 patient with thrombophlebitis, in 1 patient with exit site infection, and in 8 patients with septicemia of unknown origin. Total colonization incidence was 7%, with colonization only in 11 patients who had 16 episodes; incidence of exit site infection was 3.7%. Age > or = 18 years was the only identified risk factor for developing staphylococcal infection (P = 0.03). Despite a methicillin resistance rate of 45% and omission of vancomycin from the routine initial empiric antimicrobial regimen, the clinical course of coagulase-negative staphylococcal infections was relatively benign. A single patient, who experienced marrow rejection, died on day +31 with septicemia and only one patient experienced microbiological failure with recurrent colonization. Bacteria grown in both aerobic and anaerobic bottles were more likely true bacteremia than contaminant (P = 0.03). We conclude that the hazard of coagulase-negative staphylococcal infection does not mandate inclusion of a glycopeptide in the initial empiric antimicrobial regimen in BMT patients, even during febrile neutropenia. Hickman/Broviac-related staphylococcal infections, except for tunnel infection or

  1. 78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

    Science.gov (United States)

    2013-08-14

    ...] Consolidation of Wound Care Products Containing Live Cells AGENCY: Food and Drug Administration, HHS. ACTION... certain wound care products containing live cells from the Center for Devices and Radiological Health... CDRH and CBER. FDA believes that as more wound care products containing live cells are developed such...

  2. Cross-sectional study to identify staphylococcal species isolated from teat and inguinal skin of different-aged dairy heifers.

    Science.gov (United States)

    Adkins, P R F; Dufour, S; Spain, J N; Calcutt, M J; Reilly, T J; Stewart, G C; Middleton, J R

    2018-04-01

    The purpose of this study was to describe the prevalence and distribution of staphylococcal species on the teat and inguinal skin of dairy heifers across the various stages of the heifer life cycle. The cross-sectional study included 106 Holstein heifers with an age range of 0 d to 27 mo that were selected from 11 different groups, based on housing type and age, on a single dairy operation. A composite swabbing sample including all 4 teats and a second composite sample including both inguinal regions of each heifer were collected using gas-sterilized electrostatic dusters (Swiffers; Procter and Gamble, Cincinnati, OH). Swabbing samples were mixed with 10 mL of sterile saline, agitated, and cultured on mannitol salt agar plates. At 24 h, plates were read and up to 10 staphylococcal colonies were saved for further analysis. Staphylococcal isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or PCR amplification and partial sequencing of rpoB or tuf. The prevalence of staphylococci was compared between the inguinal and teat regions using the chi-squared or Fisher's exact test, as applicable. Logistic regression models were used to investigate the relationship between a heifer's age (treated as a quantitative continuous variable) and the probability of isolating a given staphylococcal species from a given body site (inguinal region or teats). Overall, the most common species identified were Staphylococcus haemolyticus followed by Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus devriesei, and Staphylococcus sciuri. Staphylococcus aureus was more prevalent on the teat than in the inguinal region, whereas Staphylococcus arlettae was more prevalent in the inguinal region than on the teat. All other staphylococcal species were as likely to be found on the teat skin as the inguinal region skin. Isolation from the inguinal and teat skin was associated with age for Staphylococcus agnetis, S. chromogenes

  3. Optical imaging of non-fluorescent nanoparticle probes in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Gufeng; Stender, Anthony S.; Sun, Wei; and Fang, Ning

    2009-12-17

    Precise imaging of cellular and subcellular structures and dynamic processes in live cells is crucial for fundamental research in life sciences and in medical applications. Non-fluorescent nanoparticles are an important type of optical probe used in live-cell imaging due to their photostability, large optical cross-sections, and low toxicity. Here, we provide an overview of recent developments in the optical imaging of non-fluorescent nanoparticle probes in live cells.

  4. Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli

    Directory of Open Access Journals (Sweden)

    Takamasa Ishidome

    2017-08-01

    Full Text Available Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.

  5. Mupirocin in the Treatment of Staphylococcal Infections in Chronic Rhinosinusitis: A Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Jong Seung Kim

    Full Text Available Saline irrigation of the nasal cavity is a classic and effective treatment for acute or chronic rhinosinusitis. Topical antibiotics such as mupirocin have been widely used for recalcitrant chronic rhinosinusitis. Therefore, the purpose of this study was to evaluate the effect of saline irrigation using mupirocin.A systematic literature review and meta-analysis of mupirocin saline irrigation were performed using EMBASE, MEDLINE, and Cochrane library through December 2015. Data were analyzed with R 3.2.2 software. A random effects model was used because of the diversity of included studies. Sensitivity analysis of particular tested groups and single proportion tests were also performed. The main outcome measure was residual staphylococcal infection, as confirmed by culture or PCR.Two RCTs, two prospective studies and two retrospective studies were included. A random effects model meta-analysis of the pooled data identified a relative risk of residual infection of 0.13 (95% CI: 0.06-0.26, p<0.05 with low heterogeneity (I2 = 0%. The proportion of residual staphylococcal infections after 1 month was 0.08 (95% CI: 0.04-0.16. However, this proportion increased to 0.53 at 6 months (95% CI: 0.27-0.78.The short-term use of mupirocin has a strongly reductive effect on staphylococcal infection in chronic rhinosinusitis. Although there is currently a lack of clear evidence, future studies with well-designed inclusion criteria and randomized controlled trials are needed to examine mupirocin's long-term effect on chronic rhinosinusitis.

  6. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  7. Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition.

    Directory of Open Access Journals (Sweden)

    Karin E J Rödström

    Full Text Available T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR, is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.

  8. Information management for high content live cell imaging

    Directory of Open Access Journals (Sweden)

    White Michael RH

    2009-07-01

    Full Text Available Abstract Background High content live cell imaging experiments are able to track the cellular localisation of labelled proteins in multiple live cells over a time course. Experiments using high content live cell imaging will generate multiple large datasets that are often stored in an ad-hoc manner. This hinders identification of previously gathered data that may be relevant to current analyses. Whilst solutions exist for managing image data, they are primarily concerned with storage and retrieval of the images themselves and not the data derived from the images. There is therefore a requirement for an information management solution that facilitates the indexing of experimental metadata and results of high content live cell imaging experiments. Results We have designed and implemented a data model and information management solution for the data gathered through high content live cell imaging experiments. Many of the experiments to be stored measure the translocation of fluorescently labelled proteins from cytoplasm to nucleus in individual cells. The functionality of this database has been enhanced by the addition of an algorithm that automatically annotates results of these experiments with the timings of translocations and periods of any oscillatory translocations as they are uploaded to the repository. Testing has shown the algorithm to perform well with a variety of previously unseen data. Conclusion Our repository is a fully functional example of how high throughput imaging data may be effectively indexed and managed to address the requirements of end users. By implementing the automated analysis of experimental results, we have provided a clear impetus for individuals to ensure that their data forms part of that which is stored in the repository. Although focused on imaging, the solution provided is sufficiently generic to be applied to other functional proteomics and genomics experiments. The software is available from: fhttp://code.google.com/p/livecellim/

  9. A cell transportation solution that preserves live circulating tumor cells in patient blood samples

    International Nuclear Information System (INIS)

    Stefansson, Steingrimur; Adams, Daniel L.; Ershler, William B.; Le, Huyen; Ho, David H.

    2016-01-01

    Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90 % viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs

  10. A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

    Science.gov (United States)

    Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

    2016-05-06

    Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after

  11. PeakForce Tapping resolves individual microvilli on living cells.

    Science.gov (United States)

    Schillers, Hermann; Medalsy, Izhar; Hu, Shuiqing; Slade, Andrea L; Shaw, James E

    2016-02-01

    Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

  12. Laser-Raman spectroscopy of living cells

    International Nuclear Information System (INIS)

    Webb, S.J.

    1980-01-01

    Investigations into the laser-Raman shift spectra of bacterial and mammalian cells have revealed that many Raman lines observed at 4-6 K, do not appear in the spectra of cells held at 300 K. At 300 K, Raman activity, at set frequencies, is observed only when the cells are metabolically active; however, the actual live cell spectrum, between 0 and 3400 cm -1 , has been found to alter in a specific way with time as the cells' progress through their life cycles. Lines above 300 cm -1 , from in vivo Raman active states, appear to shift to higher wave numbers whereas those below 300 cm -1 seem to shift to lower ones. The transient nature of many shift lines observed and the intensity of them when present in the spectrum indicates that, in, vivo, a metabolically induced condensation of closely related states occurs at a set time in the life of a living cell. In addition, the calculated ratio between the intensities of Stokes and anti-Stokes lines observed suggests that the metabolically induced 'collective' Raman active states are produced, in vivo, by non thermal means. It appears, therefore, that the energetics of the well established cell 'time clock' may be studied by laser-Raman spectroscopy; moreover, Raman spectroscopy may yield a new type of information regarding the physics of such biological phenomena as nutrition, virus infection and oncogenesis. (orig.)

  13. Microencapsulating and Banking Living Cells for Cell-Based Medicine

    Directory of Open Access Journals (Sweden)

    Wujie Zhang

    2011-01-01

    Full Text Available A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

  14. Plain chest film findings of staphylococcal pneumonia in children

    International Nuclear Information System (INIS)

    Kim, Sook Young; Cha, Seong Sook; Kim, Jong Deok; Chung, Duck Hwan

    1991-01-01

    Infants during the first year of life are particularly prone to the development of staphylococcal pneumonia: in fact, it is the commonest bacterial cause of death due to respiratory tract infection in this age group. Pneumatoceles within the lungs and pleural complication such as pleural effusion, empyema, pyopneumothorax and pneumothorax are characteristics in this pneumonia. Retrospectively we reviewed 22 patients of staphylococcal pneumonia in children diagnosed by pleural fluid or blood culture, throat suction cytology, or culture of pus from thigh, joint fluid or ear discharge in one or combining them during the recent 5 years from January, 1985 to December, 1989 and obtained the following results: Male to female ratio was 1:1 and peak incidence was between 1 month and 1 year of age (45.5% : 10/22). Fever, coughing and dyspnea were the predominant symptoms, and duration of symptoms was 4 to 7 dyas in 59%. Plain chest film findings on admission day: 1) 27.3% (6/22) showed the lesion almost entirely restricted to the lung with ratio of 2:1 of right and left involvement and these were treated without complication. 2) Only one case (4.5%) showed pneumatoceles combined with pneumonic infiltration and pleural effusion. 3) Pleural lesion occurred in 72.7% (16/22) : 22.7% (5/22) without pneumonic infiltration and 50% (11/22) with pneumonic infiltration. They were empyema, pyopneumothorax, and pleural effusion in descending order. Mortalty was 4.5% (1/22)

  15. AFM review study on pox viruses and living cells.

    Science.gov (United States)

    Ohnesorge, F M; Hörber, J K; Häberle, W; Czerny, C P; Smith, D P; Binnig, G

    1997-10-01

    Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

  16. Staphylococcal Bicomponent Pore-Forming Toxins: Targets for Prophylaxis and Immunotherapy

    Directory of Open Access Journals (Sweden)

    M. Javad Aman

    2014-03-01

    Full Text Available Staphylococccus aureus represents one of the most challenging human pathogens as well as a common colonizer of human skin and mucosal surfaces. S. aureus causes a wide range of diseases from skin and soft tissue infection (SSTI to debilitating and life-threatening conditions such as osteomyelitis, endocarditis, and necrotizing pneumonia. The range of diseases reflects the remarkable diversity of the virulence factors produced by this pathogen, including surface antigens involved in the establishment of infection and a large number of toxins that mediate a vast array of cellular responses. The staphylococcal toxins are generally believed to have evolved to disarm the innate immune system, the first line of defense against this pathogen. This review focuses on recent advances on elucidating the biological functions of S. aureus bicomponent pore-forming toxins (BCPFTs and their utility as targets for preventive and therapeutic intervention. These toxins are cytolytic to a variety of immune cells, primarily neutrophils, as well as cells with a critical barrier function. The lytic activity of BCPFTs towards immune cells implies a critical role in immune evasion, and a number of epidemiological studies and animal experiments relate these toxins to clinical disease, particularly SSTI and necrotizing pneumonia. Antibody-mediated neutralization of this lytic activity may provide a strategy for development of toxoid-based vaccines or immunotherapeutics for prevention or mitigation of clinical diseases. However, certain BCPFTs have been proposed to act as danger signals that may alert the immune system through an inflammatory response. The utility of a neutralizing vaccination strategy must be weighed against such immune-activating potential.

  17. Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy.

    Science.gov (United States)

    Aman, M Javad; Adhikari, Rajan P

    2014-03-04

    Staphylococccus aureus represents one of the most challenging human pathogens as well as a common colonizer of human skin and mucosal surfaces. S. aureus causes a wide range of diseases from skin and soft tissue infection (SSTI) to debilitating and life-threatening conditions such as osteomyelitis, endocarditis, and necrotizing pneumonia. The range of diseases reflects the remarkable diversity of the virulence factors produced by this pathogen, including surface antigens involved in the establishment of infection and a large number of toxins that mediate a vast array of cellular responses. The staphylococcal toxins are generally believed to have evolved to disarm the innate immune system, the first line of defense against this pathogen. This review focuses on recent advances on elucidating the biological functions of S. aureus bicomponent pore-forming toxins (BCPFTs) and their utility as targets for preventive and therapeutic intervention. These toxins are cytolytic to a variety of immune cells, primarily neutrophils, as well as cells with a critical barrier function. The lytic activity of BCPFTs towards immune cells implies a critical role in immune evasion, and a number of epidemiological studies and animal experiments relate these toxins to clinical disease, particularly SSTI and necrotizing pneumonia. Antibody-mediated neutralization of this lytic activity may provide a strategy for development of toxoid-based vaccines or immunotherapeutics for prevention or mitigation of clinical diseases. However, certain BCPFTs have been proposed to act as danger signals that may alert the immune system through an inflammatory response. The utility of a neutralizing vaccination strategy must be weighed against such immune-activating potential.

  18. Live cell microscopy of DNA damage response in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina; Gallina, Irene; Eckert-Boulet, Nadine Valerie

    2012-01-01

    live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction...

  19. Lab-on-a-chip for label free biological semiconductor analysis of Staphylococcal Enterotoxin B

    NARCIS (Netherlands)

    Yang, Minghui; Sun, Steven; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

    2010-01-01

    We describe a new lab-on-a-chip (LOC) which utilizes a biological semiconductor (BSC) transducer for label free analysis of Staphylococcal Enterotoxin B (SEB) (or other biological interactions) directly and electronically. BSCs are new transducers based on electrical percolation through a

  20. Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

    Science.gov (United States)

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

    2015-05-27

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

  1. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    Science.gov (United States)

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  2. Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

    DEFF Research Database (Denmark)

    Canovas de la Nuez, Jaime; Baldry, Mara; Bojer, Martin S

    2016-01-01

    -inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction...... between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus...... suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources...

  3. Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

    Science.gov (United States)

    Wöllert, Torsten; Langford, George M

    2016-01-01

    Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

  4. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    International Nuclear Information System (INIS)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8 + T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract SEB

  5. Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles.

    Science.gov (United States)

    Cardoso Dos Santos, M; Goetz, J; Bartenlian, H; Wong, K-L; Charbonnière, L J; Hildebrandt, N

    2018-04-18

    Fluorescent nanoparticles (NPs) have become irreplaceable tools for advanced cellular and subcellular imaging. While very bright NPs require excitation with UV or visible light, which can create strong autofluorescence of biological components, NIR-excitable NPs without autofluorescence issues exhibit much lower brightness. Here, we show the application of a new type of surface-photosensitized terbium NPs (Tb-NPs) for autofluorescence-free intracellular imaging in live HeLa cells. The combination of exceptionally high brightness, high photostability, and long photoluminecence (PL) lifetimes for highly efficient suppression of the short-lived autofluorescence allowed for time-gated PL imaging of intracellular vesicles over 72 h without toxicity and at extremely low Tb-NP concentrations down to 12 pM. Detection of highly resolved long-lifetime (ms) PL decay curves from small (∼10 μm 2 ) areas within single cells within a few seconds emphasized the unprecedented photophysical properties of Tb-NPs for live-cell imaging that extend well beyond currently available nanometric imaging agents.

  6. Direct and dynamic detection of HIV-1 in living cells.

    Directory of Open Access Journals (Sweden)

    Jonas Helma

    Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

  7. Imaging Proteolysis by Living Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2000-01-01

    Full Text Available Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549 through the use of quenchedfluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin 13-selective cysteine protease inhibitor, intracellular fluorescence was decreased ~90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ~50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1 a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2 the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

  8. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA)

    Science.gov (United States)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27,078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, ...

  9. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    Science.gov (United States)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  10. Expression of Staphylococcal Enterotoxins under Stress Encountered during Food Production and Preservation.

    Science.gov (United States)

    Schelin, Jenny; Susilo, Yusak Budi; Johler, Sophia

    2017-12-15

    Staphylococcal food poisoning (SFP) is the most prevalent cause of food-borne intoxications worldwide. Consumption of enterotoxins preformed in food causes violent vomiting and can be fatal in children and the elderly. While being repressed by competing bacteria in most matrices, Staphylococcus aureus benefits from crucial competitive advantages in foods with high osmolarity or low pH. During recent years, the long-standing belief in the feasibility of assessing SFP risk based on colony-forming units of S. aureus present in food products has been disproven. Instead, researchers and food business operators are acutely aware of the imminent threat arising from unforeseeable enterotoxin production under stress conditions. This paradigm shift led to a variety of new publications enabling an improved understanding of enterotoxin expression under stress conditions encountered in food. The wealth of data provided by these studies is extremely diverse, as it is based on different methodological approaches, staphylococcal strains, stressors, and enterotoxins. Therefore, in this review, we aggregated and critically evaluated the complex findings of these studies, to provide readers with a current overview of the state of research in the field.

  11. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

    Science.gov (United States)

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL) significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning.

  12. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

    Directory of Open Access Journals (Sweden)

    Yuko Shimamura

    Full Text Available This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA. Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning.

  13. Biomimetic silica encapsultation of living cells

    Science.gov (United States)

    Jaroch, David Benjamin

    Living cells perform complex chemical processes on size and time scales that artificial systems cannot match. Cells respond dynamically to their environment, acting as biological sensors, factories, and drug delivery devices. To facilitate the use of living systems in engineered constructs, we have developed several new approaches to create stable protective microenvironments by forming bioinspired cell-membrane-specific silica-based encapsulants. These include vapor phase deposition of silica gels, use of endogenous membrane proteins and polysaccharides as a site for silica nucleation and polycondensation in a saturated environment, and protein templated ordered silica shell formation. We demonstrate silica layer formation at the surface of pluripotent stem-like cells, bacterial biofilms, and primary murine and human pancreatic islets. Materials are characterized by AFM, SEM and EDS. Viability assays confirm cell survival, and metabolite flux measurements demonstrate normal function and no major diffusion limitations. Real time PCR mRNA analysis indicates encapsulated islets express normal levels of genetic markers for β-cells and insulin production. The silica glass encapsulant produces a secondary bone like calcium phosphate mineral layer upon exposure to media. Such bioactive materials can improve device integration with surrounding tissue upon implantation. Given the favorable insulin response, bioactivity, and long-term viability observed in silica-coated islets, we are currently testing the encapsulant's ability to prevent immune system recognition of foreign transplants for the treatment of diabetes. Such hybrid silica-cellular constructs have a wide range of industrial, environmental, and medical applications.

  14. Inhibition of biological activity of staphylococcal enterotoxin A (SEA) by apple juice and apple polyphenols.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Friedman, Mendel

    2010-05-12

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and a commercial apple polyphenol preparation (Apple Poly) to inhibit the biological activity of SEA. Dilutions of freshly prepared apple juices and Apple Poly inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

  15. A hybrid bio-jetting approach for directly engineering living cells

    International Nuclear Information System (INIS)

    Kwok, Albert; Irvine, Scott; Arumuganathar, Sumathy; Jayasinghe, Suwan N; McEwan, Jean R

    2008-01-01

    This paper reports developments on a hybrid cell-engineering protocol coupling both bio-electrosprays and aerodynamically assisted bio-jets for process-handling living cells. The current work demonstrates the ability to couple these two cell-jetting protocols for handling a wide range of cells for deposition. The post-treated cells are assessed for their viability by way of flow cytometry, which illustrates a significant population of viable cells post-treatment in comparison to those controls. This work is the first example of coupling these two protocols for the process handling of living cells. The hybrid protocol demonstrates the achievement of stable cone jetting of a cellular suspension in the single-needle configuration which was previously unachieved with single-needle bio-electrosprays. Furthermore the living cells explored in these investigations expressed GFP, thus demonstrating the ability to couple gene therapy with this hybrid protocol. Hence, this approach could one day be explored for building biologically viable tissues incorporating a therapeutic payload for combating a range of cellular/tissue-based pathologies

  16. Regional outbreak of staphylococcal scalded skin syndrome in healthy children

    Directory of Open Access Journals (Sweden)

    Hyun Jeong Do

    2010-01-01

    Full Text Available Purpose : Staphylococcal scalded skin syndrome (SSSS is a relatively uncommon superficial blistering skin disease that is due to Staphylococcus aureus. We had experienced a regional outbreak of SSSS over 3 years in healthy children. Methods : We retrospectively reviewed the medical records of those patients diagnosed as SSSS. Most of neonatal cases were nosocomial infections and excluded from the analysis. The clinical features, laboratory findings, the isolation and antibiotic resistance of S. aureus, the antibiotic management and other supportive treatments were analyzed. Results : Fifty-five patients with SSSS were admitted to our hospital from October 2001 to September 2004. The median age of patients was 3.0 years. Of the 55 patients, 9 were the generalized type, 13 were the intermediate type and 33 were the scarletiniform rash. All the patients were living in neighborhood of the Jinju area. S. aureus were isolated from 9 of the patients and all of the isolated S. aureus were methicillin resistant. All the patients except two were treated with intravenous flocloxacillin or nafcillin and/or cefotaxime. All the patients recovered during the follow-up period of 2 to 3 weeks. Conclusion : We experienced a regional outbreak of SSSS in previous healthy children. Further study for finding the carriers of S. aureus caused SSSS and preventing the spread of this disease is needed. Additionally, guidelines for treating SSSS due to methicillin resistant S. aureus should be established.

  17. Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

    Science.gov (United States)

    Chan, K L Andrew; Kazarian, Sergei G

    2013-07-21

    The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

  18. Synthetic analog computation in living cells.

    Science.gov (United States)

    Daniel, Ramiz; Rubens, Jacob R; Sarpeshkar, Rahul; Lu, Timothy K

    2013-05-30

    A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber's law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.

  19. Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

    Science.gov (United States)

    Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

    2013-01-01

    Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

  20. Neonatal staphylococcal scalded skin syndrome: clinical and outbreak containment review.

    LENUS (Irish Health Repository)

    Neylon, Orla

    2012-01-31

    Staphylococcal scalded skin syndrome (SSSS) is a toxin-mediated exfoliating skin condition predominated by desquamation and blistering. Neonatal outbreaks have already been reported; however, our outbreak highlights the potential for SSSS following neonatal health promotion measures such as intra-muscular vitamin K administration and metabolic screening (heel prick) as well as effective case containment measures and the value of staff screening. Between February and June 2007, five confirmed cases of neonatal SSSS were identified in full-term neonates born in an Irish regional maternity hospital. All infants were treated successfully. Analysis of contact and environmental screening was undertaken, including family members and healthcare workers. Molecular typing on isolates was carried out. An outbreak control team (OCT) was assembled and took successful prospective steps to prevent further cases. All five Staphylococcus aureus isolates tested positive for exfoliative toxin A, of which two distinct strains were identified on pulsed-field gel electrophoresis analysis. Two cases followed staphylococcal inoculation during preventive measures such as intra-muscular vitamin K administration and metabolic screening (heel prick). None of the neonatal isolates were methicillin resistant. Of 259 hospital staff (70% of staff) screened, 30% were colonised with S. aureus, and 6% were positive for MRSA carriage. This is the first reported outbreak of neonatal SSSS in Ireland. Effective case containment measures and clinical value of OCT is demonstrated. Results of staff screening underlines the need for vigilance and compliance in hand disinfection strategies in maternity hospitals especially during neonatal screening and preventive procedures.

  1. Functional memory B cells and long-lived plasma cells are generated after a single Plasmodium chabaudi infection in mice.

    Directory of Open Access Journals (Sweden)

    Francis Maina Ndungu

    2009-12-01

    Full Text Available Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses.

  2. Merkel cells are long-lived cells whose production is stimulated by skin injury✰

    Science.gov (United States)

    Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

    2017-01-01

    Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

  3. Merkel cells are long-lived cells whose production is stimulated by skin injury.

    Science.gov (United States)

    Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

    2017-02-01

    Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Semi-automated quantification of living cells with internalized nanostructures

    KAUST Repository

    Margineanu, Michael B.

    2016-01-15

    Background Nanostructures fabricated by different methods have become increasingly important for various applications in biology and medicine, such as agents for medical imaging or cancer therapy. In order to understand their interaction with living cells and their internalization kinetics, several attempts have been made in tagging them. Although methods have been developed to measure the number of nanostructures internalized by the cells, there are only few approaches aimed to measure the number of cells that internalize the nanostructures, and they are usually limited to fixed-cell studies. Flow cytometry can be used for live-cell assays on large populations of cells, however it is a single time point measurement, and does not include any information about cell morphology. To date many of the observations made on internalization events are limited to few time points and cells. Results In this study, we present a method for quantifying cells with internalized magnetic nanowires (NWs). A machine learning-based computational framework, CellCognition, is adapted and used to classify cells with internalized and no internalized NWs, labeled with the fluorogenic pH-dependent dye pHrodo™ Red, and subsequently to determine the percentage of cells with internalized NWs at different time points. In a “proof-of-concept”, we performed a study on human colon carcinoma HCT 116 cells and human epithelial cervical cancer HeLa cells interacting with iron (Fe) and nickel (Ni) NWs. Conclusions This study reports a novel method for the quantification of cells that internalize a specific type of nanostructures. This approach is suitable for high-throughput and real-time data analysis and has the potential to be used to study the interaction of different types of nanostructures in live-cell assays.

  5. Circumventing photodamage in live-cell microscopy

    Science.gov (United States)

    Magidson, Valentin; Khodjakov, Alexey

    2013-01-01

    Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

  6. Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping.

    Science.gov (United States)

    Gallina, S; Bianchi, D M; Bellio, A; Nogarol, C; Macori, G; Zaccaria, T; Biorci, F; Carraro, E; Decastelli, L

    2013-12-01

    In June 2011, an outbreak of Staphylococcus aureus enterotoxin food poisoning gastroenteritis occurred in Turin, Italy, following a catered dinner party at a private home. Within a few hours, 26 of the 47 guests experienced gastrointestinal illness, and 9 were hospitalized. A retrospective cohort study using a standardized questionnaire was carried out, and the risk ratios for each food item were calculated. The analysis indicated consumption of seafood salad as the most probable cause of the outbreak (risk ratio = 11.72; 95 % confidence interval, 1.75 to 78.54). Biological samples were collected from four of the hospitalized guests (stool and vomit), nasal mucosa swabs from three food handlers employed with the caterer, and available food residuals. All stool and vomit samples tested positive for enterotoxigenic S. aureus. As residues of the seafood salad were no longer available for sampling, suspected contamination could not be verified. However, no other food was found contaminated by S. aureus or its enterotoxins. All isolates from the biological samples were characterized at the genomic level by means of two multiplex PCR protocols to determine the presence of genes encoding staphylococcal enterotoxins, pulsed-field gel electrophoresis and staphylococcal protein A gene (spa) typing to describe their genetic profiles. All the isolates presented genes encoding SEA and SEI; the pulsed-field gel electrophoresis genetic profiles revealed the same pulsotype in the microorganism isolated from the hospitalized guests as in one of the isolates from a food handler's nasal mucosa, and the spa typing analysis reported two closely related spa types (t701 and t267), implicating the food handler as the most likely outbreak source.

  7. Distribution and characterization of staphylococcal interspersed repeat units (SIRUs) and potential use for strain differentiation

    DEFF Research Database (Denmark)

    Hardy, K.J.; Ussery, David; Oppenheim, B.A.

    2004-01-01

    are similar to those used in the study of the evolution of Mycobacterium tuberculosis clades. Seven VNTRs, termed staphylococcal interspersed repeat units (SIRUs), distributed around the genome are described, occurring in both unique and multiple sites, and varying in length from 48 to 159 bp. Variations...

  8. Inhibition of Biological Activity of Staphylococcal Enterotoxin A (SEA) by Apple Juice and Apple Polyphenols

    Science.gov (United States)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal entertoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, dia...

  9. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    International Nuclear Information System (INIS)

    Paulin, Sarah; Rosado, Helena; Taylor, Peter W; Jamshad, Mohammed; Dafforn, Timothy R; Garcia-Lara, Jorge; Foster, Simon J; Galley, Nicola F; Roper, David I

    2014-01-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function. (paper)

  10. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    Science.gov (United States)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  11. Varicella infection in a neonate with subsequent staphylococcal scalded skin syndrome and fatal shock.

    Science.gov (United States)

    Singh, Shakal Narayan; Tahazzul, Mohammad; Singh, Anita; Chandra, Surabhi

    2012-08-01

    A male term neonate, at day 23 of life, presented with vesicular lesions over the trunk, which spread to allover the body on the next day. Five days later, he started developing blistering of the skin over the trunk and extremities, which subsequently ruptured, leaving erythematous, tender raw areas with peeling of the skin. The mother had vesicular eruptions, which started on the second day of delivery and progressed over the next 3 days. Subsequently, similar eruptions were noticed in two of the siblings before affecting the neonate. On the basis of the exposure history and clinical picture, a diagnosis was made of varicella infection with staphylococcal scalded skin syndrome (SSSS). The blood culture and the wound surface culture grew Staphylococcus aureus. Treatment included intravenous fluid, antibiotics, acyclovir and wound care. However, after 72 h of hospitalisation, the neonate first developed shock, refractory to fluid boluses, vasopressors and catecholamine along with other supports; and he then succumbed. In all neonates, staphylococcal infection with varicella can be fatal due to SSSS, the toxic shock syndrome or septicaemia.

  12. Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

    Science.gov (United States)

    Mignacca, Sebastian Alessandro; Dore, Simone; Spuria, Liliana; Zanghì, Pietro; Amato, Benedetta; Duprè, Ilaria; Armas, Federica; Biasibetti, Elena; Camperio, Cristina; Lollai, Stefano A; Capucchio, Maria Teresa; Cannas, Eugenia Agnese; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

    2017-12-01

    Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

  13. Acid base activity of live bacteria: Implications for quantifying cell wall charge

    Science.gov (United States)

    Claessens, Jacqueline; van Lith, Yvonne; Laverman, Anniet M.; Van Cappellen, Philippe

    2006-01-01

    To distinguish the buffering capacity associated with functional groups in the cell wall from that resulting from metabolic processes, base or acid consumption by live and dead cells of the Gram-negative bacterium Shewanella putrefaciens was measured in a pH stat system. Live cells exhibited fast consumption of acid (pH 4) or base (pH 7, 8, 9, and 10) during the first few minutes of the experiments. At pH 5.5, no acid or base was required to maintain the initial pH constant. The initial amounts of acid or base consumed by the live cells at pH 4, 8, and 10 were of comparable magnitudes as those neutralized at the same pHs by intact cells killed by exposure to gamma radiation or ethanol. Cells disrupted in a French press required higher amounts of acid or base, due to additional buffering by intracellular constituents. At pH 4, acid neutralization by suspensions of live cells stopped after 50 min, because of loss of viability. In contrast, under neutral and alkaline conditions, base consumption continued for the entire duration of the experiments (5 h). This long-term base neutralization was, at least partly, due to active respiration by the cells, as indicated by the build-up of succinate in solution. Qualitatively, the acid-base activity of live cells of the Gram-positive bacterium Bacillus subtilis resembled that of S. putrefaciens. The pH-dependent charging of ionizable functional groups in the cell walls of the live bacteria was estimated from the initial amounts of acid or base consumed in the pH stat experiments. From pH 4 to 10, the cell wall charge increased from near-zero values to about -4 × 10 -16 mol cell -1 and -6.5 × 10 -16 mol cell -1 for S. putrefaciens and B. subtilis, respectively. The similar cell wall charging of the two bacterial strains is consistent with the inferred low contribution of lipopolysaccharides to the buffering capacity of the Gram-negative cell wall (of the order of 10%).

  14. Axial tomography in 3D live cell microscopy

    Science.gov (United States)

    Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

    2017-07-01

    A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

  15. Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

    Directory of Open Access Journals (Sweden)

    Neil O Carragher

    2011-04-01

    Full Text Available Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

  16. The clinical spectrum and antibiotic sensitivity patterns of staphylococcal pyodermas in the community and hospital

    Directory of Open Access Journals (Sweden)

    Shireen Furtado

    2014-01-01

    Full Text Available Context: The uncontrolled use of antibiotics has resulted in a relentless spread of multiresistant strains of Staphylococcus aureus. There are studies conducted in medical colleges in Chandigarh, Chennai, Mumbai and Vellore comparing pyodermas in the community and hospital setting based on clinical and bacteriological parameters. Aims: This study, conducted over 1½ years from March 2009 to August 2010, aimed at analyzing the clinical spectrum and antibiotic sensitivity pattern of community and hospital-associated (HA staphylococcal pyoderma. It also assessed the prevalence of methicillin-resistant S. aureus (MRSA in the community and hospital cohort settings. Subjects and Methods: The study comprised of 200 cases of staphylococcal pyodermas, derived from the community (150 cases and hospital (50 cases. Patients were evaluated based on their clinical presentation; antibiotic susceptibility was tested using the Kirby-Bauer disk diffusion method. Statistical Analysis Used: Statistical significance between individual attributes between the community and HA staphylococcal pyoderma groups was analyzed using Chi-square test and mean differences using student′s t-test. Results: Factors associated with community-associated (CA pyodermas were young age (P = 0.0021, primary pyodermas, and involvement of extremities, while those with HA pyodermas were middle age, secondary pyodermas, and significantly increased body surface involvement (P = 0.041. Incidence of CA-MRSA was 11.3%, while that of HA-MRSA was 18%. Conclusions: A high level of resistance to first-line drugs such as penicillin, ciprofloxacin and cotrimoxazole was observed, more so in the hospital strain than in the community strain. S. aureus demonstrated good susceptibility to cephalosporins. Though the two strains of MRSA differed clinically, they showed 100% sensitivity to vancomycin and linezolid.

  17. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    Energy Technology Data Exchange (ETDEWEB)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S., E-mail: prakash@mailbox.sc.edu

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

  18. The effects of atomic force microscopy upon nominated living cells

    Energy Technology Data Exchange (ETDEWEB)

    O' Hagan, Barry Michael Gerard [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)]. E-mail: bmg.ohagan@ulstser.ac.uk; Doyle, Peter [Unilever Research, Port Sunlight, The Wirral, Merseyside (United Kingdom); Allen, James M. [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); Sutton, Kerry [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); McKerr, George [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)

    2004-12-15

    This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment.

  19. The effects of atomic force microscopy upon nominated living cells

    International Nuclear Information System (INIS)

    O'Hagan, Barry Michael Gerard; Doyle, Peter; Allen, James M.; Sutton, Kerry; McKerr, George

    2004-01-01

    This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment

  20. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  1. HaloTag protein-mediated specific labeling of living cells with quantum dots

    International Nuclear Information System (INIS)

    So, Min-kyung; Yao Hequan; Rao Jianghong

    2008-01-01

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

  2. [Development of a Fluorescence Probe for Live Cell Imaging].

    Science.gov (United States)

    Shibata, Aya

    2017-01-01

     Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

  3. Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

    Science.gov (United States)

    Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

    2016-11-01

    Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

  4. Cyborg cells: functionalisation of living cells with polymers and nanomaterials.

    Science.gov (United States)

    Fakhrullin, Rawil F; Zamaleeva, Alsu I; Minullina, Renata T; Konnova, Svetlana A; Paunov, Vesselin N

    2012-06-07

    Living cells interfaced with a range of polyelectrolyte coatings, magnetic and noble metal nanoparticles, hard mineral shells and other complex nanomaterials can perform functions often completely different from their original specialisation. Such "cyborg cells" are already finding a range of novel applications in areas like whole cell biosensors, bioelectronics, toxicity microscreening, tissue engineering, cell implant protection and bioanalytical chemistry. In this tutorial review, we describe the development of novel methods for functionalisation of cells with polymers and nanoparticles and comment on future advances in this technology in the light of other literature approaches. We review recent studies on the cell viability and function upon direct deposition of nanoparticles, coating with polyelectrolytes, polymer assisted assembly of nanomaterials and hard shells on the cell surface. The cell toxicity issues are considered for many practical applications in terms of possible adverse effects of the deposited polymers, polyelectrolytes and nanoparticles on the cell surface.

  5. Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

    Science.gov (United States)

    Winter, E M; Hogers, B; van der Graaf, L M; Gittenberger-de Groot, A C; Poelmann, R E; van der Weerd, L

    2010-03-01

    Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. (c) 2010 Wiley-Liss, Inc.

  6. Weak effect of metal type and ica genes on staphylococcal infection of titanium and stainless steel implants.

    Science.gov (United States)

    Hudetz, D; Ursic Hudetz, S; Harris, L G; Luginbühl, R; Friederich, N F; Landmann, R

    2008-12-01

    Currently, ica is considered to be the major operon responsible for staphylococcal biofilm. The effect of biofilm on susceptibility to staphylococcal infection of different implant materials in vivo is unclear. The interaction of ica-positive (wild-type (WT)) and ica-negative (ica(-)) Staphylococcus aureus and Staphylococcus epidermidis strains with titanium and both smooth and rough stainless steel surfaces was studied by scanning electron microscopy in vitro and in a mouse tissue cage model during 2 weeks following perioperative or postoperative inoculation in vivo. In vitro, WT S. epidermidis adhered equally and more strongly than did WT S. aureus to all materials. Both WT strains, but not ica(-) strains, showed multilayered biofilm. In vivo, 300 CFUs of WT and ica(-)S. aureus led, in all metal cages, to an infection with a high level of planktonic CFUs and only 0.89% adherent CFUs after 8 days. In contrast, 10(6) CFUs of the WT and ica(-) strains were required for postoperative infection with S. epidermidis. In all metal types, planktonic numbers of S. epidermidis dropped to titanium cages adherent WT bacteria survived in higher numbers than ica(-) bacteria. In conclusion, the metal played a minor role in susceptibility to and persistence of staphylococcal infection; the presence of ica genes had a strong effect on biofilm in vitro and a weak effect in vivo; and S. epidermidis was more pathogenic when introduced during implantation than after implantation.

  7. Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

    Directory of Open Access Journals (Sweden)

    Akira C Saito

    Full Text Available Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC field to induce a linear cell-GUV alignment, and then a direct current (DC pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

  8. Temperature-dependent imaging of living cells by AFM

    International Nuclear Information System (INIS)

    Espenel, Cedric; Giocondi, Marie-Cecile; Seantier, Bastien; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2008-01-01

    Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 deg. C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature

  9. Teachable, high-content analytics for live-cell, phase contrast movies.

    Science.gov (United States)

    Alworth, Samuel V; Watanabe, Hirotada; Lee, James S J

    2010-09-01

    CL-Quant is a new solution platform for broad, high-content, live-cell image analysis. Powered by novel machine learning technologies and teach-by-example interfaces, CL-Quant provides a platform for the rapid development and application of scalable, high-performance, and fully automated analytics for a broad range of live-cell microscopy imaging applications, including label-free phase contrast imaging. The authors used CL-Quant to teach off-the-shelf universal analytics, called standard recipes, for cell proliferation, wound healing, cell counting, and cell motility assays using phase contrast movies collected on the BioStation CT and BioStation IM platforms. Similar to application modules, standard recipes are intended to work robustly across a wide range of imaging conditions without requiring customization by the end user. The authors validated the performance of the standard recipes by comparing their performance with truth created manually, or by custom analytics optimized for each individual movie (and therefore yielding the best possible result for the image), and validated by independent review. The validation data show that the standard recipes' performance is comparable with the validated truth with low variation. The data validate that the CL-Quant standard recipes can provide robust results without customization for live-cell assays in broad cell types and laboratory settings.

  10. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-01-01

    attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic p

  11. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity

    OpenAIRE

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly i...

  12. High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

    Science.gov (United States)

    Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

    2017-12-01

    With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Biological interaction of living cells with COSAN-based synthetic vesicles.

    Science.gov (United States)

    Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

    2015-01-15

    Cobaltabisdicarbollide (COSAN) [3,3'-Co(1,2-C2B9H11)2](-), is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes.

  14. Inhibition of the superantigenic activities of Staphylococcal enterotoxin A by an aptamer antagonist.

    Science.gov (United States)

    Wang, Kaiyu; Wu, Dong; Chen, Zhuang; Zhang, Xianhui; Yang, Xiangyue; Yang, Chaoyong James; Lan, Xiaopeng

    2016-09-01

    Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS

    Science.gov (United States)

    Deitch, Arline D.; Moses, Montrose J.

    1957-01-01

    Living chick spinal ganglion neurons grown for 19 to 25 days in vitro were photographed with a color-translating ultraviolet microscope (UV-91) at 265, 287, and 310 mµ. This instrument was unique in permitting rapid accumulation of ultraviolet information with minimal damage to the cell. In the photographs taken at 265 mµ of the living neurons, discrete ultraviolet-absorbing cytoplasmic masses were observed which were found to be virtually unchanged in appearance after formalin fixation. These were identical with the Nissl bodies of the same cells seen after staining with basic dyes. The correlation of ultraviolet absorption, ribonuclease extraction, and staining experiments with acid and basic dyes confirmed the ribonucleoprotein nature of these Nissl bodies in the living and fixed cells. No change in distribution or concentration of ultraviolet-absorbing substance was observed in the first 12 ultraviolet photographs of a neuron, and it is concluded that the cells had not been subjected to significant ultraviolet damage during the period of photography. On the basis of these observations, as well as previous findings with phase contrast microscopy, it is concluded that Nissl bodies preexist in the living neuron as discrete aggregates containing high concentrations of nucleoprotein. PMID:13438929

  16. Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

    International Nuclear Information System (INIS)

    Lindroth, S.; Niskanen, A.

    1977-01-01

    A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect a little as 2-5 ng of enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin. (orig.) [de

  17. Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR

    Science.gov (United States)

    Couto, Isabel; Pereira, Sandro; Miragaia, Maria; Sanches, Ilda Santos; de Lencastre, Hermínia

    2001-01-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems. PMID:11526135

  18. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR.

    Science.gov (United States)

    Couto, I; Pereira, S; Miragaia, M; Sanches, I S; de Lencastre, H

    2001-09-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

  19. Ultrafast nanolaser device for detecting cancer in a single live cell.

    Energy Technology Data Exchange (ETDEWEB)

    Gourley, Paul Lee; McDonald, Anthony Eugene

    2007-11-01

    Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of live tumor cells without invasive chemical reagents when coupled with ultrafast laser methods. These optically based methods are critical to advancing early detection, diagnosis, and treatment of disease. The first year goals of this project are to develop a laser-based imaging system integrated with an in- vitro, live-cell, micro-culture to study mammalian cells under controlled conditions. In the second year, the system will be used to elucidate the morphology and distribution of mitochondria in the normal cell respiration state and in the disease state for normal and disease states of the cell. In this work we designed and built an in-vitro, live-cell culture microsystem to study mammalian cells under controlled conditions of pH, temp, CO2, Ox, humidity, on engineered material surfaces. We demonstrated viability of cell culture in the microsystem by showing that cells retain healthy growth rates, exhibit normal morphology, and grow to confluence without blebbing or other adverse influences of the material surfaces. We also demonstrated the feasibility of integrating the culture microsystem with laser-imaging and performed nanolaser flow spectrocytometry to carry out analysis of the cells isolated mitochondria.

  20. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  1. Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    2010-01-01

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

  2. Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2009-11-26

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

  3. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    International Nuclear Information System (INIS)

    Glogger, M; Subota, I; Spindler, M-C; Engstler, M; Fenz, S F; Stichler, S; Bertlein, S; Teßmar, J; Groll, J

    2017-01-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μ s. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. (paper)

  4. Uranium and thorium uptake by live and dead cells of Pseudomonas Sp

    International Nuclear Information System (INIS)

    Siva Prasath, C.S.; Manikandan, N.; Prakash, S.

    2010-01-01

    This study presents uptake of uranium (U) and thorium (Th) by live and dead cells of Pseudomonas Sp. Increasing concentration of U and Tb showed decrease in absorption by Pseudomonas Sp. Dead cells of Pseudomonas Sp. exhibited same or more uptake of U and Th than living cells. Increasing temperature promotes uptake of U and Th by Pseudomonas Sp. (author)

  5. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Shawish, Reyad R.

    2016-04-01

    Full Text Available Food-borne pathogens are of high concern for public health and food safety. food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs in processed beef from Kingdom of Saudi Arabia (KSA and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of . Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates.The percentage presence of in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted.

  6. Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

    Science.gov (United States)

    Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

    2017-03-01

    Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

  7. FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

    Science.gov (United States)

    Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

    2009-06-01

    We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

  8. Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses.

    Directory of Open Access Journals (Sweden)

    Jakob Begun

    2007-04-01

    Full Text Available Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.

  9. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  10. Collective Dynamics of Intracellular Water in Living Cells

    International Nuclear Information System (INIS)

    Orecchini, A; Sebastiani, F; Paciaroni, A; Petrillo, C; Sacchetti, F; Jasnin, M; Francesco, A De; Zaccai, G; Moulin, M; Haertlein, M

    2012-01-01

    Water dynamics plays a fundamental role for the fulfillment of biological functions in living organisms. Decades of hydrated protein powder studies have revealed the peculiar dynamical properties of hydration water with respect to pure water, due to close coupling interactions with the macromolecule. In such a framework, we have studied coherent collective dynamics in protein and DNA hydration water. State-of-the-art neutron instrumentation has allowed us to observe the propagation of coherent density fluctuations within the hydration shell of the biomolecules. The corresponding dispersion curves resulted to be only slightly affected by the coupling with the macromolecules. Nevertheless, the effects of the interaction appeared as a marked increase of the mode damping factors, which suggested a destructuring of the water hydrogen-bond network. Such results were interpreted as the signature of a 'glassy' dynamical character of macromolecule hydration water, in agreement with indications from measurements of the density of vibrational states. Extending the investigations to living organisms at physiological conditions, we present here an in-vivo study of collective dynamics of intracellular water in Escherichia coli cells. The cells and water were fully deuterated to minimise the incoherent neutron scattering background. The water dynamics observed in the living cells is discussed in terms of the dynamics of pure bulk water and that of hydration water measured in powder samples.

  11. Picosecond orientational dynamics of water in living cells.

    Science.gov (United States)

    Tros, Martijn; Zheng, Linli; Hunger, Johannes; Bonn, Mischa; Bonn, Daniel; Smits, Gertien J; Woutersen, Sander

    2017-10-12

    Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions.The cytoplasm's crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

  12. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  13. Live-cell thermometry with nitrogen vacancy centers in nanodiamonds

    Science.gov (United States)

    Jayakumar, Harishankar; Fedder, Helmut; Chen, Andrew; Yang, Liudi; Li, Chenghai; Wrachtrup, Joerg; Wang, Sihong; Meriles, Carlos

    The ability to measure temperature is typically affected by a tradeoff between sensitivity and spatial resolution. Good thermometers tend to be bulky systems and hence are ill-suited for thermal sensing with high spatial localization. Conversely, the signal resulting from nanoscale temperature probes is often impacted by noise to a level where the measurement precision becomes poor. Adding to the microscopist toolbox, the nitrogen vacancy (NV) center in diamond has recently emerged as a promising platform for high-sensitivity nanoscale thermometry. Of particular interest are applications in living cells because diamond nanocrystals are biocompatible and can be chemically functionalized to target specific organelles. Here we report progress on the ability to probe and compare temperature within and between living cells using nanodiamond-hosted NV thermometry. We focus our study on cancerous cells, where atypical metabolic pathways arguably lead to changes in the way a cell generates heat, and thus on its temperature profile.

  14. An Alternative Approach to Combination Vaccines: Intradermal Administration of Isolated Components for Control of Anthrax, Botulism, Plague and Staphylococcal Toxic Shock

    National Research Council Canada - National Science Library

    Morefield, Garry L; Tammariello, Ralph F; Purcell, Bret K; Worsham, Patricia L; Chapman, Jennifer; Smith, Leonard A; Alarcon, Jason B; Mikszta, John A; Ulrich, Robert G

    2008-01-01

    ... incompatible vaccine mixtures. Intradermally administered arrays of vaccines for protection from anthrax, botulism, plague, and staphylococcal toxic shock were biocompatible in vivo, retained potent antibody responses...

  15. Role of blood culture systems in the evaluation of epidemiological features of coagulase-negative staphylococcal bloodstream infection in critically ill patients.

    Science.gov (United States)

    Oud, L; Krimerman, S; Salam, N; Srugo, I

    1999-12-01

    The impact of blood culture systems on the detection of coagulase-negative staphylococcal bloodstream infections in critically ill patients prior to and following the introduction of the Bactec 9240 blood culture system (Becton Dickinson Diagnostic Instrument Systems, USA), which replaced the Bactec NR 730 (Becton Dickinson Diagnostic Instrument Systems), was investigated over a 3-year period. Following the introduction of the new culture system, the incidence of bloodstream infections doubled (P<0.001). Patient demographics, severity of illness, and mortality remained unchanged, while the annual standardized mortality ratio decreased significantly. These data suggest that blood culture systems may have a major impact on the perceived incidence of coagulase-negative staphylococcal bloodstream infections in this population.

  16. Real-Time Gene Expression Profiling of Live Shewanella Oneidensis Cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiaoliang Sunney Xie

    2009-03-30

    The overall objective of this proposal is to make real-time observations of gene expression in live Shewanella oneidensis cells with high sensitivity and high throughput. Gene expression, a central process to all life, is stochastic because most genes often exist in one or two copies per cell. Although the central dogma of molecular biology has been proven beyond doubt, due to insufficient sensitivity, stochastic protein production has not been visualized in real time in an individual cell at the single-molecule level. We report the first direct observation of single protein molecules as they are generated, one at a time in a single live E. coli cell, yielding quantitative information about gene expression [Science 2006; 311: 1600-1603]. We demonstrated a general strategy for live-cell single-molecule measurements: detection by localization. It is difficult to detect single fluorescence protein molecules inside cytoplasm - their fluorescence is spread by fast diffusion to the entire cell and overwhelmed by the strong autofluorescence. We achieved single-molecule sensitivity by immobilizing the fluorescence protein on the cell membrane, where the diffusion is much slowed. We learned that under the repressed condition protein molecules are produced in bursts, with each burst originating from a stochastically-transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. We also simultaneously published a paper reporting a different method using β-glactosidase as a reporter [Nature 440, 358 (2006)]. Many important proteins are expressed at low levels, inaccessible by previous proteomic techniques. Both papers allowed quantification of protein expression with unprecedented sensitivity and received overwhelming acclaim from the scientific community. The Nature paper has been identified as one of the most-cited papers in the past year [http://esi-topics.com/]. We have also an analytical framework describing the

  17. Live Cells Decreased Methane Production in Intestinal Content of Pigs

    Directory of Open Access Journals (Sweden)

    Y. L. Gong

    2013-06-01

    Full Text Available An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4 production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells, control (no live yeast cells and yeast (YST supplementation groups (supplemented with live yeast cells, YST1 or YST2. The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2, and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM basis. Total gas and CH4 production decreased (p<0.05 with supplementation of yeast. The methane production reduction potential (MRP was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD and total volatile fatty acids (VFA concentration increased (p<0.05 in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05, but that of acetate decreased (p<0.05, which led to a decreased (p<0.05 acetate: propionate (A: P ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05 with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05 with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic

  18. Live-cell imaging: new avenues to investigate retinal regeneration

    Directory of Open Access Journals (Sweden)

    Manuela Lahne

    2017-01-01

    Full Text Available Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish (Danio rerio possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

  19. Effects of high-gradient magnetic fields on living cell machinery

    Czech Academy of Sciences Publication Activity Database

    Zablotskyy, V.; Lunov, O.; Kubinová, Šárka; Polyakova, T.; Syková, Eva; Dejneka, A.

    2016-01-01

    Roč. 49, č. 2016 (2016), s. 493003 ISSN 0022-3727 R&D Projects: GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: FP - Other Medical Disciplines Impact factor: 2.588, year: 2016

  20. Short communication: Pasteurization as a means of inactivating staphylococcal enterotoxins A, B, and C in milk.

    Science.gov (United States)

    Necidova, Lenka; Bogdanovicova, Katerina; Harustiakova, Danka; Bartova, Katerina

    2016-11-01

    Our aim was to assess the effect of pasteurization temperature on inactivation of staphylococcal enterotoxins (SE). Milk samples were inoculated with log 4.38 to 5.18cfu/mL of 40 different Staphylococcus aureus strains having the ability to produce types A, B, or C SE and incubated at 37°C for 24h to develop SE. This incubation was followed by heat treatment for 15 s at 72, 85, and 92°C. Samples were analyzed for Staph. aureus count by plate method and, specifically, for SE presence. An enzyme-linked immunofluorescent assay on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semiquantitatively based on test values. The Staph. aureus count in milk before pasteurization did not affect the amount of SE. Before pasteurization, SEB was detected in the lowest amount compared with other SE types. Staphylococcal enterotoxins were markedly reduced with pasteurization and inactivated at pasteurization temperatures to an extent depending on the amount in the sample before pasteurization. After pasteurization at 72°C, SE were detected in 87.5% of samples (35/40), after pasteurization at 85°C in 52.5% of samples (21/40), and after pasteurization at 92°C in 45.0% of samples (18/40). We determined that SE may still persist in milk even when Staph. aureus bacteria are inactivated through pasteurization. Although pasteurization may partially inactivate SE in milk, a key measure in the prevention of staphylococcal enterotoxicosis linked to pasteurized milk consumption is to avoid any cold chain disruption during milk production and processing. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Novel types of staphylococcal cassette chromosome mec elements identified in clonal complex 398 methicillin-resistant Staphylococcus aureus strains.

    NARCIS (Netherlands)

    Li, S.; Skov, R.L.; Han, X.; Larsen, A.R.; Larsen, J.; Sorum, M.; Wulf, M.; Voss, A.; Hiramatsu, K.; Ito, T.

    2011-01-01

    The structures of staphylococcal cassette chromosome mec (SCCmec) elements carried by 31 clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from the participants at a conference were analyzed. The SCCmecs were classified into novel types, namely, IX, X,

  2. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  3. Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.

    Science.gov (United States)

    De, Abhijit; Gambhir, Sanjiv Sam

    2005-12-01

    This study demonstrates a significant advancement of imaging of a distance-dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge-coupled device (CCD) camera. A CCD camera-based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof-of-principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled-CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein-protein interactions directly at limited depths in living mice.

  4. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  5. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-01-01

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  6. Design of microdevices for long-term live cell imaging

    International Nuclear Information System (INIS)

    Chen, Huaying; Nordon, Robert E; Rosengarten, Gary; Li, Musen

    2012-01-01

    Advances in fluorescent live cell imaging provide high-content information that relates a cell's life events to its ancestors. An important requirement to track clonal growth and development is the retention of motile cells derived from an ancestor within the same microscopic field of view for days to weeks, while recording fluorescence images and controlling the mechanical and biochemical microenvironments that regulate cell growth and differentiation. The aim of this study was to design a microwell device for long-term, time-lapse imaging of motile cells with the specific requirements of (a) inoculating devices with an average of one cell per well and (b) retaining progeny of cells within a single microscopic field of view for extended growth periods. A two-layer PDMS microwell culture device consisting of a parallel-plate flow cell bonded on top of a microwell array was developed for cell capture and clonal culture. Cell deposition statistics were related to microwell geometry (plate separation and well depth) and the Reynolds number. Computational fluid dynamics was used to simulate flow in the microdevices as well as cell–fluid interactions. Analysis of the forces acting upon a cell was used to predict cell docking zones, which were confirmed by experimental observations. Cell–fluid dynamic interactions are important considerations for design of microdevices for long-term, live cell imaging. The analysis of force and torque balance provides a reasonable approximation for cell displacement forces. It is computationally less intensive compared to simulation of cell trajectories, and can be applied to a wide range of microdevice geometries to predict the cell docking behavior. (paper)

  7. Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

    Science.gov (United States)

    Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

    2014-01-01

    Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

  8. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    Science.gov (United States)

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  9. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    Science.gov (United States)

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  10. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  11. Modeling the effects of a Staphylococcal Enterotoxin B (SEB on the apoptosis pathway

    Directory of Open Access Journals (Sweden)

    Hammamieh Rasha

    2006-05-01

    Full Text Available Abstract Background The lack of detailed understanding of the mechanism of action of many biowarfare agents poses an immediate challenge to biodefense efforts. Many potential bioweapons have been shown to affect the cellular pathways controlling apoptosis 1234. For example, pathogen-produced exotoxins such as Staphylococcal Enterotoxin B (SEB and Anthrax Lethal Factor (LF have been shown to disrupt the Fas-mediated apoptotic pathway 24. To evaluate how these agents affect these pathways it is first necessary to understand the dynamics of a normally functioning apoptosis network. This can then serve as a baseline against which a pathogen perturbed system can be compared. Such comparisons can expose both the proteins most susceptible to alteration by the agent as well as the most critical reaction rates to better instill control on a biological network. Results We explore this through the modeling and simulation of the Fas-mediated apoptotic pathway under normal and SEB influenced conditions. We stimulated human Jurkat cells with an anti-Fas antibody in the presence and absence of SEB and determined the relative levels of seven proteins involved in the core pathway at five time points following exposure. These levels were used to impute relative rate constants and build a quantitative model consisting of a series of ordinary differential equations (ODEs that simulate the network under both normal and pathogen-influenced conditions. Experimental results show that cells exposed to SEB exhibit an increase in the rate of executioner caspase expression (and subsequently apoptosis of 1 hour 43 minutes (± 14 minutes, as compared to cells undergoing normal cell death. Conclusion Our model accurately reflects these results and reveals intervention points that can be altered to restore SEB-influenced system dynamics back to levels within the range of normal conditions.

  12. Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

    Directory of Open Access Journals (Sweden)

    Jaime Canovas

    2016-11-01

    Full Text Available Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci

  13. Direct Visualization of De novo Lipogenesis in Single Living Cells

    Science.gov (United States)

    Li, Junjie; Cheng, Ji-Xin

    2014-10-01

    Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

  14. Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.

    Directory of Open Access Journals (Sweden)

    Jan M Bruder

    Full Text Available Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1 localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.

  15. Quantification of GPCR internalization by single-molecule microscopy in living cells.

    NARCIS (Netherlands)

    Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

    2011-01-01

    Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells.

  16. Rhinosinusitis derived Staphylococcal enterotoxin B possibly associates with pathogenesis of ulcerative colitis

    Directory of Open Access Journals (Sweden)

    An Zi-Yuan

    2005-09-01

    Full Text Available Abstract Background During clinical practice, we noticed that some patients with both ulcerative colitis (UC and chronic rhinosinusitis (CRS showed amelioration of UC after treatment of CRS. This study was designed to identify a possible association between CRS and UC. Methods Thirty-two patients with both CRS and UC received treatment with functional endoscopic sinus surgery (FESS for CRS. Clinical symptom scores for CRS and UC, as well as serum levels of anti-Staphylococcal enterotoxin B (SEB were evaluated at week 0 and week 12. Sinus wash fluid SEB content was measured with enzyme-linked immunosorbent assay (ELISA. The surgically removed tissues were cultured to identify growth of Staphylococcus. aureus (S. aureus. Immunohistochemistry was employed to identify anti-SEB positive cells in the colonic mucosa. Colonic biopsies were obtained and incubated with SEB. Mast cell activation in the colonic mucosa in response to incubation with SEB was observed with electron microscopy and immunoassay. Results The clinical symptom scores of CRS and UC severe scores (UCSS were significantly reduced in the UC-CRS patients after FESS. The number of cultured S. aureus colonies from the surgically removed sinus mucosa significantly correlated with the decrease in UCSS. High levels of SEB were detected in the sinus wash fluids of the patients with UC-CRS. Histamine and tryptase release was significantly higher in the culture supernate in the patients with UC-CRS than the patients with UC-only and normal controls. Anti-SEB positive cells were located in the colonic mucosa. Conclusion The pathogenesis of UC in some patients may be associated with their pre-existing CRS by a mechanism of swallowing sinusitis-derived SEB. We speculate that SEB initiates inappropriate immune reactions and inflammation in the colonic mucosa that further progresses to UC.

  17. Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

    Science.gov (United States)

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

    Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics.

  18. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    Science.gov (United States)

    Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

    2017-02-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μs. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

  19. Axial tomography in live cell laser microscopy

    Science.gov (United States)

    Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

    2017-09-01

    Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

  20. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    Science.gov (United States)

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  1. Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

    International Nuclear Information System (INIS)

    Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

    2002-01-01

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins

  2. Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

    2002-07-25

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins.

  3. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    International Nuclear Information System (INIS)

    Ribot, E.J.; Foster, P.J.

    2012-01-01

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  4. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    Energy Technology Data Exchange (ETDEWEB)

    Ribot, E.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); Foster, P.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); University of Western Ontario, Department of Medical Biophysics, London, ON (Canada)

    2012-09-15

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  5. Magnetic Bead and Fluorescent Silica Nanoparticles Based Optical Immunodetection of Staphylococcal Enterotoxin B (SEB in Bottled Water

    Directory of Open Access Journals (Sweden)

    Shiva K. RASTOGI

    2009-10-01

    Full Text Available Staphylococcal enterotoxins (SEs are a major cause of food-borne diseases, most commonly SEs assayed immunologically with ELISA. An immunoassay based on fluorescein dye doped silica dioxide nanoparticles (F-SiNPs and magnetic bead (MB is described here for the detection of staphylococcal enterotoxin B (SEB. F-SiNPs have unique optical properties which make them attractive for biosensing. The water-in-oil (W/O reverse microemulsion method was used for the synthesis of F-SiNPs (~ 95 nm of diameter. The F-SiNPs were characterized using SEM, TEM and FTIR spectroscopy. The detection of SEB is preformed in PBS buffer, and bottled drinking water using sandwich immunoassay format. Target analytes were captured using MBs modified with the antigen-specific “capture” antibody, and detected using F-SiNP labeled secondary antigen-specific antibody. We report a limit of detection down to 1 ng/mL SEB spiked sample in less than 2 hr assay time using fluorocount method. This study demonstrates the bio warfare agent SEB capture by magnetic beads and detection using F-SiNPs.

  6. Effects of high-gradient magnetic fields on living cell machinery

    Czech Academy of Sciences Publication Activity Database

    Zablotskyy, Vitaliy A.; Lunov, Oleg; Kubinová, Šárka; Polyakova, Tetyana; Syková, E.; Dejneka, Alexandr

    2016-01-01

    Roč. 49, č. 49 (2016), s. 1-23, č. článku 493003. ISSN 0022-3727 R&D Projects: GA MŠk LO1409 Grant - others:FUNBIO(XE) CZ.2.16/3.1.00/21568 Institutional support: RVO:68378271 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: BO - Biophysics Impact factor: 2.588, year: 2016

  7. Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

    Science.gov (United States)

    Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

    2018-03-15

    Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. The lived experiences of adolescents with sickle cell disease in Kingston, Jamaica

    Directory of Open Access Journals (Sweden)

    Andrea Brown Forrester

    2015-09-01

    Full Text Available Aim: To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. Method: A descriptive qualitative design was used for this research. In-depth interviews were conducted with six adolescents with sickle cell disease at a Sickle Cell Unit operated by the University of the West Indies. Interviews were audiotaped, transcribed, and thematically analyzed. Results: The majority of the adolescents demonstrated a positive self-concept. They reported strong family, school, and peer support which made them feel accepted. All were actively engaged in social activities such as parties, but had challenges participating in sporting activities. Various coping strategies were utilized to address challenges of the disease including praying, watching television, and surfing the Internet. Conclusion: Sickle cell disease can be very challenging for the adolescent, but with positive self-concept and increased social support, especially from family and peers, these adolescents were able to effectively cope with their condition and live productive lives.

  9. The lived experiences of adolescents with sickle cell disease in Kingston, Jamaica.

    Science.gov (United States)

    Forrester, Andrea Brown; Barton-Gooden, Antoinette; Pitter, Cynthia; Lindo, Jascinth L M

    2015-01-01

    To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. A descriptive qualitative design was used for this research. In-depth interviews were conducted with six adolescents with sickle cell disease at a Sickle Cell Unit operated by the University of the West Indies. Interviews were audiotaped, transcribed, and thematically analyzed. The majority of the adolescents demonstrated a positive self-concept. They reported strong family, school, and peer support which made them feel accepted. All were actively engaged in social activities such as parties, but had challenges participating in sporting activities. Various coping strategies were utilized to address challenges of the disease including praying, watching television, and surfing the Internet. Sickle cell disease can be very challenging for the adolescent, but with positive self-concept and increased social support, especially from family and peers, these adolescents were able to effectively cope with their condition and live productive lives.

  10. Live cell imaging combined with high-energy single-ion microbeam

    Science.gov (United States)

    Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

    2016-03-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

  11. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    International Nuclear Information System (INIS)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille; Rochel, Natacha; Mély, Yves; Didier, Pascal; Weiss, Etienne

    2013-01-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins

  12. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  13. Study of action of biological aerococcus аutosymbiont on the model of staphylococcal infection

    Directory of Open Access Journals (Sweden)

    Stepanskyi D.O.

    2017-06-01

    Full Text Available The paper presents data on the study of the antagonistic effect of aerococcus autosymbionts in experimental models of infections caused by staphylococci. To study the antagonistic action of aerococcus autosymbiont on staphylococcus, a model of chronic staphylococcal infection in white mice and rabbits was used. In staphylococcal experimental infection, aerococcus antagonistic action against staphylococcus was tested by subcutaneous injection on white mice. Aerococci survival under the skin was studied. Aerococcus autosymbionts introduced under the skin to staphylococcus in 5 hours and 3 hours after administration of the latter cause antagonist effect and inhibit the development of infiltrates. Similar results were obtained when introducing aerococcus autosymbionts in the focus of infection in 5 minutes, 5 minutes and 3 hours, 1 and 5 hours after infection. Aerococci introduced after 24 and 48 hours shortened terms of disease manifestations as compared to the control group of mice. The therapeutic effect of aerococcus autosymbionts was tested on the experimental model of burns and wounds infected with staphylococcus. In animals with wounds treated with aerococcus autostrains the number of pathogenic staphylococci was 10 times less than in the control group. According to our observations aerococcus autosymbionts showed no irritant effect when applied on the wound surface, helped its healing, sharply reduced the percentage of staphylococcus inoculation from wound secretions.

  14. Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

    Science.gov (United States)

    Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

    2012-06-05

    Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

  15. Label-free evanescent microscopy for membrane nano-tomography in living cells.

    Science.gov (United States)

    Bon, Pierre; Barroca, Thomas; Lévèque-Fort, Sandrine; Fort, Emmanuel

    2014-11-01

    We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model. We apply this nano-axial tomography of living cell membranes to retrieve quantitative information on membrane invagination dynamics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  16. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    Science.gov (United States)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  17. Living labeling techniques of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Dong Qingyu; Chen Li

    2007-01-01

    Mesenchymal stem cells (MSCs) are well known for their self-renew and multi- differentiation potentiality. With the transplantation of the MSCs which can promote the regeneration and repair of the injured tissue, a new route for the treatment of dieases is hopeful to be effective. To trace the distribution, migration, proliferation and differentiation of the implanted MSCs, there need effective labeling techniques, especially living labeling techniques. (authors)

  18. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    Science.gov (United States)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  19. Molecular beacon nanosensors for live cell detection and tracking differentiation and reprogramming

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

    open to closed state within living cells. Using MBs targeting pluripotent stem cell markers we demonstrated reverse into a more immature state of LUHMES induced by neurosphere-like growth conditions. Moreover, we have been able to trace localisation of this particular population during differentiation...... in separation of fluorophore from quencher and thereby emission of a fluorescent signal that can be detected. In this project the usability and applicability of MBs for live cell detection and tracing of gene expression was demonstrated. MBs library targeting gene markers for pluripotent stem cells as well...... and thus demonstrate the usability of MBs for monitoring cell behaviour within 3D clusters. Finally, MBs detection of expression of human pluripotent markers after reprograming of adult somatic cells with plasmid codding for mouse transcription factors was demonstrated. In conclusion, the method of using...

  20. [Non-invasive analysis of proteins in living cells using NMR spectroscopy].

    Science.gov (United States)

    Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

    2015-01-01

    NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

  1. Detecting infrared luminescence and non-chemical signaling of living cells: single cell mid-IR spectroscopy in cryogenic environments

    Science.gov (United States)

    Pereverzev, Sergey

    2017-02-01

    Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.

  2. Atomic force microscopy as a tool for the investigation of living cells.

    Science.gov (United States)

    Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

    2013-01-01

    Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

  3. Live cell imaging combined with high-energy single-ion microbeam

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Guo, Jinlong [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Northwest Normal University, Lanzhou (China); Chen, Hao [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Institute of Nuclear Science and Technology, University of Lanzhou, Lanzhou (China)

    2016-03-15

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10{sup −3} s{sup −1} and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10{sup −2} s{sup −1}.

  4. Live cell imaging combined with high-energy single-ion microbeam

    International Nuclear Information System (INIS)

    Guo, Na; Du, Guanghua; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe; Guo, Jinlong; Chen, Hao

    2016-01-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10"−"3 s"−"1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10"−"2 s"−"1.

  5. Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

    Science.gov (United States)

    Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

    2016-02-01

    Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

  6. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

    Science.gov (United States)

    Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

    2015-10-01

    Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development

  7. Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology.

    Science.gov (United States)

    Sundaram, S K; Sacksteder, Colette A; Weber, Thomas J; Riley, Brian J; Addleman, R Shane; Harrer, Bruce J; Peterman, John W

    2013-01-01

    A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live cells interact with an external stimulus, such as a nanosized particle, and the toxicity and broad risk associated with these stimuli. It is difficult to capture the complexity and dynamics of these interactions by following omics-based approaches exclusively, which can be expensive and time-consuming. Attenuated total reflectance-Fourier transform infrared spectroscopy is well suited to provide noninvasive live-cell monitoring of cellular responses to potentially toxic nanosized particles or other stimuli. This alternative approach provides the ability to carry out rapid toxicity screenings and nondisruptive monitoring of live-cell cultures. We review the technical basis of the approach, the instrument configuration and interface with the biological media, the various effects that impact the data, subsequent data analysis and toxicity, and present some preliminary results on live-cell monitoring.

  8. [Methods of substances and organelles introduction in living cell for cell engineering technologies].

    Science.gov (United States)

    Nikitin, V A

    2007-01-01

    We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell.

  9. Neutron scattering to study membrane systems: from lipid vesicles to living cells.

    Energy Technology Data Exchange (ETDEWEB)

    Nickels, Jonathan D. [ORNL; Chatterjee, Sneha [ORNL; Stanley, Christopher B. [ORNL; Qian, Shuo [ORNL; Cheng, Xiaolin [ORNL; Myles, Dean A A [ORNL; Standaert, Robert F. [ORNL; Elkins, James G. [ORNL; Katsaras, John [ORNL

    2017-03-01

    The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

  10. Thermodynamics of protein destabilization in live cells.

    Science.gov (United States)

    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

    2015-10-06

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

  11. Live cell CRISPR-imaging in plants reveals dynamic telomere movements

    KAUST Repository

    Dreissig, Steven

    2017-05-16

    Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm within 30 minutes during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for imaging of multiple genomic loci in live plants cells. CRISPR-imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

  12. Real-time monitoring of caspase cascade activation in living cells.

    Science.gov (United States)

    Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

    2012-10-10

    We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

  13. Profilaxis, immunoprophylaxis and therapy of staphylococcal mastitis

    Directory of Open Access Journals (Sweden)

    Vakanjac Slobodanka

    2006-01-01

    Full Text Available Inflammation of the mammary gland, mastitis, in cows presents one of the most acute problems in intensive milk production, incurring huge economic losses which reach from 20-80% even in developed countries in the past decade. On the grounds of the programme of the respective Ministry, the Veterinary Service of the Republic of Serbia is obliged to monitor, detect, curb and control infective inflammation of the mammary gland caused by staphylococcus and streptococcus. Long-term different approaches to mastitis treatment did not result in the required solution, so that the problem of mastitis is still present and acute. In order to prevent the infiltration of the pathogenic cause into the mammary gland, its populating and multiplication, it is necessary to maintain constant and regular controls of milk, as well as undertake of preventive and therapeutic measures aimed at reducing the incidence of mastitis. Immunization and immunotherapy of mastitis are new and very interesting areas for scientific investigations and work. In the recent decades vaccines against staphylococcal mastitis are being successfully developed, whose success is reflected in the reduced incidence of clinical and subcilinical mastitis.

  14. Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells

    Science.gov (United States)

    Saito, Masako; Nagasawa, Masayuki; Takada, Hidetoshi; Hara, Toshiro; Tsuchiya, Shigeru; Agematsu, Kazunaga; Yamada, Masafumi; Kawamura, Nobuaki; Ariga, Tadashi; Tsuge, Ikuya; Nonoyama, Shigeaki; Karasuyama, Hajime

    2011-01-01

    Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by recurrent staphylococcal infections and atopic dermatitis associated with elevated serum IgE levels. Although defective differentiation of IL-17–producing CD4+ T cells (Th17) partly accounts for the susceptibility to staphylococcal skin abscesses and pneumonia, the pathogenesis of atopic manifestations in HIES still remains an enigma. In this study, we examined the differentiation and function of Th1, Th2, regulatory T cells (Treg cells), and dendritic cells (DCs) in HIES patients carrying either STAT3 or TYK2 mutations. Although the in vitro differentiation of Th1 and Th2 cells and the number and function of Treg cells in the peripheral blood were normal in HIES patients with STAT3 mutations, primary and monocyte-derived DCs showed defective responses to IL-10 and thus failed to become tolerogenic. When treated with IL-10, patient DCs showed impaired up-regulation of inhibitory molecules on their surface, including PD-L1 and ILT-4, compared with control DCs. Moreover, IL-10–treated DCs from patients displayed impaired ability to induce the differentiation of naive CD4+ T cells to FOXP3+ induced Treg cells (iTreg cells). These results suggest that the defective generation of IL-10–induced tolerogenic DCs and iTreg cells may contribute to inflammatory changes in HIES. PMID:21300911

  15. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.

    Directory of Open Access Journals (Sweden)

    Marzia Massignani

    Full Text Available BACKGROUND: Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. PRINCIPAL FINDINGS: We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. SIGNIFICANCE: We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

  16. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  17. Visualization and targeted disruption of protein interactions in living cells

    Science.gov (United States)

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  18. Live-cell visualization of gasdermin D-driven pyroptotic cell death.

    Science.gov (United States)

    Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

    2017-09-01

    Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

    Science.gov (United States)

    Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

    2009-11-01

    Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

  20. Staphylococcal superantigen-specific IgE antibodies: degree of sensitization and association with severity of asthma

    OpenAIRE

    Elabras Filho, José; Mello, Fernanda Carvalho de Queiroz; Lupi, Omar; Bica, Blanca Elena Rios Gomes; Papi, José Angelo de Souza; França, Alfeu Tavares

    2016-01-01

    ABSTRACT Objective: To determine the presence of staphylococcal superantigen-specific IgE antibodies and degree of IgE-mediated sensitization, as well as whether or not those are associated with the severity of asthma in adult patients. Methods: This was a cross-sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of Rio de Janeiro, Brazil. Consecutive patients were divided into two groups according to the severity of asthma ba...

  1. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  2. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Directory of Open Access Journals (Sweden)

    Federico Baltar

    2018-01-01

    Full Text Available Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached, and dissolved (i.e., cell-free enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100% of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

  3. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Science.gov (United States)

    Baltar, Federico

    2018-01-01

    Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

  4. Vital Autofluorescence: Application to the Study of Plant Living Cells

    Directory of Open Access Journals (Sweden)

    Victoria V. Roshchina

    2012-01-01

    approach to study the autofluorescence of plant living cells—from cell diagnostics up to modelling the cell-cell contacts and cell interactions with fluorescent biologically active substances. It bases on the direct observations of secretions released from allelopathic and medicinal species and the cell-donor interactions with cell-acceptors as biosensors (unicellular plant generative and vegetative microspores. Special attention was paid to the interactions with pigmented and fluorescing components of the secretions released by the cells-donors from plant species. Colored components of secretions are considered as histochemical dyes for the analysis of cellular mechanisms at the cell-cell contacts and modelling of cell-cell interactions. The fluorescence of plant biosensors was also recommended for the testing of natural plant excretions as medical drugs.

  5. Effects of Rhodomyrtus tomentosa Leaf Extract on Staphylococcal Adhesion and Invasion in Bovine Udder Epidermal Tissue Model

    Directory of Open Access Journals (Sweden)

    Auemphon Mordmuang

    2015-10-01

    Full Text Available Bovine mastitis is one of the most important infectious diseases in dairy herds, and staphylococci are the most important etiologic agents of this disease. Antibiotics and chemical agents used in livestock for prevention and cure of the disease can accumulate in milk and give rise to food safety concerns. Rhodomyrtus tomentosa leaf extract was studied as an alternative approach to reduce the bacterial infections. The ethanolic extract of this plant demonstrated antibacterial activity with minimum inhibitory concentration (MIC values as low as 16–64 μg/mL against staphylococcal isolates. In addition, the extract had an effect on the bacterial cell surface properties by increasing its hydrophobicity in a concentration dependent manner. To further extend the antibacterial efficacy, silver nanoparticles synthesized with the extract, a pure rhodomyrtone, and liposomal encapsulated rhodomyrtone were applied and their inhibitory effects on bacterial adhesion and invasion were determined by ex vivo study in a bovine udder epidermal tissue model. These agents exerted remarkable antibacterial activity against staphylococci and decreased the adhesion of the bacterial cells to the tissues. These results supported that R. tomentosa ethanolic extract could be applied as an alternative agent for bovine udder care in dairy farms.

  6. Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

    International Nuclear Information System (INIS)

    Mas, Josep; Berg-Sørensen, Kirstine; Richardson, Andrew C; Reihani, S Nader S; Oddershede, Lene B

    2013-01-01

    With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods. (paper)

  7. Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

    Science.gov (United States)

    You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2017-07-01

    Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

  8. Modulation of protein properties in living cells using nanobodies.

    Science.gov (United States)

    Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S; Cardoso, M Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich

    2010-01-01

    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

  9. An integrated on-line irradiation and in situ live cell imaging system

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen, E-mail: gen.yang@pku.edu.cn; Wang, Yugang

    2015-09-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO{sub 2}, O{sub 2} concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

  10. An integrated on-line irradiation and in situ live cell imaging system

    International Nuclear Information System (INIS)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

    2015-01-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO 2 , O 2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia

  11. An integrated on-line irradiation and in situ live cell imaging system

    Science.gov (United States)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

    2015-09-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO2, O2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

  12. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  13. Examining live cell cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy

    Science.gov (United States)

    Khmaladze, Alexander

    2017-11-01

    Cellular apoptosis is a unique, organized series of events, leading to programmed cell death. In this work, we present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy measurements of live cell cultures yield information about cell shape and volume, changes to which are indicative of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopic measurements provide complementary information about cells, such as protein, lipid and nucleic acid content, and the spectral signatures associated with structural changes in molecules. Our work indicates that the chemical changes in proteins, which were detected by Raman measurements, preceded morphological changes, which were seen with digital holographic microscopy.

  14. Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

    Science.gov (United States)

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

    2012-05-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

  15. Raman tweezers spectroscopy of live, single red and white blood cells.

    Directory of Open Access Journals (Sweden)

    Aseefhali Bankapur

    Full Text Available An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC and white blood cells (WBC under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW. Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

  16. Energy, control and DNA structure in the living cell

    DEFF Research Database (Denmark)

    Wijker, J.E.; Jensen, Peter Ruhdal; Gomes, A. Vaz

    1995-01-01

    Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either...

  17. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

  18. Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

    Energy Technology Data Exchange (ETDEWEB)

    Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Shafran, Yana [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel); Langer, Klaus; Zlatev, Iavor [Westfälischen Wilhelms-Universität Münster, Institut für Pharmazeutische Technologie und Biopharmazie (Germany); Wronski, Robert; Windisch, Manfred [QPS Austria GmbH (Austria); Briesen, Hagen von [Fraunhofer Institute for Biomedical Engineering IBMT, Department of Cell Biology & Applied Virology (Germany); Schmidt, Reinhold [Medical University of Graz, Department of Neurology (Austria); Pietrzik, Claus [University Medical Center of the Johannes Gutenberg University of Mainz, Institute of Pathobiochemistry (Germany); Deutsch, Mordechai, E-mail: motti.jsc@gmail.com [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel)

    2015-12-15

    Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells.

  19. Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

    Directory of Open Access Journals (Sweden)

    Huiqiang Wang

    Full Text Available Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV. In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

  20. Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

    Science.gov (United States)

    Yerramilli, V Siddartha; Kim, Kyung Hyuk

    2018-03-16

    RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

  1. Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2012-04-01

    Full Text Available Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA, the electric cell-substrate impedance sensing (ECIS technique, and the light addressable potentiometric sensor (LAPS. The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology.

  2. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  3. On strain and stress in living cells

    Science.gov (United States)

    Cox, Brian N.; Smith, David W.

    2014-11-01

    Recent theoretical simulations of amelogenesis and network formation and new, simple analyses of the basic multicellular unit (BMU) allow estimation of the order of magnitude of the strain energy density in populations of living cells in their natural environment. A similar simple calculation translates recent measurements of the force-displacement relation for contacting cells (cell-cell adhesion energy) into equivalent volume energy densities, which are formed by averaging the changes in contact energy caused by a cell's migration over the cell's volume. The rates of change of these mechanical energy densities (energy density rates) are then compared to the order of magnitude of the metabolic activity of a cell, expressed as a rate of production of metabolic energy per unit volume. The mechanical energy density rates are 4-5 orders of magnitude smaller than the metabolic energy density rate in amelogenesis or bone remodeling in the BMU, which involve modest cell migration velocities, and 2-3 orders of magnitude smaller for innervation of the gut or angiogenesis, where migration rates are among the highest for all cell types. For representative cell-cell adhesion gradients, the mechanical energy density rate is 6 orders of magnitude smaller than the metabolic energy density rate. The results call into question the validity of using simple constitutive laws to represent living cells. They also imply that cells need not migrate as inanimate objects of gradients in an energy field, but are better regarded as self-powered automata that may elect to be guided by such gradients or move otherwise. Thus Ġel=d/dt 1/2 >[(C11+C12)ɛ02+2μγ02]=(C11+C12)ɛ0ɛ˙0+2μγ0γ˙0 or Ġel=ηEɛ0ɛ˙0+η‧Eγ0γ˙0 with 1.4≤η≤3.4 and 0.7≤η‧≤0.8 for Poisson's ratio in the range 0.2≤ν≤0.4 and η=1.95 and η‧=0.75 for ν=0.3. The spatial distribution of shear strains arising within an individual cell as cells slide past one another during amelogenesis is not known

  4. TimeLapseAnalyzer: Multi-target analysis for live-cell imaging and time-lapse microscopy

    DEFF Research Database (Denmark)

    Huth, Johannes; Buchholz, Malte; Kraus, Johann M.

    2011-01-01

    The direct observation of cells over time using time-lapse microscopy can provide deep insights into many important biological processes. Reliable analyses of motility, proliferation, invasive potential or mortality of cells are essential to many studies involving live cell imaging and can aid in...... counting and tube formation analysis in high throughput screening of live-cell experiments. TimeLapseAnalyzer is freely available (MATLAB, Open Source) at http://www.informatik.uniulm. de/ni/mitarbeiter/HKestler/tla......., we developed TimeLapseAnalyzer. Apart from general purpose image enhancements and segmentation procedures, this extensible, self-contained, modular cross-platform package provides dedicated modalities for fast and reliable analysis of multi-target cell tracking, scratch wound healing analysis, cell...

  5. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

    Science.gov (United States)

    Nia, Yacine; Mutel, Isabelle; Assere, Adrien; Lombard, Bertrand; Auvray, Frederic; Hennekinne, Jacques-Antoine

    2016-04-13

    Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

  6. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices

    Directory of Open Access Journals (Sweden)

    Yacine Nia

    2016-04-01

    Full Text Available Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE only in cheese and dairy products, European Food Safety Authority (EFSA data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT to evaluate the competency of the European countries’ National Reference Laboratories (NRLs to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013–2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98% and specificity (100% of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

  7. The radiation effects on the living cell

    International Nuclear Information System (INIS)

    Sage, E.; Dutrillaux, B.; Soussi, Th.; Boiteux, S.; Lopez, B.; Feunteun, J.

    1999-06-01

    This publication is a presentation of particular points discussed during the colloquium of the 15-18 june 1999, for which scientific researches are performed at the CEA/CNRS. They deal with the radiobiology, for the radiation effects on living matter; with the DNA, for the knowledge and repair mechanisms on cells submitted to ionizing radiations; with the exposition to UV in correlation with neoplasms; with the P53 gene which is a tumour suppressor. (A.L.B.)

  8. Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

    Science.gov (United States)

    Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

    2015-02-11

    Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  9. Living with a diagnosis of non-small cell lung cancer: patients' lived experiences.

    LENUS (Irish Health Repository)

    McCarthy, Ita

    2012-01-31

    The aim of this study was to explore patients\\' experience of living with non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC know that their treatment is not with curative intent and can expect distressing symptoms. In this phenomenological study, six adults with a diagnosis of NSCLC were interviewed. Data was analysed guided by van Manen\\'s six-step process. Four main themes were interpreted: \\'Maintaining my life\\'; \\'The enemy within\\'; \\'Staying on the train\\

  10. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  11. Secondary Metabolite Localization by Autofluorescence in Living Plant Cells

    Directory of Open Access Journals (Sweden)

    Pascale Talamond

    2015-03-01

    Full Text Available Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla. This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

  12. In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

    Science.gov (United States)

    Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

    2005-08-01

    Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

  13. The live cell irradiation and observation setup at SNAKE

    Energy Technology Data Exchange (ETDEWEB)

    Hable, V. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)], E-mail: volker.hable@unibw.de; Greubel, C.; Bergmaier, A.; Reichart, P. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany); Hauptner, A.; Kruecken, R. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Strickfaden, H.; Dietzel, S.; Cremer, T. [Department Biologie II, LMU-Muenchen, 82152 Martinsried (Germany); Drexler, G.A.; Friedl, A.A. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Muenchen (Germany); Dollinger, G. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)

    2009-06-15

    We describe a new setup at the ion microprobe SNAKE (Superconducting Nanoscope for Applied nuclear (Kern-) physics Experiments) at the Munich 14 MV Tandem accelerator that facilitates both living cell irradiation with sub micrometer resolution and online optical imaging of the cells before and after irradiation by state of the art phase contrast and fluorescence microscopy. The cells are kept at standard cell growth conditions at 37 {sup o}C in cell culture medium. After irradiation it is possible to switch from single ion irradiation conditions to cell observation within 0.5 s. First experiments were performed targeting substructures of a cell nucleus that were tagged by TexasRed labeled nucleotides incorporated in the cellular DNA by 55 MeV single carbon ion irradiation. In addition we show first online sequences of short time kinetics of Mdc1 protein accumulation in the vicinity of double strand breaks after carbon ion irradiation.

  14. Castration-Resistant Lgr5+ Cells Are Long-Lived Stem Cells Required for Prostatic Regeneration

    Directory of Open Access Journals (Sweden)

    Bu-er Wang

    2015-05-01

    Full Text Available The adult prostate possesses a significant regenerative capacity that is of great interest for understanding adult stem cell biology. We demonstrate that leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5 is expressed in a rare population of prostate epithelial progenitor cells, and a castration-resistant Lgr5+ population exists in regressed prostate tissue. Genetic lineage tracing revealed that Lgr5+ cells and their progeny are primarily luminal. Lgr5+ castration-resistant cells are long lived and upon regeneration, both luminal Lgr5+ cells and basal Lgr5+ cells expand. Moreover, single Lgr5+ cells can generate multilineage prostatic structures in renal transplantation assays. Additionally, Lgr5+ cell depletion revealed that the regenerative potential of the castrated adult prostate depends on Lgr5+ cells. Together, these data reveal insights into the cellular hierarchy of castration-resistant Lgr5+ cells, indicate a requirement for Lgr5+ cells during prostatic regeneration, and identify an Lgr5+ adult stem cell population in the prostate.

  15. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    Science.gov (United States)

    Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-08-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

  16. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

  17. Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

    Science.gov (United States)

    MacLean, Lorna; Price, Helen; O'Toole, Peter

    2016-01-01

    Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

  18. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    Science.gov (United States)

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

  19. A new fluorescent pH probe for imaging lysosomes in living cells.

    Science.gov (United States)

    Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

    2014-01-15

    A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Fully synthetic phage-like system for screening mixtures of small molecules in live cells.

    Science.gov (United States)

    Byk, Gerardo; Partouche, Shirly; Weiss, Aryeh; Margel, Shlomo; Khandadash, Raz

    2010-05-10

    A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.

  1. Imaging proteolytic activity in live cells and animal models.

    Directory of Open Access Journals (Sweden)

    Stefanie Galbán

    Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

  2. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

    Science.gov (United States)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

    2017-10-01

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  3. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

    2013-01-01

    and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were...... confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations....

  4. Use of luciferase probes to measure ATP in living cells and animals.

    Science.gov (United States)

    Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

    2017-08-01

    ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

  5. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  6. Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

    Science.gov (United States)

    Rodrigo-Navarro, Aleixandre; Rico, Patricia; Saadeddin, Anas; Garcia, Andres J.; Salmeron-Sanchez, Manuel

    2014-07-01

    Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications.

  7. Open-dish incubator for live cell imaging with an inverted microscope.

    Science.gov (United States)

    Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

    2003-10-01

    Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

  8. Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

    Science.gov (United States)

    Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

    2009-08-13

    It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

  9. Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

    Science.gov (United States)

    MacIntyre, Hugh L; Cullen, John J

    2016-08-01

    Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

  10. Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

    Science.gov (United States)

    Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

    2017-04-26

    Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

  11. Analysis of surface properties of fixed and live cells using derivatized agarose beads.

    Science.gov (United States)

    Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

    2002-01-01

    A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

  12. Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

    Science.gov (United States)

    Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

    2016-01-01

    Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

  13. Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

    Science.gov (United States)

    Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

    2017-03-27

    Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

  14. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  15. Immune evasion by a staphylococcal inhibitor of myeloperoxidase

    Science.gov (United States)

    de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.; Fevre, Cindy; Voyich, Jovanka M.; McCarthy, Alex J.; Garcia, Brandon L.; van Kessel, Kok P. M.; van Strijp, Jos A. G.; Geisbrecht, Brian V.; Haas, Pieter-Jan A.

    2017-01-01

    Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing. PMID:28808028

  16. Immune evasion by a staphylococcal inhibitor of myeloperoxidase

    Energy Technology Data Exchange (ETDEWEB)

    de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.; Nijland, Reindert; Fevre, Cindy; Voyich, Jovanka M.; McCarthy, Alex J.; Garcia, Brandon L.; van Kessel, Kok P. M.; van Strijp, Jos A. G.; Geisbrecht, Brian V.; Haas, Pieter-Jan A.

    2017-08-14

    Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

  17. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    Science.gov (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  18. A combined optical and atomic force microscope for live cell investigations

    International Nuclear Information System (INIS)

    Madl, Josef; Rhode, Sebastian; Stangl, Herbert; Stockinger, Hannes; Hinterdorfer, Peter; Schuetz, Gerhard J.; Kada, Gerald

    2006-01-01

    We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors

  19. Raman microscopy of individual living human embryonic stem cells

    Science.gov (United States)

    Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

    2010-04-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

  20. Raman spectroscopy for grading of live osteosarcoma cells.

    Science.gov (United States)

    Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

    2015-04-18

    Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

  1. Lives of a Cell: 40 Years Later, A Third Interpretation

    Centers for Disease Control (CDC) Podcasts

    2015-06-16

    Reginald Tucker reads an abridged version of the article Lives of a Cell: 40 Years Later, A Third Interpretation.  Created: 6/16/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 6/18/2015.

  2. Live cell CRISPR-imaging in plants reveals dynamic telomere movements

    KAUST Repository

    Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F.; Puchta, Holger; Houben, Andreas

    2017-01-01

    of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking

  3. Magnetogenetic control of protein gradients inside living cells with high spatial and temporal resolution.

    Science.gov (United States)

    Etoc, Fred; Vicario, Chiara; Lisse, Domenik; Siaugue, Jean-Michel; Piehler, Jacob; Coppey, Mathieu; Dahan, Maxime

    2015-05-13

    Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

  4. The effects of living distantly from peritoneal dialysis units on peritonitis risk, microbiology, treatment and outcomes: a multi-centre registry study

    Directory of Open Access Journals (Sweden)

    Cho Yeoungjee

    2012-06-01

    Full Text Available Abstract Background The aim of the study was to determine whether distance between residence and peritoneal dialysis (PD unit influenced peritonitis occurrence, microbiology, treatment and outcomes. Methods The study included all patients receiving PD between 1/10/2003 and 31/12/2008, using ANZDATA Registry data. Results 365 (6% patients lived ≥100 km from their nearest PD unit (distant group, while 6183 (94% lived S. aureus peritonitis (adjusted odds ratio [OR] 1.64, 95% CI 1.09-2.47. Distant patients with first peritonitis episodes were less likely to be hospitalised (64% vs 73%, p = 0.008 and receive antifungal prophylaxis (4% vs 10%, p = 0.01, but more likely to receive vancomycin-based antibiotic regimens (52% vs 42%, p  Conclusions Living ≥100 km away from a PD unit was associated with increased risk of S. aureus peritonitis, modified approaches to peritonitis treatment and peritonitis outcomes that were comparable to, or better than patients living closer to a PD unit. Staphylococcal decolonisation should receive particular consideration in remote living patients.

  5. Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries.

    Science.gov (United States)

    Ruppé, Etienne; Barbier, François; Mesli, Yasmine; Maiga, Aminata; Cojocaru, Radu; Benkhalfat, Mokhtar; Benchouk, Samia; Hassaine, Hafida; Maiga, Ibrahim; Diallo, Amadou; Koumaré, Abdel Karim; Ouattara, Kalilou; Soumaré, Sambou; Dufourcq, Jean-Baptiste; Nareth, Chhor; Sarthou, Jean-Louis; Andremont, Antoine; Ruimy, Raymond

    2009-02-01

    In staphylococci, methicillin (meticillin) resistance (MR) is mediated by the acquisition of the mecA gene, which is carried on the size and composition variable staphylococcal cassette chromosome mec (SCCmec). MR has been extensively studied in Staphylococcus aureus, but little is known about MR coagulase-negative staphylococci (MR-CoNS). Here, we describe the diversity of SCCmec structures in MR-CoNS from outpatients living in countries with contrasting environments: Algeria, Mali, Moldova, and Cambodia. Their MR-CoNS nasal carriage rates were 29, 17, 11, and 31%, respectively. Ninety-six MR-CoNS strains, comprising 75 (78%) Staphylococcus epidermidis strains, 19 (20%) Staphylococcus haemolyticus strains, 1 (1%) Staphylococcus hominis strain, and 1 (1%) Staphylococcus cohnii strain, were analyzed. Eighteen different SCCmec types were observed, with 28 identified as type IV (29%), 25 as type V (26%), and 1 as type III (1%). Fifteen strains (44%) were untypeable for their SCCmec. Thirty-four percent of MR-CoNS strains contained multiple ccr copies. Type IV and V SCCmec were preferentially associated with S. epidermidis and S. haemolyticus, respectively. MR-CoNS constitute a widespread and highly diversified MR reservoir in the community.

  6. A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

    Science.gov (United States)

    1999-07-01

    and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

  7. Engineering Multifunctional Living Paints: Thin, Convectively-Assembled Biocomposite Coatings of Live Cells and Colloidal Latex Particles Deposited by Continuous Convective-Sedimentation Assembly

    Science.gov (United States)

    Jenkins, Jessica Shawn

    Advanced composite materials could be revolutionized by the development of methods to incorporate living cells into functional materials and devices. This could be accomplished by continuously and rapidly depositing thin ordered arrays of adhesive colloidal latex particles and live cells that maintain stability and preserve microbial reactivity. Convective assembly is one method of rapidly assembling colloidal particles into thin (advantages over thicker randomly ordered composites, including enhanced cell stability and increased reactivity through minimized diffusion resistance to nutrients and reduced light scattering. This method can be used to precisely deposit live bacteria, cyanobacteria, yeast, and algae into biocomposite coatings, forming reactive biosensors, photoabsorbers, or advanced biocatalysts. This dissertation developed new continuous deposition and coating characterization methods for fabricating and characterizing 90 hours) photohydrogen production under anoxygenic conditions. Nutrient reduction slows cell division, minimizing coating outgrowth, and promotes photohydrogen generation, improving coating reactivity. Scanning electron microscopy of microstructure revealed how coating reactivity can be controlled by the size and distribution of the nanopores in the biocomposite layers. Variations in colloid microsphere size and suspension composition do not affect coating reactivity, but both parameters alter coating microstructure. Porous paper coated with thin coatings of colloidal particles and cells to enable coatings to be used in a gas-phase without dehydration may offer higher volumetric productivity for hydrogen production. Future work should focus on optimization of cell density, light intensity, media cycling, and acetate concentration.

  8. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

    Science.gov (United States)

    Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

    2013-01-01

    Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

  9. Hemi-fused structure mediates and controls fusion and fission in live cells.

    Science.gov (United States)

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

  10. Hepatic Artery Mycotic Aneurysm Associated with Staphylococcal Endocarditis with Successful Treatment: Case Report with Review of the Literature

    Directory of Open Access Journals (Sweden)

    Dhara Chaudhari

    2013-01-01

    Full Text Available Mycotic hepatic artery aneurysm is a vascular pathology associated with bacterial endocarditis. It is rare in occurrence after the introduction of effective antibiotics. We present a young patient with injection drug abuse associated staphylococcal endocarditis which was successfully treated with antibiotics and valve replacement who presented with abdominal pain. He was found to have mycotic aneurysm of hepatic artery which was successfully treated with coil embolization.

  11. Molecular Detection of Inducible Clindamycin Resistance among Staphylococcal Strains Isolated from Hospital Patients

    Directory of Open Access Journals (Sweden)

    Shadiyeh Abdollahi

    2013-04-01

    Full Text Available Background & Objectives: Macrolide, lincosamide and streptogramin B (MLSB antimicrobial agents are used in the treatment of staphylococcal infections. They prevent the microbial protein synthesis system through binding to 23 S rRNA. The aim of this study was to apply molecular methods to detect inducible clindamycin resistance genes among staphylococcal strains isolated from clinical specimens.   Methods : Two hundred staphylococcus strains were isolated from nose and throat swabs of patients in Toohid and Besat hospitals in Sanandaj . Antimicrobial susceptibilities of isolates were determined using disc diffusion method, agar screen test and D-Test. A multiplex PCR was performed using primers specific for erm (A, B, C, TR genes.   Results: Out of 200 isolates, 18.5 % were MRSA and 32% were MRCNS (methicillin resistant coagulase negative staphylococci. Of 80 erythromycin resistant isolates, 48 were coagulase negative and 32 were S. aureus. Among the 48 coagulase negative staphylococci (CONS isolates, 11.63% expressed the MLSB-inducible phenotypes. Using PCR, the frequency of different genes in the collection of isolates were as follows: ermA 5.41 % , erm B 5.41 % , and erm C 3.13%. The ermTR gene was negative in all isolates. Among the 32 S. aureus isolates, 9.38% expressed the MLSB-nducible phenotype. Using PCR, these isolates harbored erm A (2.22%, ermB (2.22%, ermC (2.22% and ermTR (2.22% .   Conclusion: This is the first study to show the rate of inducible clindamycin clinical isolates of staphylococci harboring erm genes in Sananadaj. It also demonstrated the frequency of erm genes was higher among CONS isolates than S. aureus. This data suggested the transfer of resistance gene from nonpathogenic to pathogenic strains is likely to happen. Therefore, screening and control of these resistance genes is recommended at clinical laboratories.

  12. Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

    Science.gov (United States)

    Karau, Melissa J; Tilahun, Mulualem E; Krogman, Ashton; Osborne, Barbara A; Goldsby, Richard A; David, Chella S; Mandrekar, Jayawant N; Patel, Robin; Rajagopalan, Govindarajan

    2017-10-03

    Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

  13. Noncontiguous Finished Genome Sequence of Staphylococcus aureus KLT6, a Staphylococcal Enterotoxin B-Positive Strain Involved in a Food Poisoning Outbreak in Switzerland

    Science.gov (United States)

    Tobes, Raquel; Manrique, Marina; Brozynska, Marta; Stephan, Roger; Pareja, Eduardo

    2013-01-01

    We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B. PMID:23704175

  14. Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products.

    Science.gov (United States)

    Yang, S E; Yu, R C; Chou, C C

    2001-01-22

    In this study, growth and survival of Salmonella spp. and Staphylococcus aureus in steamed egg and scrambled egg held at 5, 18, 22, 37, 55 and 60 degrees C are investigated. The production of staphylococcal enterotoxin in steamed egg is also examined. Results reveal that Salmonella spp. and Staph. aureus in the egg products multiply best at 37 degrees C, followed closely by 22 and 18 degrees C. Neither pathogen showed growth in the egg products held at 5 degrees C. Initial inoculation dose, holding temperature and holding time affected the population of both organisms found in the egg products. Staphylococcal enterotoxin A (SEA) and B (SEB) are detected only in the egg products held at 37 or 22 degrees C. After holding at 37 degrees C for 36 h, scrambled egg inoculated with ca. 5.0 log cfu/g Staph. aureus contains the highest levels of SEA (> 64 ng/g) and SEB (> 64 ng/g). Although Salmonella spp. and Staph. aureus grow better in steamed eggs than in scrambled eggs, production of staphylococcal enterotoxin, in general, was higher in scrambled eggs than in steamed eggs. On the other hand, a repaid destruction of the test organisms in steamed eggs held at 60 degrees C was observed. Holding the steamed eggs at 60 degrees C, Salmonella spp. and Staph. aureus with an initial population of ca. 5.9 and 5.6 log cfu/g, respectively, reduced to a non-detectable level in 1 h.

  15. Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB.

    Directory of Open Access Journals (Sweden)

    Mulualem E Tilahun

    Full Text Available Staphylococcal enterotoxin B (SEB is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.

  16. Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

    Science.gov (United States)

    Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D; Chorro, Laurent; Carlin, Leo M; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S

    2013-02-19

    Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

  17. Effect of infrared light on live blood cells: Role of β-carotene.

    Science.gov (United States)

    Barkur, Surekha; Bankapur, Aseefhali; Chidangil, Santhosh; Mathur, Deepak

    2017-06-01

    We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells. Copyright © 2017. Published by Elsevier B.V.

  18. Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

    Science.gov (United States)

    Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

    2015-02-01

    Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. © FASEB.

  19. Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

    Science.gov (United States)

    Okuno, Masanari; Hamaguchi, Hiro-o

    2010-12-15

    We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

  20. Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia.

    Science.gov (United States)

    Zhao, Xi; Huang, Xiaomeng; Wang, Xinmei; Wu, Yun; Eisfeld, Ann-Kathrin; Schwind, Sebastian; Gallego-Perez, Daniel; Boukany, Pouyan E; Marcucci, Guido I; Lee, Ly James

    2015-12-01

    A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

  1. Labeling proteins on live mammalian cells using click chemistry.

    Science.gov (United States)

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

  2. In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

    Science.gov (United States)

    Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

    2015-08-04

    Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

  3. DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

    Science.gov (United States)

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

  4. A combined optical and atomic force microscope for live cell investigations

    Energy Technology Data Exchange (ETDEWEB)

    Madl, Josef [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Rhode, Sebastian [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Stangl, Herbert [Institute for Medical Chemistry, Medical University Vienna, Waehringerstr. 10, 1090 Vienna (Austria); Stockinger, Hannes [Department of Molecular Immunology, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Lazarettgasse 19, 1090 Vienna (Austria); Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Schuetz, Gerhard J. [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kada, Gerald [Scientec, Mitterbauerweg 4, 4020 Linz (Austria)]. E-mail: gerald_kada@agilent.com

    2006-06-15

    We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors.

  5. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  6. Applications of chitosan-based thermo-sensitive copolymers for harvesting living cell sheet

    International Nuclear Information System (INIS)

    Chen, J.-P.; Yang, T.-F.

    2008-01-01

    A thermo-sensitive chitosan-based copolymer hydrogel was used for harvesting living cell sheets. The hydrogel was tested for harvesting 3T3 cells after carrying out cell culture at 37 deg. C and incubating the confluent cells at 20 deg. C for spontaneous detachment of cell sheets from hydrogel surface without enzyme treatment. Results from cell viability assay and microscopy observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was about 80%. The detached cell sheet retained high viability and could proliferate again after transferred to a new culture surface

  7. Quantitative control of mitochondria transfer between live single cells using a microfluidic device

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

    Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

  8. Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

    Science.gov (United States)

    Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

    2010-05-01

    The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

  9. A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

    NARCIS (Netherlands)

    E. Ghaznavi Rad (Ehsanollah); N.S. Mariana (Nor Shamsudin); Z. Sekawi (Zamberi); A.F. van Belkum (Alex); V. Neela (Vasanthakumari)

    2010-01-01

    textabstractA multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus

  10. A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

    Science.gov (United States)

    Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

    2017-03-01

    Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

  11. Short- versus long-duration levofloxacin plus rifampicin for acute staphylococcal prosthetic joint infection managed with implant retention: a randomised clinical trial.

    Science.gov (United States)

    Lora-Tamayo, Jaime; Euba, Gorane; Cobo, Javier; Horcajada, Juan Pablo; Soriano, Alex; Sandoval, Enrique; Pigrau, Carles; Benito, Natividad; Falgueras, Luis; Palomino, Julián; Del Toro, María Dolores; Jover-Sáenz, Alfredo; Iribarren, José Antonio; Sánchez-Somolinos, Mar; Ramos, Antonio; Fernández-Sampedro, Marta; Riera, Melchor; Baraia-Etxaburu, Josu Mirena; Ariza, Javier

    2016-09-01

    Levofloxacin plus rifampicin (L+R) is the treatment of choice for acute staphylococcal prosthetic joint infection (PJI) managed with debridement and implant retention (DAIR). Long courses have been empirically recommended, but some studies have suggested that shorter treatments could be as effective. Our aim was to prove that a short treatment schedule was non-inferior to the standard long schedule. An open-label, multicentre, randomised clinical trial (RCT) was performed. Patients with an early post-surgical or haematogenous staphylococcal PJI, managed with DAIR and initiated on L+R were randomised to receive 8 weeks of treatment (short schedule) versus a long schedule (3 months or 6 months for hip or knee prostheses, respectively). The primary endpoint was cure rate. From 175 eligible patients, 63 were included (52% women; median age, 72 years): 33 patients (52%) received the long schedule and 30 (48%) received the short schedule. There were no differences between the two groups except for a higher rate of polymicrobial infection in the long-schedule group (27% vs. 7%; P = 0.031). Median follow-up was 540 days. In the intention-to-treat analysis, cure rates were 58% and 73% in patients receiving the long and short schedules, respectively (difference -15.7%, 95% CI -39.2% to 7.8%). Forty-four patients (70%) were evaluable per-protocol: cure rates were 95.0% and 91.7% for the long and short schedules, respectively (difference 3.3%, 95% CI -11.7% to 18.3%). This is the first RCT suggesting that 8 weeks of L+R could be non-inferior to longer standard treatments for acute staphylococcal PJI managed with DAIR. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  12. Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed.

    Science.gov (United States)

    Godinho, Luis F; Schrader, Michael

    2017-01-01

    Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.

  13. Fluorescent tags of protein function in living cells.

    Science.gov (United States)

    Whitaker, M

    2000-02-01

    A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000. Copyright 2000 John Wiley & Sons, Inc.

  14. Automated analysis of invadopodia dynamics in live cells

    Directory of Open Access Journals (Sweden)

    Matthew E. Berginski

    2014-07-01

    Full Text Available Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

  15. Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

    KAUST Repository

    Elafandy, Rami T.; AbuElela, Ayman; Mishra, Pawan; Janjua, Bilal; Oubei, Hassan M.; Buttner, Ulrich; Majid, Mohammed Abdul; Ng, Tien Khee; Merzaban, Jasmeen; Ooi, Boon S.

    2016-01-01

    Knowledge of materials' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes' emission spectrally shift based on the material's thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

  16. Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

    KAUST Repository

    Elafandy, Rami T.

    2016-11-23

    Knowledge of materials\\' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes\\' emission spectrally shift based on the material\\'s thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

  17. Increasing the frequency of hand washing by healthcare workers does not lead to commensurate reductions in staphylococcal infection in a hospital ward

    Directory of Open Access Journals (Sweden)

    Kerr Kevin G

    2008-09-01

    Full Text Available Abstract Background Hand hygiene is generally considered to be the most important measure that can be applied to prevent the spread of healthcare-associated infection (HAI. Continuous emphasis on this intervention has lead to the widespread opinion that HAI rates can be greatly reduced by increased hand hygiene compliance alone. However, this assumes that the effectiveness of hand hygiene is not constrained by other factors and that improved compliance in excess of a given level, in itself, will result in a commensurate reduction in the incidence of HAI. However, several researchers have found the law of diminishing returns to apply to hand hygiene, with the greatest benefits occurring in the first 20% or so of compliance, and others have demonstrated that poor cohorting of nursing staff profoundly influences the effectiveness of hand hygiene measures. Collectively, these findings raise intriguing questions about the extent to which increasing compliance alone can further reduce rates of HAI. Methods In order to investigate these issues further, we constructed a deterministic Ross-Macdonald model and applied it to a hypothetical general medical ward. In this model the transmission of staphylococcal infection was assumed to occur after contact with the transiently colonized hands of HCWs, who, in turn, acquire contamination only by touching colonized patients. The aim of the study was to evaluate the impact of imperfect hand cleansing on the transmission of staphylococcal infection and to identify, whether there is a limit, above which further hand hygiene compliance is unlikely to be of benefit. Results The model demonstrated that if transmission is solely via the hands of HCWs, it should, under most circumstances, be possible to prevent outbreaks of staphylococcal infection from occurring at a hand cleansing frequencies Conclusion Although our study confirmed hand hygiene to be an effective control measure, it demonstrated that the law of

  18. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-01-01

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  19. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-01-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

  20. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-03

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  1. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

    International Nuclear Information System (INIS)

    Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

    2009-01-01

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  2. Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

    Science.gov (United States)

    Carlson, Alicia L; Fujisaki, Joji; Wu, Juwell; Runnels, Judith M; Turcotte, Raphaël; Spencer, Joel A; Celso, Cristina Lo; Scadden, David T; Strom, Terry B; Lin, Charles P

    2013-01-01

    We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

  3. Live birth potential of good morphology and vitrified blastocysts presenting abnormal cell divisions

    DEFF Research Database (Denmark)

    Azzarello, Antonino; Høst, Thomas; Hay-Schmidt, Anders

    2017-01-01

    a lower live birth rate (17.0%) than blastocyst with solely regular cell divisions (29.3%). ACDs could occur at more than one cell division in the same good morphology blastocyst. Reported as independent events, we observed ACDs occurring more frequently at the later cell cycles (1st: 1.3%; 2nd: 8.0%; 3rd...

  4. Raman tweezers in microfluidic systems for analysis and sorting of living cells

    Science.gov (United States)

    Pilát, Zdeněk.; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

    2014-12-01

    We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

  5. Cocompartmentation of proteins and K+ within the living cell

    International Nuclear Information System (INIS)

    Kellermayer, M.; Ludany, A.; Jobst, K.; Szucs, G.; Trombitas, K.; Hazlewood, C.F.

    1986-01-01

    Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins [ 35 S]methionine-labelled and 42 K-labelled K + were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brij 58. In Triton X-100-treated cells, the loss of K + was complete within 2 min. The loss of K + from the Brij 58-treated cells was complete only after 10 min and the mobilization of K + showed sigmoid-type release kinetics. These results support the view that most of K + and diffusible proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell

  6. Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

    Science.gov (United States)

    Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

    2017-08-01

    To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

  7. Tabebuia avellanedae naphthoquinones: activity against methicillin-resistant staphylococcal strains, cytotoxic activity and in vivo dermal irritability analysis

    Directory of Open Access Journals (Sweden)

    Giambiagi-deMarval Marcia

    2006-03-01

    Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA and coagulase-negative staphylococcus infections are a worldwide concern. Currently, these isolates have also shown resistance to vancomycin, the last therapy used in these cases. It has been observed that quinones and other related compounds exhibit antibacterial activity. This study evaluated the antibacterial activity, toxicity and in vivo dermal irritability of lapachol extracted from Tabebuia avellanedae and derivatives against methicillin-resistant staphylococcal isolates. In addition, its mechanism of action was also analyzed. Methods The compounds β-lapachone, 3-hydroxy β N lapachone and α-lapachone were tested to determine the MIC values against methicillin-resistant S. aureus, S. epidermidis and S. haemolyticus strains, being the two last ones hetero-resistant to vancomycin. Experiments of protein synthesis analysis to investigate the naphthoquinones action were assessed. In vitro toxicity to eukaryotic BSC-40 African Green Monkey Kidney cell cultures and in vivo primary dermal irritability in healthy rabbits were also performed. Results The compounds tested showed antibacterial activity (MICs of 8, 4/8 and 64/128 μg/mL to β-lapachone, 3-hydroxy β N lapachone and α-lapachone, respectively, but no bactericidal activity was observed (MBC > 512 μg/mL for all compounds. Although it has been observed toxic effect in eukaryotic cells, the compounds were shown to be atoxic when applied as topic preparations in healthy rabbits. No inhibition of proteins synthesis was observed. Conclusion Our results suggest that quinones could be used in topic preparations against wound infections caused by staphylococci, after major investigation of the pharmacological properties of the compounds. Studies about the use of these compounds on tumoral cells could be carried on, due to their effect in eukaryotic cells metabolism.

  8. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    Energy Technology Data Exchange (ETDEWEB)

    Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  9. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    International Nuclear Information System (INIS)

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-01-01

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

  10. A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

    Science.gov (United States)

    Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

    2017-06-01

    As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

  11. Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing

    DEFF Research Database (Denmark)

    Horvath, R.; Pedersen, H.C.; Skivesen, N.

    2005-01-01

    The effect of the attachment and spreading of living cells on the modes of a grating coupled reverse symmetry waveguide sensor is investigated in real time. The reverse symmetry design has an increased probing depth into the sample making it well suited for the monitoring of cell morphology....... As a result, significant changes in the incoupling peak height and peak shape were observed during cell attachment and spreading. It is suggested that the area under the incoupling peaks reflects the initial cell attachment process, while the mean peak position is mostly governed by the spreading of the cells...

  12. Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

    Science.gov (United States)

    Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

    2018-02-01

    Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

  13. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

  14. New quantitative image analysis of staphylococcal biofilms on the surfaces of nontranslucent metallic biomaterials.

    Science.gov (United States)

    Adachi, Kouichi; Tsurumoto, Toshiyuki; Yonekura, Akihiko; Nishimura, Seisuke; Kajiyama, Shiro; Hirakata, Yoichi; Shindo, Hiroyuki

    2007-03-01

    Implant-related infection after orthopedic surgery is difficult to cure. One of the causes of infection is the bacterial biofilm that forms around biomaterials used during surgery. Therefore, it is necessary to investigate bacterial biofilms extensively to resolve the problems of these postoperative infections. However, no established culture method or quantification system exists for bacterial biofilms grown on the surface of the metallic biomaterials used in orthopedics, which are nonradiolucent. The purpose of this study was to develop a quantitative method to evaluate the difference in resistance of stainless steel versus titanium to staphylococcal biofilms and the efficacy of antibiotics against biofilms. The bacterial strains used in this study were three Staphylococcus aureus stains: strain Seattle 1945 and two clinical strains cultured from postoperative infections. Staphylococcal biofilms were formed on stainless steel washers (SUS304) and titanium washers (pure titanium). They were stained with crystal violet and were examined with a digital microscope to calculate the bacterial coverage rate (BCR) by NIH imaging. The BCR of S. aureus biofilms formed on stainless steel and titanium washers increased over time. At 24, 48, and 72 h after cultivation, the amount of biofilm on the surface of the stainless steel washers was significantly greater or tended to be greater than that on the titanium. Cefazolin was applied to the obtained biofilms of two clinically isolated S. aureus strains. Cefazolin did not eradicate the biofilms but significantly reduced the biofilm of one strain. The newly developed quantitative method (static microtube culture and measurement system) was useful for assessing the amount of bacterial biofilms on the surface of nontranslucent biomaterial. We found that titanium may be more resistant to bacterial infection than stainless steel. To control implant-related severe infections, the biomaterials should be assessed from the viewpoint of

  15. Digital photocontrol of the network of live excitable cells

    Science.gov (United States)

    Erofeev, I. S.; Magome, N.; Agladze, K. I.

    2011-11-01

    Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

  16. Determination of the nano-scaled contact area of staphylococcal cells.

    Science.gov (United States)

    Spengler, Christian; Thewes, Nicolas; Jung, Philipp; Bischoff, Markus; Jacobs, Karin

    2017-07-20

    Bacterial adhesion is a crucial step during the development of infections as well as the formation of biofilms. Hence, fundamental research of bacterial adhesion mechanisms is of utmost importance. So far, less is known about the size of the contact area between bacterial cells and a surface. This gap will be filled by this study using a single-cell force spectroscopy-based method to investigate the contact area between a single bacterial cell of Staphylococcus aureus and a solid substrate. The technique relies on the strong influence of the hydrophobic interaction on bacterial adhesion: by incrementally crossing a very sharp hydrophobic/hydrophilic interface while performing force-distance curves with a single bacterial probe, the bacterial contact area can be determined. Assuming circular contact areas, their radii - determined in our experiments - are in the range from tens of nanometers to a few hundred nanometers. The contact area can be slightly enlarged by a larger load force, yet does not resemble a Hertzian contact, rather, the enlargement is a property of the individual bacterial cell. Additionally, Staphylococcus carnosus has been probed, which is less adherent than S. aureus, yet both bacteria exhibit a similar contact area size. This corroborates the notion that the adhesive strength of bacteria is not a matter of contact area, but rather a matter of which and how many molecules of the bacterial species' cell wall form the contact. Moreover, our method of determining the contact area can be applied to other microorganisms and the results might also be useful for studies using nanoparticles covered with soft, macromolecular coatings.

  17. High content live cell imaging for the discovery of new antimalarial marine natural products

    Directory of Open Access Journals (Sweden)

    Cervantes Serena

    2012-01-01

    Full Text Available Abstract Background The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. Methods A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Results Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Conclusion Collectively, our results show that high-content live cell-imaging (HCLCI can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials.

  18. High content live cell imaging for the discovery of new antimalarial marine natural products.

    Science.gov (United States)

    Cervantes, Serena; Stout, Paige E; Prudhomme, Jacques; Engel, Sebastian; Bruton, Matthew; Cervantes, Michael; Carter, David; Tae-Chang, Young; Hay, Mark E; Aalbersberg, William; Kubanek, Julia; Le Roch, Karine G

    2012-01-03

    The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. © 2011 Cervantes et al; licensee BioMed Central Ltd.

  19. Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

    Directory of Open Access Journals (Sweden)

    Laetitia Kurzawa

    Full Text Available Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

  20. Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

    International Nuclear Information System (INIS)

    Noguchi, M.; Yokoya, A.; Narita, A.; Fujii, K.; Kanari, Y.

    2015-01-01

    Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death. (authors)

  1. Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Al-Shammari AM

    2014-05-01

    Full Text Available Ahmed M Al-Shammari,1 Farah E Ismaeel,2 Shahlaa M Salih,2 Nahi Y Yaseen11Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Researches, Mustansiriya University, 2Departments of Biotechnology, College of Science, Al-Nahrain University, Baghdad, IraqAbstract: Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5 was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5 cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05 on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72

  2. Imaging in living cells using νB-H Raman spectroscopy: monitoring COSAN uptake.

    Science.gov (United States)

    Tarrés, Màrius; Canetta, Elisabetta; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

    2014-03-28

    The boron-rich cobaltabisdicarbollide (COSAN) and its 8,8'-I2 derivative (I2-COSAN), both of purely inorganic nature, are shown to accumulate within living cells, where they can be detected using νB-H Raman microspectroscopy. This demonstrates an alternative method for cell labelling and detection.

  3. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  4. A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging.

    Science.gov (United States)

    Koo, Chi-Kin; Wong, Ka-Leung; Man, Cornelia Wing-Yin; Lam, Yun-Wah; So, Leo King-Yan; Tam, Hoi-Lam; Tsao, Sai-Wah; Cheah, Kok-Wai; Lau, Kai-Chung; Yang, Yang-Yi; Chen, Jin-Can; Lam, Michael Hon-Wah

    2009-02-02

    The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

  5. Electrical pulse – mediated enhanced delivery of silver nanoparticles into living suspension cells for surface enhanced Raman spectroscopy

    International Nuclear Information System (INIS)

    Lin, J; Li, B; Feng, S; Chen, G; Li, Y; Huang, Z; Chen, R; Yu, Y; Huang, H; Lin, S; Li, C; Su, Y; Zeng, H

    2012-01-01

    Electrical pulse-mediated enhanced silver nanoparticles delivery is a much better method for intracellular surface-enhanced Raman spectroscopy (SERS) measurements of suspension cells. Robust and high-quality SERS spectra of living suspension cells were obtained based on an electroporation-SERS method, which can overcomes the shortcoming of non-uniform distribution of silver nanoparticles localized in the cell cytoplasm after electroporation and reduces the amount variance of silver nanoparticles delivered into different cells. The electroporation parameters include three 150 V (375 V/cm) electric pulses of 1, 5, and 5 ms durations respectively. Our results indicate that considerable amount of silver nanoparticles can be rapidly delivered into the human promyelocytic leukemia HL60 cells, and the satisfied SERS spectra were obtained while the viability of the treated cells was highly maintained (91.7%). The electroporation-SERS method offers great potential approach in delivering silver nanoparticles into living suspension cells, which is useful for widely biomedical applications including the real-time intracellular SERS analysis of living cells

  6. Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection.

    Science.gov (United States)

    Real, Fernando; Sennepin, Alexis; Ganor, Yonatan; Schmitt, Alain; Bomsel, Morgane

    2018-05-08

    During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa. Copyright © 2018. Published by Elsevier Inc.

  7. Self-adhesive microculture system for extended live cell imaging.

    Science.gov (United States)

    Skommer, J; McGuinness, D; Wlodkowic, D

    2011-06-01

    Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.

  8. Fluorescent CdSe/ZnS nanocrystal-peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells

    International Nuclear Information System (INIS)

    Chen, Fanqing; Gerion, Daniele

    2004-01-01

    One of the biggest challenges in cell biology is the imaging of living cells. For this purpose, the most commonly used visualization tool is fluorescent markers. However, conventional labels, such as organic fluorescent dyes or green fluorescent proteins (GFP), lack the photostability to allow the tracking of cellular events that happen over minutes to days. In addition, they are either toxic to cells (dyes), or difficult to construct and manipulate (GFP). We report here the use of a new class of fluorescent labels, silanized CdSe/ZnS nanocrystal-peptide conjugates, for imaging the nuclei of living cells. CdSe/ZnS nanocrystals, or so called quantum dots (qdots), are extremely photostable, and have been used extensively in cellular imaging of fixed cells. However, most of the studies about living cells so far have been concerned only with particle entry into the cytoplasm or the localization of receptors on the cell membrane. Specific targeting of qdots to the nucleus of living cells ha s not been reported in previous studies, due to the lack of a targeting mechanism and proper particle size. Here we demonstrate for the first time the construction of a CdSe/ZnS nanocrystal-peptide conjugate that carries the SV40 large T antigen nuclear localization signal (NLS), and the transfection of the complex into living cells. By a novel adaptation of commonly used cell transfection techniques for qdots, we were able to introduce and retain the NLS-qdots conjugate in living cells for up to a week without detectable negative cellular effects. Moreover, we can visualize the movement of the CdSe/ZnS nanocrystal-peptide conjugates from cytoplasm to the nucleus, and the accumulation of the complex in the cell nucleus, over a long observation time period. This report opens the door for using qdots to visualize long-term biological events that happen in the cell nucleus, and provides a new nontoxic, long-term imaging platform for cell nuclear processes

  9. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Ji Hye; Joo, Sang-Woo [Department of Chemistry, Soongsil University, Seoul 156-743 (Korea, Republic of); Cho, Keunchang [Logos Biosystems, Incorporated, Anyang 431-070 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr, E-mail: sjoo@ssu.ac.kr [Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2011-06-10

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  10. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    International Nuclear Information System (INIS)

    Seo, Ji Hye; Joo, Sang-Woo; Cho, Keunchang; Lee, So Yeong

    2011-01-01

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  11. Weak Ergodicity Breaking of Receptor Motion in Living Cells Stemming from Random Diffusivity

    Science.gov (United States)

    Manzo, Carlo; Torreno-Pina, Juan A.; Massignan, Pietro; Lapeyre, Gerald J.; Lewenstein, Maciej; Garcia Parajo, Maria F.

    2015-01-01

    Molecular transport in living systems regulates numerous processes underlying biological function. Although many cellular components exhibit anomalous diffusion, only recently has the subdiffusive motion been associated with nonergodic behavior. These findings have stimulated new questions for their implications in statistical mechanics and cell biology. Is nonergodicity a common strategy shared by living systems? Which physical mechanisms generate it? What are its implications for biological function? Here, we use single-particle tracking to demonstrate that the motion of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a receptor with unique pathogen-recognition capabilities, reveals nonergodic subdiffusion on living-cell membranes In contrast to previous studies, this behavior is incompatible with transient immobilization, and, therefore, it cannot be interpreted according to continuous-time random-walk theory. We show that the receptor undergoes changes of diffusivity, consistent with the current view of the cell membrane as a highly dynamic and diverse environment. Simulations based on a model of an ordinary random walk in complex media quantitatively reproduce all our observations, pointing toward diffusion heterogeneity as the cause of DC-SIGN behavior. By studying different receptor mutants, we further correlate receptor motion to its molecular structure, thus establishing a strong link between nonergodicity and biological function. These results underscore the role of disorder in cell membranes and its connection with function regulation. Because of its generality, our approach offers a framework to interpret anomalous transport in other complex media where dynamic heterogeneity might play a major role, such as those found, e.g., in soft condensed matter, geology, and ecology.

  12. Distinction between Pore Assembly by Staphylococcal α-Toxin versus Leukotoxins

    Directory of Open Access Journals (Sweden)

    Olivier Joubert

    2007-01-01

    Full Text Available The staphylococcal bipartite leukotoxins and the homoheptameric α-toxin belong to the same family of β-barrel pore-forming toxins despite slight differences. In the α-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the β-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST and bridging of the N-terminal extremity to the adjacent β-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and α-toxin.

  13. Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

    Science.gov (United States)

    Haas, Beth L.; Matson, Jyl S.; DiRita, Victor J.; Biteen, Julie S.

    2015-01-01

    Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. PMID:25123183

  14. Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents.

    Science.gov (United States)

    Schillaci, Domenico; Cusimano, Maria Grazia; Cunsolo, Vincenzo; Saletti, Rosaria; Russo, Debora; Vazzana, Mirella; Vitale, Maria; Arizza, Vincenzo

    2013-06-24

    The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

  15. Live-Cell MicroRNA Imaging through MnO2 Nanosheet-Mediated DD-A Hybridization Chain Reaction.

    Science.gov (United States)

    Ou, Min; Huang, Jin; Yang, Xiaohai; He, Xiaoxiao; Quan, Ke; Yang, Yanjing; Xie, Nuli; Li, Jing; Wang, Kemin

    2018-01-18

    Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live-cell miRNA imaging using a biodegradable MnO 2 nanosheet-mediated DD-A FRET hybridization chain reaction (HCR). The MnO 2 nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO 2 nanosheets are degraded by cellular GSH. Then, the target miR-21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (donor) and one TAMRA (acceptor) into close proximity to generate significantly enhanced DD-A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Characterization of the residual structure in the unfolded state of the Delta 131 Delta fragment of staphylococcal nuclease

    DEFF Research Database (Denmark)

    Francis, C. J.; Lindorff-Larsen, Kresten; Best, R. B.

    2006-01-01

    dynamics simulations to characterise the residual structure of the 131 fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that 131 under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild......The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular...

  17. Labeling proteins inside living cells using external fluorophores for microscopy.

    Science.gov (United States)

    Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

    2016-12-09

    Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

  18. Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

    Energy Technology Data Exchange (ETDEWEB)

    Puricelli, Luca; Galluzzi, Massimiliano; Schulte, Carsten; Podestà, Alessandro, E-mail: alessandro.podesta@mi.infn.it; Milani, Paolo [CIMaINa and Department of Physics, Università degli Studi di Milano, Via Celoria 16, 20133 Milano (Italy)

    2015-03-15

    Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

  19. Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

    Science.gov (United States)

    Heine, Jörn; Wurm, Christian A.; Keller-Findeisen, Jan; Schönle, Andreas; Harke, Benjamin; Reuss, Matthias; Winter, Franziska R.; Donnert, Gerald

    2018-05-01

    Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 μm.

  20. Silicon nanocrystals and nanodiamonds in live cells: photoluminescence characteristics, cytotoxicity and interaction with cell cytoskeleton

    Czech Academy of Sciences Publication Activity Database

    Fučíková, A.; Valenta, J.; Pelant, Ivan; Hubálek Kalbáčová, M.; Brož, A.; Rezek, Bohuslav; Kromka, Alexander; Bakaeva, Zulfiya

    2014-01-01

    Roč. 4, č. 20 (2014), s. 10334-10342 ISSN 2046-2069 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GA202/09/2078 Institutional support: RVO:68378271 ; RVO:61389013 Keywords : silicon nanocrystals * nanodiamonds * live cells * photoluminescence Subject RIV: BO - Biophysics Impact factor: 3.840, year: 2014

  1. The spatio-temporal organization of DNA-repair: a live cell study

    NARCIS (Netherlands)

    D. Hoogstraten (Deborah)

    2003-01-01

    textabstractThe aim of the work outlined in this thesis is to gain more insight into the organization, dynamic properties and differential reaction kinetics of NER factors within living mammalian cell nuclei. To accomplish this, we made use of the green fluorescent protein technology to study TFIIH

  2. Structural model of radiation effects in living cells

    International Nuclear Information System (INIS)

    Neyman, J.; Puri, P.S.

    1976-01-01

    The chance mechanism of cell damage and of repair in the course of irradiation involves two details familiar to biologists that thus far seem to have been overlooked in mathematical treatment. One of these details is that, generally, the passage of a single ''primary'' radiation particle generates a ''cluster'' of secondaries which can produce ''hits'' that damage the living cell. With high linear energy transfer, each cluster contains very many secondary particles. With low linear energy transfer, the number of secondaries per cluster is generally small. The second overlooked detail of the chance mechanism is concerned with what may be called the time scales of radiation damage and of the subsequent repair. The generation of a cluster of secondary particles and the possible hits occur so rapidly that, for all practical purposes, they may be considered as occurring instantly. On the other hand, the subsequent changes in the damaged cells appear to require measurable amounts of time. The constructed stochastic model embodies these details, the clustering of secondary particles and the time scale difference. The results explain certain details of observed phenomena

  3. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    OpenAIRE

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultipl...

  4. Staphylococcal superantigen-specific IgE antibodies: degree of sensitization and association with severity of asthma.

    Science.gov (United States)

    Elabras, José; Mello, Fernanda Carvalho de Queiroz; Lupi, Omar; Bica, Blanca Elena Rios Gomes; Papi, José Angelo de Souza; França, Alfeu Tavares

    2016-01-01

    To determine the presence of staphylococcal superantigen-specific IgE antibodies and degree of IgE-mediated sensitization, as well as whether or not those are associated with the severity of asthma in adult patients. This was a cross-sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of Rio de Janeiro, Brazil. Consecutive patients were divided into two groups according to the severity of asthma based on the Global Initiative for Asthma criteria: mild asthma (MA), comprising patients with mild intermittent or persistent asthma; and moderate or severe asthma (MSA). We determined the serum levels of staphylococcal toxin-specific IgE antibodies, comparing the results and performing a statistical analysis. The study included 142 patients: 72 in the MA group (median age = 46 years; 59 females) and 70 in the MSA group (median age = 56 years; 60 females). In the sample as a whole, 62 patients (43.7%) presented positive results for staphylococcal toxin-specific IgE antibodies: staphylococcal enterotoxin A (SEA), in 29 (20.4%); SEB, in 35 (24.6%); SEC, in 33 (23.2%); and toxic shock syndrome toxin (TSST), in 45 (31.7%). The mean serum levels of IgE antibodies to SEA, SEB, SEC, and TSST were 0.96 U/L, 1.09 U/L, 1.21 U/L, and 1.18 U/L, respectively. There were no statistically significant differences between the two groups in terms of the qualitative or quantitative results. Serum IgE antibodies to SEA, SEB, SEC, and TSST were detected in 43.7% of the patients in our sample. However, neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma. Determinar a presença de anticorpos IgE específicos para superantígenos estafilocócicos e o grau de sensibilização mediada por esses, assim como se esses estão associados à gravidade da asma em pacientes adultos. Estudo transversal incluindo asmáticos adultos em acompanhamento ambulatorial em

  5. The lived experience of autologous stem cell-transplanted patients: Post-transplantation and before discharge.

    Science.gov (United States)

    Alnasser, Qasem; Abu Kharmah, Salahel Deen; Attia, Manal; Aljafari, Akram; Agyekum, Felicia; Ahmed, Falak Aftab

    2018-04-01

    To explore the lived experience of the patients post-haematopoietic stem cell transplantation and specifically after engraftment and before discharge. Patients post-stem cell transplantation experience significant changes in all life aspects. Previous studies carried out by other researchers focused mainly on the postdischarge experience, where patients reported their perceptions that have always been affected by the life post-transplantation and influenced by their surroundings. The lived experience of patients, specifically after engraftment and prior to discharge (the "transition" phase), has not been adequately explored in the literature. Doing so might provide greater insight into the cause of change post-haematopoietic stem cell transplantation. This study is a phenomenological description of the participants' perception about their lived experience post-haematopoietic stem cell transplantation. The study used Giorgi's method of analysis. Through purposive sampling, 15 post-haematopoietic stem cell transplantation patients were recruited. Data were collected by individual interviews. Data were then analysed based on Giorgi's method of analysis to reveal the meaning of a phenomenon as experienced through the identification of essential themes. The analysis process revealed 12 core themes covered by four categories that detailed patients lived experience post-haematopoietic stem cell transplantation. The four categories were general transplant experience, effects of transplantation, factors of stress alleviation and finally life post-transplantation. This study showed how the haematopoietic stem cell transplantation affected the patients' physical, psychological and spiritual well-being. Transplantation also impacted on the patients' way of thinking and perception of life. Attending to patients' needs during transplantation might help to alleviate the severity of the effects and therefore improve experience. Comprehensive information about transplantation needs

  6. Following the Dynamics of pH in Endosomes of Live Cells with SERS Nanosensors

    DEFF Research Database (Denmark)

    Kneipp, J.; Kneipp, Harald; Wittig, B.

    2010-01-01

    The surface enhanced Raman scattering (SERS) spectrum of a reporter molecule attached to gold or silver nanostructures, which is pH-sensitive, can deliver information on the local pH in the environment of the nanostructure. Here, we demonstrate the use of a mobile SERS nanosensor made from gold...... nanaoaggregates and 4-mercaptobenzoic acid (pMBA) attached as a reporter for monitoring changes in local pH of the cellular compartments of living NIH/3T3 cells. We show that SERS nanosensors enable the dynamics of local pH in individual live cells to be followed at subendosomal resolution in a timeline...

  7. A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging

    Science.gov (United States)

    Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

    2013-04-01

    Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

  8. Use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging.

    Science.gov (United States)

    Tam, Jenny M; Castro, Carlos E; Heath, Robert J W; Mansour, Michael K; Cardenas, Michael L; Xavier, Ramnik J; Lang, Matthew J; Vyas, Jatin M

    2011-07-28

    Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system(1, 2); however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis(3) have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture. Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions(4). Radiation pressure was first observed and applied to optical tweezer systems in 1970(5, 6), and was first used to control biological specimens in 1987(7). Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena(8-13). We describe a method(14) that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals(15, 16) (e.g. AIDS

  9. Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

    Science.gov (United States)

    Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

    2010-08-01

    In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent

  10. A new image correction method for live cell atomic force microscopy

    International Nuclear Information System (INIS)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X

    2007-01-01

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane

  11. Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

    Science.gov (United States)

    Zaric, Marija; Becker, Pablo Daniel; Hervouet, Catherine; Kalcheva, Petya; Ibarzo Yus, Barbara; Cocita, Clement; O'Neill, Lauren Alexandra; Kwon, Sung-Yun; Klavinskis, Linda Sylvia

    2017-12-28

    The generation of tissue resident memory (T RM ) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8 + T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8 + T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8 + T cell expression of CXCR3 + , CD103 +, CD49a + , CD69 + , CD127 + homing, retention and survival markers. Furthermore, memory CD8 + T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8 + T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Integrated dual-tomography for refractive index analysis of free-floating single living cell with isotropic superresolution.

    Science.gov (United States)

    B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern

    2018-04-13

    Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.

  13. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    Science.gov (United States)

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-07

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  14. Gold nanoparticles delivery in mammalian live cells: a critical review

    Directory of Open Access Journals (Sweden)

    Raphaël Lévy

    2010-02-01

    the University of Liverpool as a Post-doctoral Marie Curie Research Fellow. In 2006, he obtained a prestigious David Phillips Fellowship, to develop single particle-based imaging in living cells (photothermal microscopy. His research interests include the design and characterization of nanomaterials and their interactions with living cells. Umbreen Shaheen completed her Master in Zoology and then lectured at the University of Balochistan. She studied biotechnology at the National Institute of Biotechnology and Genetic Engineering (NIBGE, Pakistan and is currently doing her PhD at the University of Liverpool, on intracellular delivery of peptide-capped gold nanoparticles. Yann Cesbron is a PhD student at the University of Liverpool, developing photothermal microscopy for biological imaging. He graduated at the University Louis Pasteur (Strasbourg, France with a Master of Science in Condensed Matter Physics and a second Master of Science in Polymer Materials. He moved to Liverpool in 2006 to start his PhD. Violaine Sée is a BBSRC David Phillips Research Fellow at the University of Liverpool. She graduated in Chemistry and Molecular and Cellular Biology at the University Louis Pasteur in Strasbourg (France. After a Master in Pharmacology, in 2001 she obtained her PhD in Pharmacology and Neurobiology at the University Louis Pasteur. She was then assistant lecturer and subsequently moved to the University of Liverpool as a Post-doctoral Research Fellow. In 2005, she obtained a prestigious David Phillips Fellowship, to develop her work on intracellular signaling dynamics. She is focusing on the imaging of single living cells in order to understand regulation of gene transcription and cell fate. She has recently been interested in using new techniques for single molecule imaging in live cells based on the use of gold nanoparticles.

  15. Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

    Science.gov (United States)

    2012-07-27

    56 stock solution and 75 ml of the 16 dilution buffer was mixed with 25 ml of the supernatant, incubated for 30 min at 65 oC to inactivate endogenous ...shock by staphylococcal pyrogenic exotoxin type C. Infect Immun 36: 123–128. Meier survival analysis and log-rank tests were performed to compare

  16. LONG-LIVED BONE MARROW PLASMA CELLS DURING IMMUNE RESPONSE TO ALPHA (1→3 DEXTRAN

    Directory of Open Access Journals (Sweden)

    I. N. Chernyshova

    2015-01-01

    Full Text Available Production kinetics and some functional properties of long-lived marrow plasma cells were studied in mice immunized with T-independent type 2 antigens. Alpha (1→3 dextran was used as an antigen for immunization. The mice were immunized by dextran, and the numbers of IgM antibody producing cells were determined by ELISPOT method. The cell phenotype was determined by cytofluorimetric technique. In the area of normal bone marrow lymphocytes ~4% of T and ~85% of B cells were detected. About 35% of the cells expressed a plasmocyte marker (CD138; 3% were CD138+IgM+, and about 6% of the lymphocytes were double-positive for CD138+IgA+. Among spleen lymphocytes, 50% of T and 47% of B cells were detected. About 1.5% lymphocytes were CD138+, and 0.5% were positive for CD138 and IgM. Time kinetics of antibody-producing cells in bone marrow and spleen was different. In spleen populations, the peak amounts of antibody-secreting cells have been shown on the day 4; the process abated by the day 28. Vice versa, the numbers of the antibody-producing cells in bone marrow started to increase on the day 4. The process reached its maximum on day 14, and after 28th day became stationary. The in vitro experiments have shown that supplementation of bone marrow cells from immune mice with dextran did not influence their functional activity. It was previously shown for cells responding to T-dependent antigens only. A specific marker for the long-lived plasma cells is still unknown. However, these cells possess a common CD138 marker specific for all plasma cells. A method for isolation of bone marrow CD138+ cells was developed. The CD138+ cells were of 87-97% purity, being enriched in long-lived bone marrow cells, and produced monospecific antibodies.

  17. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  18. Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

    Science.gov (United States)

    Lee, Il-Hyung; Saha, Suvrajit; Polley, Anirban; Huang, Hector; Mayor, Satyajit; Rao, Madan; Groves, Jay T

    2015-03-26

    Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

  19. Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

    Directory of Open Access Journals (Sweden)

    Smija M Kurian

    Full Text Available Fusarium oxysporum exhibits conidial anastomosis tube (CAT fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

  20. Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

    DEFF Research Database (Denmark)

    Mas, Josep; Richardson, Andrew Callum; Reihani, S. Nader S.

    2013-01-01

    is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes......With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under...... correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm...

  1. Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

    International Nuclear Information System (INIS)

    Wang, Y.-H.; Chen, C.-P.; Chan, M.-H.; Chang, M.; Hou, Y.-W.; Chen, H.-H.; Hsu, H.-R.; Liu, Kevin; Lee, H.-J.

    2006-01-01

    Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or β-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo

  2. Effects of high-gradient magnetic fields on living cell machinery

    International Nuclear Information System (INIS)

    Zablotskii, V; Lunov, O; Kubinova, S; Polyakova, T; Dejneka, A; Sykova, E

    2016-01-01

    A general interest in biomagnetic effects is related to fundamental studies of the influence of magnetic fields on living objects on the cellular and whole organism levels. Emerging technologies offer new directions for the use of high-gradient magnetic fields to control cell machinery and to understand the intracellular biological processes of the emerging field of nanomedicine. In this review we aim at highlighting recent advances made in identifying fundamental mechanisms by which magnetic gradient forces act on cell fate specification and cell differentiation. The review also provides an analysis of the currently available magnetic systems capable of generating magnetic fields with spatial gradients of up to 10 MT m −1 , with the focus on their suitability for use in cell therapy. Relationships between experimental factors and underlying biophysical mechanisms and assumptions that would ultimately lead to a deeper understanding of cell machinery and the development of more predictive models for the evaluation of the effects of magnetic fields on cells, tissue and organisms are comprehensively discussed. (topical review)

  3. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

  4. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    DEFF Research Database (Denmark)

    Hao, Xian; Wu, Jiazhen; Shan, Yuping

    2012-01-01

    the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (M beta CD...

  5. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  6. Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

    Science.gov (United States)

    Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

    2015-03-01

    This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

  7. Direct imaging of APP proteolysis in living cells

    Directory of Open Access Journals (Sweden)

    Niccoló Parenti

    2017-04-01

    Full Text Available Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP. Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the

  8. Direct imaging of APP proteolysis in living cells.

    Science.gov (United States)

    Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

    2017-01-01

    Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing

  9. Mechanism of stimulation of antibody-forming ability of bone marrow cells of mice immunized with staphylococci

    International Nuclear Information System (INIS)

    Lyashchenko, K.P.; Golovanova, T.A.; Bobrovnik, S.A.

    1987-01-01

    The purpose of this paper is to study the formation of the ability of the bone marrow cells of mice immunized with staphylococci to create antibodies to this antigen. The research includes a study of the effect of the irradiation in vitro of the bone marrow cells on their stimulating activity and the role played by the thymus and spleen in the formation of this activity. Experiments were carried out on CBA and BALB/c mice as well as on mice with congenital absence of the thymus. The bone marrow cell donors were immunized intravenously with staphylococcal corpuscular antigen. Receptor mice were irradiated with cobalt 60 gamma radiation and injected intravenously with bone marrow cell extract from the immunized donors and were immunized with the antigen. Spleen cells were labelled with chromium 51 and injected intravenously into intact syngeneic recipients together with as well as without the antigen. Three days later the level of radioactivity in the spleen and femora of the animals was determined by scintillation counting. Total radioactivity of the bone marrow was calculated. Irradiation of the bone marrow cells of immunized animals was shown to abolish their stimulating effect on the humoral immune response of intact syngeneic recipients to the staphylococcal corpuscular antigen. Consequently, the immunostimulating effect of bone marrow cells is realized through the proliferating and radiosensitive lymphoid cells rather than through the macrophages

  10. A new image correction method for live cell atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X [College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China)

    2007-04-21

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane.

  11. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

    Directory of Open Access Journals (Sweden)

    Bjoern Traenkle

    2017-08-01

    Full Text Available Single-domain antibodies (sdAbs have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

  12. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

    Science.gov (United States)

    Traenkle, Bjoern; Rothbauer, Ulrich

    2017-01-01

    Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

  13. Pharmacokinetic/pharmacodynamic evaluation of linezolid for the treatment of staphylococcal infections in critically ill patients.

    Science.gov (United States)

    Dong, Haiyan; Xie, Jiao; Wang, Taotao; Chen, Lihong; Zeng, Xiaoyan; Sun, Jinyao; Wang, Xue; Dong, Yalin

    2016-09-01

    Several studies have demonstrated that the ideal therapeutic effect of linezolid cannot be achieved in critically ill patients with the recommended standard dosing regimen of 600 mg every 12 h (q12h). Moreover, the optimal strategy for successful treatment is still lacking. This study analysed factors influencing the efficacy of linezolid treatment and determined the target for successful treatment by logistic regression in 27 critically ill patients with staphylococcal infection who received linezolid 600 mg q12h. The results showed that only the 24-h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) ratio was significantly associated with staphylococcal eradication. Reaching 80% bacterial eradication required an AUC24/MIC of 120.5, defining the therapeutic target. Different dosing regimens were evaluated using Monte Carlo simulation to determine the optimal dosage strategy for linezolid. Although the probability of target attainment (PTA) was high (>99.9%) for the standard dosing regimen at MIC ≤ 1 mg/L, the PTA was almost 0 at MIC = 2 mg/L, thus the dosing regimen required adjustment. In addition, if the dosing regimen was adjusted to 600 mg every 8 h or 600 mg every 6 h, the major staphylococci (except for MRSA and MSSA) exhibited a cumulative fraction of response of >80%, showing a higher treatment success. These findings indicate that a strategy of high linezolid dosage may be needed to increase the probability of successful treatment at MIC > 1 mg/L. The role of therapeutic drug monitoring should be encouraged for optimising linezolid exposure in critically ill patients. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  14. Knowledge and Perceptions Regarding Community-Acquired Staphylococcal Infections Among Health Care Workers in Hawai‘i

    Science.gov (United States)

    Katz, Alan; Hurwitz, Eric; Tice, Alan

    2013-01-01

    Introduction Since the early 1990s, national rates of methicillin-resistant Staphylococcus aureus (MRSA) infections have increased dramatically. Initially identified in health care settings, community-acquired MRSA is now a major public health concern. With Hawai‘i's strikingly high incidence and prevalence of MRSA infections, a high level of knowledge and awareness among health care workers is essential to successfully controlling this evolving epidemic. Methods Health care and related workers were surveyed to assess their knowledge and perceptions about staphylococcal and MRSA infections. Knowledge was estimated by demonstrated ability to correctly identify risk factors including diabetes, obesity, pets, and seawater exposure as well as understanding the seriousness of antibiotic resistance. Perceptions were estimated by demonstrated awareness of the severity and elevated incidence and prevalence of S. aureus and MRSA infections. Results This study identified that occupation (advance clinical practitioner, nurse, public health professional, athletic trainers, and non-medical workers) as well as work location (community vs hospital) influence knowledge and perceptions regarding the epidemiology, severity, and risk factors of S. aureus and MRSA infections. Additionally, despite a well-documented global crisis with antibiotic resistance, Hawai‘i's community health care workers were less inclined to correctly identify the threat of antibiotic resistance as compared to their hospital-based colleagues. Conclusion Trends were observed in knowledge and perceptions with level of medical education. Differences were also noted according to work location. Overall, health care and related workers in the community were less likely to understand basic principles associated with S. aureus infections as well as misperceive this imminent threat. These findings provide compelling evidence for focused educational interventions targeting community health care and related workers

  15. Anti-staphylococcal properties of Eichhornia crassipes, Pistacia vera, and Ziziphus amole leaf extracts: Isolates from cattle and rabbits.

    Science.gov (United States)

    Gutiérrez-Morales, Andrea; Velázquez-Ordoñez, Valente; Khusro, Ameer; Salem, Abdelfattah Z M; Estrada-Zúñiga, María Elena; Salem, Mohamed Z M; Valladares-Carranza, Benjamin; Burrola-Aguilar, Cristina

    2017-12-01

    The desideratum aim of the present context was to assess the biopotency of methanolic extracts of Eichhornia crassipes (E. crassipes), Pistacia vera (P. vera), and Ziziphus amole (Z. amole) leaves against various staphylococcal strains, and to quantify the phenolics as well as saponin content in them. The antibacterial activity of various concentrations (62.5-1000 μg/mL) of plant extracts was tested against control clinical strains (Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, and S. aureus ATCC 43300), methicillin-resistant S. aureus (MRSA1 and MRSA2), oxacillin sensitive S. aureus (SOSA1 and SOSA2), and coagulase-negative Staphylococcus epidermidis (CoNS1, CoNS2, and CoNS3) using disc diffusion assay. Leaf extracts of the three plants exhibited pronounced growth inhibitory characteristics against staphylococci in a dose dependent manner. E. crassipes extract depicted the highest relative percentage inhibition values against control clinical strains (68.6 ± 0.5%), while P. vera (68.6 ± 0.3%) and Z. amole (74.79 ± 0.3%) extracts showed pronounced relative inhibition values against staphylococcal strains isolated from cattle. Total phenols and saponin content of leaf extracts were investigated by standard in vitro methods. The methanolic extracts of these plants were found to comprise substantial content of phenolics and saponin at varying levels. The highest value of phenolics was estimated in P. vera extract (60.0 ± 1.3 mg gallic acid/g extract), followed by Z. amole (33.6 ± 1.4 mg gallic acid/g extract), and E. crassipes (23.0 ± 1.3 mg gallic acid/g extract). Saponin content for P. vera, Z. amole, and E. crassipes extracts were estimated as 41.0 ± 1.3, 35.8 ± 1.3, and 25.0 ± 1.2 mg diosgenin/g extract, respectively. The outcome of this study suggested the exploitation of methanolic extract of P. vera, Z. amole, and E. crassipes leaves for their possible application in ethnomedicine, particularly as drugs preparation

  16. Non-Rigid Contour-Based Registration of Cell Nuclei in 2-D Live Cell Microscopy Images Using a Dynamic Elasticity Model.

    Science.gov (United States)

    Sorokin, Dmitry V; Peterlik, Igor; Tektonidis, Marco; Rohr, Karl; Matula, Pavel

    2018-01-01

    The analysis of the pure motion of subnuclear structures without influence of the cell nucleus motion and deformation is essential in live cell imaging. In this paper, we propose a 2-D contour-based image registration approach for compensation of nucleus motion and deformation in fluorescence microscopy time-lapse sequences. The proposed approach extends our previous approach, which uses a static elasticity model to register cell images. Compared with that scheme, the new approach employs a dynamic elasticity model for the forward simulation of nucleus motion and deformation based on the motion of its contours. The contour matching process is embedded as a constraint into the system of equations describing the elastic behavior of the nucleus. This results in better performance in terms of the registration accuracy. Our approach was successfully applied to real live cell microscopy image sequences of different types of cells including image data that was specifically designed and acquired for evaluation of cell image registration methods. An experimental comparison with the existing contour-based registration methods and an intensity-based registration method has been performed. We also studied the dependence of the results on the choice of method parameters.

  17. Contribution of T cell-mediated immunity to the resistance to staphlococcal infection

    International Nuclear Information System (INIS)

    Tsuda, S.; Sasai, Y.; Minami, K.; Nomoto, K.

    1978-01-01

    Abscess formation in nude mice after subcutaneous inoculation of Staphylococcus aureus (S. aureus) was more extensive and prolonged as compared with that in phenotypically normal littermates. Abscess formation in nude mice was augmented markedly by whole-body irradiation. Not only T cell-mediated immunity but also radiosensitive, nonimmune phagocytosis appear to contribute to the resistance against staphylococcal infection

  18. Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach

    Science.gov (United States)

    Martinez-Torres, Cristina; Laperrousaz, Bastien; Berguiga, Lotfi; Boyer-Provera, Elise; Elezgaray, Juan; Nicolini, Franck E.; Maguer-Satta, Veronique; Arneodo, Alain; Argoul, Françoise

    2015-09-01

    The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

  19. Comparative Effects of Food Preservatives on the Production of Staphylococcal Enterotoxin I from Staphylococcus aureus Isolate

    Directory of Open Access Journals (Sweden)

    Yanying Zhao

    2017-01-01

    Full Text Available Staphylococcal enterotoxin I (SEI is associated with staphylococcal food poisoning, but little is known about different food preservatives on the production of SEI. In this study, the effect of different food preservatives (sodium nitrite, polylysine, chitosan, and tea catechin on the bacteria growth, sei gene expression, and extracellular SEI production of Staphylococcus aureus isolate H4 was detected in tryptone soya broth (TSB culture. Our results showed that all of these preservatives depressed S. aureus H4 growth and the order of inhibitory effect was 0.8 g/L tea catechin > 6 g/L chitosan > 0.25 g/L polylysine > 0.4 g/L tea catechin > 0.15 g/L sodium nitrite. Furthermore, 0.25 g/L polylysine or 0.15 g/L sodium nitrite did not significantly alter sei gene transcription, while 6 g/L chitosan obviously increased the relative mRNA level of sei gene expression. 0.4 g/L tea catechin remarkably inhibited sei gene transcription. In addition, 0.15 g/L sodium nitrite and 6 g/L chitosan significantly enhanced SEI secretion. 0.25 g/L polylysine, especially 0.4 g/L tea catechin, sharply inhibited the level of SEI secretion. The results indicated that tea catechin not only suppressed Staphylococcus aureus growth, but also inhibited SEI production and secretion, suggesting that tea catechin may be better than sodium nitrite, polylysine, or chitosan for keeping the food from the contamination of SEI. These investigations would be useful for food industry to provide safer food products due to S. aureus enterotoxins-related control strategy.

  20. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  1. A Molecular Probe for the Detection of Polar Lipids in Live Cells.

    Science.gov (United States)

    Bader, Christie A; Shandala, Tetyana; Carter, Elizabeth A; Ivask, Angela; Guinan, Taryn; Hickey, Shane M; Werrett, Melissa V; Wright, Phillip J; Simpson, Peter V; Stagni, Stefano; Voelcker, Nicolas H; Lay, Peter A; Massi, Massimiliano; Plush, Sally E; Brooks, Douglas A

    2016-01-01

    Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular

  2. Antigen modulation of the immune response. III. Evaluation of the hypothetical short-lived memory cell

    International Nuclear Information System (INIS)

    Feldbush, T.L.; Lande, I.; Bryan, B.; O'Neill, E.

    1974-01-01

    The putative short-lived memory cells, whose existence has been suggested by the results of secondary adoptive transfer experiments, were investigated. On the basis of the following evidences we have concluded that the short-lived memory cell is probably an artifact of the adoptive transfer technique: when immune thoracic duct lymphocytes, known to consist predominantly of long-lived memory cells, were transferred to irradiated recipients and challenged at various times after transfer, approximately 80 to 90 percent of the initial response was absent by Day 14 challenge; preirradiating adoptive recipients with increasing dose of x-irradiation tended to lengthen the observed half life of memory cells; single or multiple treatments of immune donors with 0.3 mg Vinblastin before transfer resulted in neither a depression of the initial secondary response nor an alteration in the rate of decline of the memory potential; reconstitution of irradiated hosts with normal spleen cells one day before transfer of memory cells and challenge resulted in inhibition of the adoptive secondary response; and the transfer of memory cells to antigen free intermediate hosts, in which they were allowed to reside for one day or fourteen days before transfer to irradiated recipients, resulted in only a slight decline in their capacity to respond. We propose that the rapid decline of memory potential in adoptive recipients challenged at various times after transfer is due to modulating effects by the hosts as it recovers from irradiation. These effects may be the result of cell crowding or the loss of irradiation-produced stimulatory factors. The relevance of these findings to adoptive transfer systems in general and the secondary response of intact animals is discussed

  3. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  4. An improved procedure for subcellular spatial alignment during live-cell CLEM.

    Directory of Open Access Journals (Sweden)

    Benjamin S Padman

    Full Text Available Live-cell correlative light and electron microscopy (CLEM offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

  5. The electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a liquid medium and on a solid agar

    NARCIS (Netherlands)

    Kiers, PJM; van der Mei, HC; Busscher, HJ; Bos, R.R.M.

    Many Staphylococcus epidermidis strains possess capsule or slime layers and consequently the staphylococcal cell surface should be regarded as a soft, polyelectrolyte layer allowing electrophoretic fluid flow through a layer of fixed charges. The presence of such a soft layer decreases the energy

  6. Comprehensive studies on the interactions between chitosan nanoparticles and some live cells

    International Nuclear Information System (INIS)

    Zheng Aiping; Liu Huixue; Yuan Lan; Meng Meng; Wang Jiancheng; Zhang Xuan; Zhang Qiang

    2011-01-01

    As more and more oral formulations of nanoparticles are used in clinical contexts, a comprehensive study on the mechanisms of interaction between polymer nanoparticles and live cells seems merited. Such a study was conducted and the results were compared to the polymer itself in order to demonstrate different kinds of effects that are brought into the cell by polymer and its nanoparticles, especially the effects on the biomembrane. Several techniques, including surface plasmon resonance (SPR), Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, fluorescence polarization spectroscopy (FP), flow cytometry (FCM) with quantitative analysis, and confocal images with antibody staining were employed toward this end. The cytotoxicity in vitro was also evaluated. Chitosan (CS), a polycationic polymer, was used to prepare the nanoparticles. We demonstrate that chitosan nanoparticles (CS-NP) induce strong alterations in the distribution of membrane proteins, fluidity of membrane lipids, and general membrane structure. Furthermore, the uptake of CS-NP into Caco-2 cells was found to have a similar mechanism to that of CS molecules, but the differences in degree were noted. These results indicate that positive charge and nanoscale size were the factors that most significantly affected the interactions between the nanoparticles of polycationic polymers and live cells. However, no difference in cytotoxicity toward the Caco-2 cells was found between CS and CS-NP. This supports the idea that CS-NP is an effective and safe carrier for oral drug delivery.

  7. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    Science.gov (United States)

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-02-01

    Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

  8. A novel MSCRAMM sub-family in Coagulase negative staphylococcal species

    Directory of Open Access Journals (Sweden)

    Srishtee eArora

    2016-04-01

    Full Text Available Coagulase negative staphylococci are important opportunistic pathogens. Staphylococcus epidermidis, a coagulase negative staphylococcus, is the third leading cause of nosocomial infections in the US. Surface proteins like Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs are major virulence factors of pathogenic gram positive bacteria. Here, we identified a new chimeric protein; SesJ in S. epidermidis, which represents a prototype of a new subfamily of MSCRAMMs. Structural predictions show that SesJ has structural features characteristic of a MSCRAMM along with a N-Terminal repeat region and an Aspartic acid containing repeat region, features that have not been previously observed in staphylococcal MSCRAMMs but have been found in other surface proteins from gram positive bacteria. We identified and analyzed structural homologs of SesJ in three other coagulase negative staphylococci. These homologs of SesJ have an identical structural organization but varying sequence identities within the domains. Using flow cytometry, we also show that SesJ is expressed constitutively on the surface of a representative S. epidermidis strain, from early exponential to stationary growth phase. Thus SesJ is positioned to interact with protein targets in the environment and play a role in S. epidermidis virulence.

  9. Interaction of multi-functional silver nanoparticles with living cells

    International Nuclear Information System (INIS)

    Sur, Ilknur; Cam, Dilek; Kahraman, Mehmet; Culha, Mustafa; Baysal, Asli

    2010-01-01

    Silver nanoparticles (AgNPs) are widely used in household products and in medicine due to their antibacterial and to wound healing properties. In recent years, there is also an effort for their use in biomedical imaging and photothermal therapy. The primary reason behind the effort for their utility in biomedicine and therapy is their unique plasmonic properties and easy surface chemistry for a variety of functionalizations. In this study, AgNPs modified with glucose, lactose, oligonucleotides and combinations of these ligands are investigated for their cytotoxicity and cellular uptake in living non-cancer (L929) and cancer (A549) cells. It is found that the chemical nature of the ligand strongly influences the toxicity and cellular uptake into the model cells. While the lactose-and glucose-modified AgNPs enter the L929 cells at about the same rate, a significant increase in the rate of lactose-modified AgNPs into the A549 cells is observed. The binding of oligonucleotides along with the carbohydrate on the AgNP surfaces influences the differential uptake rate pattern into the cells. The cytotoxicity study with the modified AgNPs reveals that only naked AgNPs influence the viability of the A549 cells. The findings of this study may provide the key to developing effective applications in medicine such as cancer therapy.

  10. Turn-on fluorogenic and chromogenic detection of Fe(III) and its application in living cell imaging

    International Nuclear Information System (INIS)

    Sivaraman, Gandhi; Sathiyaraja, Vijayaraj; Chellappa, Duraisamy

    2014-01-01

    Two rhodamine-based sensors RDI-1, RDI-2 was designed and synthesized by incorporation of the rhodamine 6G fluorophore and 2-formyl imidazole as the recognizing unit via the imine linkages. RDI-1, RDI-2 exhibits very high selectivity and an excellent sensitivity towards Fe(III) ions in aqueous buffer solution on compared with other probes. The color change from colorless to pink and turn-on fluorescence after binding with iron (III) was observed. Based on jobs plot and ESI-MS studies, the 1:1 binding mode was proposed. Live cell imaging experiments with each probe showed that these probes widely applicable to detect Fe 3+ in living cells. -- Highlights: • Two rhodamine based probes was synthesized and used to recognize iron (III). • The chemosensors can be applied to detect iron(III) ions by color and turn-on fluorescent changes. • The very low detection limit was reported. • The applicability of these probes for live cell fluorescence imaging was studied

  11. Turn-on fluorogenic and chromogenic detection of Fe(III) and its application in living cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sivaraman, Gandhi; Sathiyaraja, Vijayaraj; Chellappa, Duraisamy, E-mail: dcmku123@gmail.com

    2014-01-15

    Two rhodamine-based sensors RDI-1, RDI-2 was designed and synthesized by incorporation of the rhodamine 6G fluorophore and 2-formyl imidazole as the recognizing unit via the imine linkages. RDI-1, RDI-2 exhibits very high selectivity and an excellent sensitivity towards Fe(III) ions in aqueous buffer solution on compared with other probes. The color change from colorless to pink and turn-on fluorescence after binding with iron (III) was observed. Based on jobs plot and ESI-MS studies, the 1:1 binding mode was proposed. Live cell imaging experiments with each probe showed that these probes widely applicable to detect Fe{sup 3+} in living cells. -- Highlights: • Two rhodamine based probes was synthesized and used to recognize iron (III). • The chemosensors can be applied to detect iron(III) ions by color and turn-on fluorescent changes. • The very low detection limit was reported. • The applicability of these probes for live cell fluorescence imaging was studied.

  12. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

    Science.gov (United States)

    Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

    2017-01-01

    Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

  13. Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria M; Della Ventura, Bartolomeo; Vinciguerra, Roberto; Chino, Marco; Gesuele, Felice; Lombardi, Angelina; Velotta, Raffaele; Altucci, Carlo; Birolo, Leila; Piccoli, Renata; Arciello, Angela

    2016-02-01

    A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

  14. g-force induced giant efficiency of nanoparticles internalization into living cells

    Science.gov (United States)

    Ocampo, Sandra M.; Rodriguez, Vanessa; de La Cueva, Leonor; Salas, Gorka; Carrascosa, Jose. L.; Josefa Rodríguez, María; García-Romero, Noemí; Luis, Jose; Cuñado, F.; Camarero, Julio; Miranda, Rodolfo; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2015-10-01

    Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

  15. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

    Directory of Open Access Journals (Sweden)

    Catarina C F Homem

    Full Text Available The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  16. Calibration and quantification of fast intracellular motion (FIM) in living cells using correlation analysis

    Czech Academy of Sciences Publication Activity Database

    Veselý, Pavel; Mikš, A.; Novák, J.; Boyde, A.

    2003-01-01

    Roč. 25, - (2003), s. 230-239 ISSN 0161-0457 R&D Projects: GA ČR GA304/99/0368 Institutional research plan: CEZ:AV0Z5052915 Keywords : fast intracellular motion * living cell ů video rate confocal laser scanning microscopy Subject RIV: EA - Cell Biology Impact factor: 0.733, year: 2003

  17. Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of Staphylococcal Enterotoxin B

    Czech Academy of Sciences Publication Activity Database

    Schlosser, G.; Kačer, P.; Kuzma, Marek; Szilágyi, Z.; Sorrentino, Z.; Manzo, C.; Pizzano, R.; Malorni, L.; Poscfalvi, G.

    2007-01-01

    Roč. 73, č. 21 (2007), s. 6945-6952 ISSN 0099-2240 Grant - others:IT(IT) project SIFORTI (MIUR-CNR, Legge 449/97); XE(XE) 94.05.09.103, ARINCO 94.IT.16.028 Institutional research plan: CEZ:AV0Z50200510 Keywords : mass spectrometry * staphylococcal enterotoxin b * bacterial protein toxins Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2007

  18. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

  19. Localization of mitochondria in living cells with rhodamine 123.

    Science.gov (United States)

    Johnson, L V; Walsh, M L; Chen, L B

    1980-01-01

    The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

  20. Continuous depth-sensing nano-mechanical characterization of living, fixed and dehydrated cells attached on a glass substrate

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yun-Ta; Liao, Jiunn-Der; Chang, Chia-Wei [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Lin, Chou-Ching K [Department of Neurology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Ju, Ming-Shaung, E-mail: jdliao@mail.ncku.edu.tw [Department of Mechanical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China)

    2010-07-16

    Continuous depth-sensing nano-indentation on living, fixed and dehydrated fibroblast cells was performed using a dynamic contact module and vertically measured from a pre-contact state to the glass substrate. The nano-indentation tip-on-cell approaches took advantage of finding a contact surface, followed by obtaining a continuous nano-mechanical profile along the nano-indentation depths. In the experiment, serial indentations from the leading edge, i.e., the lamellipodium to nucleus regions of living, fixed and dehydrated fibroblast cells were examined. Nano-indentations on a living cell anchored upon glass substrate were competent in finding the tip-on-cell contact surfaces and cell heights. For the result on the fixed and the dehydrated cells, cellular nano-mechanical properties were clearly characterized by continuous harmonic contact stiffness (HCS) measurements. The relations of HCS versus measured displacement, varied from the initial tip-on-cell contact to the glass substrate, were presumably divided into three stages, respectively induced by cellular intrinsic behavior, the substrate-dominant property, and the substrate property. This manifestation is beneficial to elucidate how the underlying substrate influences the interpretation of the nano-mechanical property of thin soft matter on a hard substrate. These findings, based upon continuous depth-sensing nano-indentations, are presumably valuable as a reference to related work, e.g., accomplished by atomic force microscopy.

  1. Continuous depth-sensing nano-mechanical characterization of living, fixed and dehydrated cells attached on a glass substrate

    International Nuclear Information System (INIS)

    Yang, Yun-Ta; Liao, Jiunn-Der; Chang, Chia-Wei; Lin, Chou-Ching K; Ju, Ming-Shaung

    2010-01-01

    Continuous depth-sensing nano-indentation on living, fixed and dehydrated fibroblast cells was performed using a dynamic contact module and vertically measured from a pre-contact state to the glass substrate. The nano-indentation tip-on-cell approaches took advantage of finding a contact surface, followed by obtaining a continuous nano-mechanical profile along the nano-indentation depths. In the experiment, serial indentations from the leading edge, i.e., the lamellipodium to nucleus regions of living, fixed and dehydrated fibroblast cells were examined. Nano-indentations on a living cell anchored upon glass substrate were competent in finding the tip-on-cell contact surfaces and cell heights. For the result on the fixed and the dehydrated cells, cellular nano-mechanical properties were clearly characterized by continuous harmonic contact stiffness (HCS) measurements. The relations of HCS versus measured displacement, varied from the initial tip-on-cell contact to the glass substrate, were presumably divided into three stages, respectively induced by cellular intrinsic behavior, the substrate-dominant property, and the substrate property. This manifestation is beneficial to elucidate how the underlying substrate influences the interpretation of the nano-mechanical property of thin soft matter on a hard substrate. These findings, based upon continuous depth-sensing nano-indentations, are presumably valuable as a reference to related work, e.g., accomplished by atomic force microscopy.

  2. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  3. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    International Nuclear Information System (INIS)

    Cheng, Xiaofei; Deng, Ping; Cui, Hongguang; Wang, Aiming

    2015-01-01

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  4. HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

    Science.gov (United States)

    Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

    2016-03-01

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

  5. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  6. Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

    Science.gov (United States)

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-10-17

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Delivery of Optical Contrast Agents using Triton-X100, Part 1: Reversible permeabilization of live cells for intracellular labeling

    OpenAIRE

    van de Ven, Anne L; Adler-Storthz, Karen; Richards-Kortum, Rebecca

    2009-01-01

    Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility...

  8. Colorimetric detection of endogenous hydrogen sulfide production in living cells

    Science.gov (United States)

    Ahn, Yong Jin; Lee, Young Ju; Lee, Jaemyeon; Lee, Doyeon; Park, Hun-Kuk; Lee, Gi-Ja

    2017-04-01

    Hydrogen sulfide (H2S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H2S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H2S concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of H2S has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective H2S detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO3 concentration, as well as the pH of the Na2S (H2S donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na2S standard solution both at room temperature and at 37 °C in a 5% CO2 incubator. As a result, the sensitivity of the colorimetric assay for H2S at 37 °C in the incubator (0.0056 Abs./μM Na2S, R2 = 0.9948) was similar to that at room temperature (0.0055 Abs./μM Na2S, R2 = 0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, L-cysteine (Cys), and dithiothreitol than to the H2S from Na2S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H2S concentrations in live C6 glioma cells by s-(5‧-adenosyl)-L-methionine stimulation with and without Cys and L-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver

  9. Peripheral blood cells among community residents living near nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yuan-Teh; Hsu, Hsiu-Ching; Chien, Kuo-Liong; Yang, Chi-Yu; Chen, Wen Jone [Department of Internal Medicine, National Taiwan University College of Medicine, No. 7 Chungshan South Road, 10020 Taipei (Taiwan, Province of China); Sung, Fung C. [Institute of Environmental Health, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China); Lin, Ruey S. [Institute of Epidemiology, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China)

    2001-12-03

    Information about hematopoieses as a result of exposure to very low levels of radiation is scarce. To investigate the human hematopoietic effect of very low level radiation exposure, measurements of peripheral blood components were performed among 3602 men and women, aged 35 and above, living in a community near two nuclear power installations in Chinshan, Taiwan. The radiation level that each individual was exposed to was represented by a surrogate level, '+', a transformed distance from each individual's residence to the two power plants D{sub 1} and D{sub 2}. In addition to comparing average hematology measurements, multiple regression analyses were done to include age, gender, smoking, drinking status and the surrogate radiation exposure level as independent variables. Univariate and bivariate analyses showed that the hematology measurements had significant associations with age, gender, smoking or drinking. The multiple regression analyses revealed that significant positive associations with '+' were found for hemoglobin, hematocrit, platelet, white blood cell and red blood cell. The platelet count might increase for 208.7x10{sup 3}/{mu}l if the exposure from the nuclear plants increased by one exposure unit. This type of association implies that those who lived closer to the nuclear power installation had a higher blood cell count; we suspect that this could be a type of radiation hormesis.

  10. Live cell imaging at the Munich ion microbeam SNAKE - a status report.

    Science.gov (United States)

    Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A

    2015-02-18

    Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.

  11. Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

    Science.gov (United States)

    Hong, Senlian; Chen, Tao; Zhu, Yuntao; Li, Ang; Huang, Yanyi; Chen, Xing

    2014-06-02

    Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

    Science.gov (United States)

    Sung, Myong-Hee

    2013-01-01

    Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. PMID:24709701

  13. Noninvasive micromanipulation of live HIV-1 infected cells via laser light

    Science.gov (United States)

    Mthunzi, Patience

    2015-12-01

    Live mammalian cells from various tissues of origin can be aseptically and noninvasively micromanipulated via lasers of different regimes. Laser-driven techniques are therefore paving a path toward the advancement of human immuno-deficiency virus (HIV-1) investigations. Studies aimed at the interaction of laser light, nanomaterials, and biological materials can also lead to an understanding of a wealth of disease conditions and result in photonics-based therapies and diagnostic tools. Thus, in our research, both continuous wave and pulsed lasers operated at varying wavelengths are employed, as they possess special properties that allow classical biomedical applications. This paper discusses photo-translocation of antiretroviral drugs into HIV-1 permissive cells and preliminary results of low-level laser therapy (LLLT) in HIV-1 infected cells.

  14. Biosorption of diethyl phthalate ester by living and nonliving Burkholderia cepacia and the role of its cell surface components.

    Science.gov (United States)

    Luo, Si; Li, Langlang; Chen, Anwei; Zeng, Qingru; Xia, Hao; Gu, Ji-Dong

    2017-07-01

    In this study, the dibutyl phthalate (DBP) binding properties of a DBP-tolerant bacterium (B. cepacia) were characterized in terms of adsorption kinetics and isotherm. Living and nonliving cells both exhibited rapid removal of DBP, achieving more than 80% of maximum sorption within 30 min of contact and reached the equilibrium after 3 h. The adsorption isotherms were well fitted with the Sips model and the nonliving cells have greater biosorption capacity and affinity for DBP than the living cells. Furthermore, the absence of an active mechanism dependent on metabolism implied that the DBP bioaccumulation by living cells was mainly attribute to passive surface binding. The optimum pH for DBP adsorption by living and nonliving cells were both observed to be 6.0. The biosorptive mechanism of DBP binding by B. cepacia was further confirmed by FTIR analysis and various chemical treatments. FTIR results indicated that the phosphate and CH 2 groups on B. cepacia were the main bounding sites for DBP. Furthermore, 2.28, 2.15, 1.93 and 0.87 g of pretreated cells were obtained from 2.40 g of native cells via extracellular polymeric substances (EPS), superficial layer-capsule, lipids components and cell membrane removal treatments, respectively. Total binding amount of DBP on the native cells, EPS-removed cells, capsule-removed cells, lipids-extracted cells and membrane-removed cells were 26.69, 24.84, 24.93, 16.11 and 10.80 mg, respectively, suggesting that the cell wall lipids, proteins or peptidoglycan might play important roles in the sorption of DBP by B. cepacia. The information could be applied in understanding on the mobility, transport and ultimate fate of PAEs in soil and related environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Synchrotron infrared spectromicroscopy as a novel bioanalytical microprobe for individual living cells: Cytotoxicity considerations

    Energy Technology Data Exchange (ETDEWEB)

    Holman, Hoi-Ying N.; Bjornstad, Kathleen A.; McNamara, Morgan P.; Martin, Michael C.; McKinney, Wayne R.; Blakely, Eleanor A.

    2001-12-12

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR)spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, ''Does the intense synchrotron beam induce any cytotoxic effects in living cells?'' In this work, we present the results from a series of standard biological assays to evaluate any short-and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye-exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using an 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5-, 10-, and 20-minute SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable chemistry changes between control and exposed cells.

  16. Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

    Science.gov (United States)

    Sipieter, François; Cappe, Benjamin; Gonzalez Pisfil, Mariano; Spriet, Corentin; Bodart, Jean-François; Cailliau-Maggio, Katia; Vandenabeele, Peter; Héliot, Laurent; Riquet, Franck B.

    2015-01-01

    Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues. PMID:26517832

  17. Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

    Science.gov (United States)

    Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K; Omoe, Katsuhiko; Sugai, Motoyuki

    2015-11-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Measurement of in-plane elasticity of live cell layers using a pressure sensor embedded microfluidic device

    Science.gov (United States)

    Lin, Chien-Han; Wang, Chien-Kai; Chen, Yu-An; Peng, Chien-Chung; Liao, Wei-Hao; Tung, Yi-Chung

    2016-11-01

    In various physiological activities, cells experience stresses along their in-plane direction when facing substrate deformation. Capability of continuous monitoring elasticity of live cell layers during a period is highly desired to investigate cell property variation during various transformations under normal or disease states. This paper reports time-lapsed measurement of live cell layer in-plane elasticity using a pressure sensor embedded microfluidic device. The sensor converts pressure-induced deformation of a flexible membrane to electrical signals. When cells are cultured on top of the membrane, flexural rigidity of the composite membrane increases and further changes the output electrical signals. In the experiments, human embryonic lung fibroblast (MRC-5) cells are cultured and analyzed to estimate the in-plane elasticity. In addition, the cells are treated with a growth factor to simulate lung fibrosis to study the effects of cell transformation on the elasticity variation. For comparison, elasticity measurement on the cells by atomic force microscopy (AFM) is also performed. The experimental results confirm highly anisotropic configuration and material properties of cells. Furthermore, the in-plane elasticity can be monitored during the cell transformation after the growth factor stimulation. Consequently, the developed microfluidic device provides a powerful tool to study physical properties of cells for fundamental biophysics and biomedical researches.

  19. Study of metal bioaccumulation by nuclear microprobe analysis of algae fossils and living algae cells

    International Nuclear Information System (INIS)

    Guo, P.; Wang, J.; Li, X.; Zhu, J.; Reinert, T.; Heitmann, J.; Spemann, D.; Vogt, J.; Flagmeyer, R.-H.; Butz, T.

    2000-01-01

    Microscopic ion-beam analysis of palaeo-algae fossils and living green algae cells have been performed to study the metal bioaccumulation processes. The algae fossils, both single cellular and multicellular, are from the late Neoproterozonic (570 million years ago) ocean and perfectly preserved within a phosphorite formation. The biosorption of the rare earth element ions Nd 3+ by the green algae species euglena gracilis was investigated with a comparison between the normal cells and immobilized ones. The new Leipzig Nanoprobe, LIPSION, was used to produce a proton beam with 2 μm size and 0.5 nA beam current for this study. PIXE and RBS techniques were used for analysis and imaging. The observation of small metal rich spores (<10 μm) surrounding both of the fossils and the living cells proved the existence of some specific receptor sites which bind metal carrier ligands at the microbic surface. The bioaccumulation efficiency of neodymium by the algae cells was 10 times higher for immobilized algae cells. It confirms the fact that the algae immobilization is an useful technique to improve its metal bioaccumulation

  20. Observations of radiation-induced chromosome fragment loss in live mammalian cells in culture, and its effect on colony-forming ability

    International Nuclear Information System (INIS)

    Grote, S.J.; Joshi, G.P.; Revell, S.H.; Shaw, C.A.

    1981-01-01

    A preceding paper (Grote, Joshi, Revell and Shaw 1981) describe a method for the direct scrutiny of live cultured mammalian cells with a microscope, and reported that all diploid Syrian hamster cells (BHK 21 C13) of a sample given 1.4 Gy of 220 kV X-rays in G1 reached post-radiation mitosis without discernible abnormality, but then diverged in observed behaviour: descendant cells from some first mitoses continued to proliferate normally while cells from other first mitoses behaved abnormally and produced either slow-growth or stop-growth colonies. This paper completes the study of the same irradiated cell sample, and shows that these post-mitotic differences in clonogenic ability were related to acentric chromosome fragment losses at post-radiation mitosis, which were detected in live daughter-cell pairs as micronuclei. The proportion of live daughter-cell pairs scored as deficient was at least 80 per cent of the proportion of comparable fixed-and-stained mitoses with detected acentric fragments. (author)

  1. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

    Energy Technology Data Exchange (ETDEWEB)

    Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E.; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R.; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J.; Pinho, Mariana G.; Schneider, Tanja; Roemer, Terry (Merck); (Bonn); (FCT/UNL)

    2016-05-04

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

  2. Sub-15 fs multiphoton lithography of three-dimensional structures for live cell applications

    International Nuclear Information System (INIS)

    Licht, Martin; Uchugonova, Aisada; König, Karsten; Straub, Martin

    2012-01-01

    Development, morphology and intratissue location of cells are influenced by the 3D nano- and microenvironment. In this paper we demonstrate multiphoton photopolymerization to generate three-dimensional structures for cell culture applications with micro- and nanotopographic features using SU-8 photoresist and mr-NIL 6000 nanoimprint resist. Moving the focal spot of high-repetition rate near-infrared sub-15 fs pulsed laser light by a galvanometric beam scanner in combination with a piezoelectric vertical stage, nearly arbitrary trajectories of polymerized photoresist were generated. This technique can be used to generate cage structures with submicron interior features for live cell applications. Preliminary experiments with PC-3 and HT-1080 cells indicate the influence of the structures on cell behavior. (paper)

  3. Skin vaccination with live virus vectored microneedle arrays induce long lived CD8(+) T cell memory.

    Science.gov (United States)

    Becker, Pablo D; Hervouet, Catherine; Mason, Gavin M; Kwon, Sung-Yun; Klavinskis, Linda S

    2015-09-08

    A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8(+) T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8(+) T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8(+) T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8(+) T cells expressing an effector memory (CD62L(-)CD127(+)) and central memory (CD62L(+)CD127(+)) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8(+) memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8(+) T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Supramolecular oligothiophene microfibers spontaneously assembled on surfaces or coassembled with proteins inside live cells.

    Science.gov (United States)

    Barbarella, Giovanna; Di Maria, Francesca

    2015-08-18

    During the last few decades, multifunctional nano- and microfibers made of semiconducting π-conjugated oligomers and polymers have generated much interest because of a broad range of applications extending from sensing to bioelectronic devices and (opto)electronics. The simplest technique for the fabrication of these anisotropic supramolecular structures is to let the molecules do the work by spontaneous organization driven by the information encoded in their molecular structure. Oligothiophenes-semiconducting and fluorescent compounds that have been extensively investigated for applications in thin-film field-effect transistors and solar cells and to a lesser extent as dyes for fluorescent labeling of proteins, DNA, and live cells-are particularly suited as building blocks for supramolecular architectures because of the peculiar properties of the thiophene ring. Because of the great polarizability of sulfur outer-shell electrons and the consequent facile geometric deformability and adaptability of the ring to the environment, thiophene can generate multiple nonbonding interactions to promote non-covalent connections between blocks. Furthermore, sulfur can be hypervalent, i.e., it can accommodate more than the eight electrons normally associated with s and p shells. Hypervalent oligothiophene-S,S-dioxides whose oxygen atoms can be involved in hydrogen bonding have been synthesized. These compounds are amphiphilic, and some of them are able to spontaneously cross the membrane of live cells. Hypervalent nonbonding interactions of divalent sulfur, defined as weak coordination to a proximate nitrogen or oxygen, have also been invoked in the solid-state packing of many organic molecules and in the architecture of proteins. In this Account, we describe two different types of thiophene-based building blocks that can induce the spontaneous formation of nanostructured microfibers in very different environments. The first, based on the synthesis of "sulfur

  5. The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

    Science.gov (United States)

    Xu, Jia; Tan, Xiao; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2015-11-01

    Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese

  6. Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

    2017-10-07

    The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Antibiotic resistance, ability to form biofilm and susceptibility to copper alloys of selected staphylococcal strains isolated from touch surfaces in Polish hospital wards

    Directory of Open Access Journals (Sweden)

    Anna Różańska

    2017-08-01

    Full Text Available Abstract Background Despite the employment of sanitary regimes, contact transmission of the aetiological agents of hospital infections is still exceedingly common. The issue of microbe transmission becomes particularly important when facing multidrug-resistant microorganisms such as methicillin-resistant staphylococci. In the case of deficiencies in cleaning and disinfection procedures, hospital equipment made of copper alloys can play an important role, complementing traditional hospital hygiene procedures. The objective of this study was to characterize staphylococcal strains isolated from touch surfaces in Polish hospital wards in terms of their drug resistance, ability to form biofilm and susceptibility to antimicrobial activity of copper alloys. Methods The materials for the study were 95 staphylococcal strains isolated from touch surfaces in 13 different hospital wards from Małopolska province (the south of Poland. Phenotypic and genotypic antibiotic resistance were checked for erythromycin, clindamycin, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and mupirocin. Biofilm formation ability for the tested strains was checked with the use of culture on Congo red agar. Susceptibility to copper, tin bronze, brass and new silver was tested using a modification of the Japanese standard. Results Over 67% of the analysed staphylococcal strains were methicillin-resistant (MR. Four strains were resistant to all of the tested antibiotics, and 14 were resistant to all except mupirocin. Strains classified as MR had significantly increased resistance to the remaining antibiotic groups. About one-third of the analysed strains revealed biofilm-forming ability. Among the majority of species, biofilm-forming and non-biofilm-forming strains were distributed evenly; in the case of S. haemolyticus only, negative strains accounted for 92.8%. Susceptibility to copper alloys was different between strains and rather lower than in the case of the SA

  8. Biothermodynamics of live cells: a tool for biotechnology and biochemical engineering

    Science.gov (United States)

    von Stockar, Urs

    2010-12-01

    The aim of this contribution is to review the application of thermodynamics to live cultures of microbial and other cells and to explore to what extent this may be put to practical use. A major focus is on energy dissipation effects in industrially relevant cultures, both in terms of heat and Gibbs energy dissipation. The experimental techniques for calorimetric measurements in live cultures are reviewed and their use for monitoring and control is discussed. A detailed analysis of the dissipation of Gibbs energy in chemotrophic growth shows that it reflects the entropy production by metabolic processes in the cells and thus also the driving force for growth and metabolism. By splitting metabolism conceptually up into catabolism and biosynthesis, it can be shown that this driving force decreases as the growth yield increases. This relationship is demonstrated by using experimental measurements on a variety of microbial strains. On the basis of these data, several literature correlations were tested as tools for biomass yield prediction. The prediction of other culture performance characteristics, including product yields for biorefinery planning, energy yields for biofuel manufacture, maximum growth rates, maintenance requirements, and threshold concentrations is also briefly reviewed.

  9. Live imaging of spindle pole disorganization in docetaxel-treated multicolor cells

    International Nuclear Information System (INIS)

    Sakaushi, Shinji; Nishida, Kumi; Minamikawa, Harumi; Fukada, Takashi; Oka, Shigenori; Sugimoto, Kenji

    2007-01-01

    Treatment of cells with docetaxel at low concentrations induces aberrant bipolar spindles of which two centrosomes stay at only one pole, and also induces multipolar spindles. To gain insight into the relations between centrosome impairment and structural defects of the spindle, live-cell imaging was performed on a human MDA Auro/imp/H3 cell line in which centrosomes/mitotic spindles, nuclear membrane and chromatin were simultaneously visualized by fluorescent proteins. In the presence of docetaxel at IC 50 concentration, the centrosomes did not segregate, and multiple aster-like structures ectopically arose around the disappearing nuclear membrane. Those ectopic structures formed an acentrosomal pole opposing to the two-centrosomes-containing pole. In late metaphase, one pole often fragmented into multiple spindle poles, leading multipolar division. These results suggest that spindle pole fragility may be induced by centrosome impairment, and collapse of the pole may contribute to induction of aneuploid daughter cells

  10. Biofilm growth program and architecture revealed by single-cell live imaging

    Science.gov (United States)

    Yan, Jing; Sabass, Benedikt; Stone, Howard; Wingreen, Ned; Bassler, Bonnie

    Biofilms are surface-associated bacterial communities. Little is known about biofilm structure at the level of individual cells. We image living, growing Vibrio cholerae biofilms from founder cells to ten thousand cells at single-cell resolution, and discover the forces underpinning the architectural evolution of the biofilm. Mutagenesis, matrix labeling, and simulations demonstrate that surface-adhesion-mediated compression causes V. cholerae biofilms to transition from a two-dimensional branched morphology to a dense, ordered three-dimensional cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture, and this growth pattern is controlled by a single gene. Competition analyses reveal the advantages of the dense growth mode in providing the biofilm with superior mechanical properties. We will further present continuum theory to model the three-dimensional growth of biofilms at the solid-liquid interface as well as solid-air interface.

  11. Real-time dynamics of RNA Polymerase II clustering in live human cells

    Science.gov (United States)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  12. Real-time Molecular Study of Bystander Effects of Low dose Low LET radiation Using Living Cell Imaging and Nanoparticale Optics

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, Mohan [UT Health Science Center at San Antonio; Xu, Nancy R [Old Dominion University; Mohan, Sumathy [UT Health Science Center at San Antonio

    2013-06-03

    In this study two novel approaches are proposed to investigate precisely the low dose low LET radiation damage and its effect on bystander cells in real time. First, a flow shear model system, which would provide us a near in vivo situation where endothelial cells in the presence of extra cellular matrix experiencing continuous flow shear stress, will be used. Endothelial cells on matri-gel (simulated extra cellular matrix) will be subjected to physiological flow shear (that occurs in normal blood vessels). Second, a unique tool (Single nano particle/single live cell/single molecule microscopy and spectroscopy; Figure A) will be used to track the molecular trafficking by single live cell imaging. Single molecule chemical microscopy allows one to single out and study rare events that otherwise might be lost in assembled average measurement, and monitor many target single molecules simultaneously in real-time. Multi color single novel metal nanoparticle probes allow one to prepare multicolor probes (Figure B) to monitor many single components (events) simultaneously and perform multi-complex analysis in real-time. These nano-particles resist to photo bleaching and hence serve as probes for unlimited timeframe of analysis. Single live cell microscopy allows one to image many single cells simultaneously in real-time. With the combination of these unique tools, we will be able to study under near-physiological conditions the cellular and sub-cellular responses (even subtle changes at one molecule level) to low and very low doses of low LET radiation in real time (milli-second or nano-second) at sub-10 nanometer spatial resolution. This would allow us to precisely identify, at least in part, the molecular mediators that are responsible of radiation damage in the irradiated cells and the mediators that are responsible for initiating the signaling in the neighboring cells. Endothelial cells subjected to flow shear (2 dynes/cm2 or 16 dynes/cm2) and exposed to 0.1, 1 and 10

  13. Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan); Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)

    2012-06-01

    Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

  14. Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

    International Nuclear Information System (INIS)

    Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

    2012-01-01

    Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO 2 -hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

  15. A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

    Science.gov (United States)

    Jing, Chaoran

    2013-01-01

    Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR) tagged protein displaces the quencher. Thus, we began by building a non-fluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins, but also less abundant, more dynamic cytoplasmic proteins. These results suggest that fluorogenic TMP-tag can significantly impact highresolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering. PMID:23745575

  16. Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

    Science.gov (United States)

    Polonsky, Michal; Chain, Benjamin; Friedman, Nir

    2016-03-01

    Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

  17. Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage

    DEFF Research Database (Denmark)

    Sørensen, T U; Gram, G J; Nielsen, S D

    1999-01-01

    BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells...... culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument....

  18. Simulations of living cell origins using a cellular automata model.

    Science.gov (United States)

    Ishida, Takeshi

    2014-04-01

    Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

  19. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  20. Direct measurement of catalase activity in living cells and tissue biopsies.

    Science.gov (United States)

    Scaglione, Christine N; Xu, Qijin; Ramanujan, V Krishnan

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  2. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan); Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011 (Japan); Adachi, Makoto; Hashimoto, Shigetaka [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan)

    2015-10-07

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  3. Determination of cell cycle phases in live B16 melanoma cells using IRMS.

    Science.gov (United States)

    Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

    2013-07-21

    The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

  4. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  5. Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency

    Science.gov (United States)

    Squirrell, Jayne M.; Fong, Jimmy J.; Ariza, Carlos A.; Mael, Amber; Meyer, Kassondra; Shevde, Nirupama K.; Roopra, Avtar; Lyons, Gary E.; Kamp, Timothy J.; Eliceiri, Kevin W.; Ogle, Brenda M.

    2012-01-01

    The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications. PMID:22952742

  6. Developing new optical imaging techniques for single particle and molecule tracking in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei [Iowa State Univ., Ames, IA (United States)

    2010-01-01

    Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells. The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian

  7. Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

    Science.gov (United States)

    Youn, Jung-Ho; Ahn, Kuk Ju; Lim, Suk-Kyung

    2011-01-01

    The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital. PMID:21897094

  8. Live celloidosome structures based on the assembly of individual cells by colloid interactions.

    Science.gov (United States)

    Fakhrullin, Rawil F; Brandy, Marie-Laure; Cayre, Olivier J; Velev, Orlin D; Paunov, Vesselin N

    2010-10-14

    A new class of colloid structures, celloidosomes, has been developed which represent hollow microcapsules whose membranes consist of a single monolayer of living cells. Two routes for producing these structures were designed based on templating of: (i) air bubbles and (ii) anisotropic microcrystals of calcium carbonate with living cells, which allowed us to fabricate celloidosomes of spherical, rhombohedral and needle-like morphologies. Air microbubbles were templated by yeast cells coated with poly(allylamine hydrochloride) (PAH), then coated with carboxymethylcellulose and rehydrated resulting in the formation of spherical multicellular structures. Similarly, calcium carbonate microcrystals of anisotropic shapes were coated with several consecutive layers of oppositely charged polyelectrolytes to obtain a positive surface charge which was used to immobilise yeast cells coated with anionic polyelectrolyte of their surfaces. After dissolving of sacrificial cores, hollow multicellular structures were obtained. The viability of the cells in the produced structures was confirmed by using fluorescein diacetate. In order to optimize the separation of celloidosomes from free cells magnetic nanoparticles were immobilised onto the surface of templates prior to the cells deposition, which greatly facilitated the separation using a permanent magnet. Two alternative approaches were developed to form celloidosome structures using magnetically functionalised core-shell microparticles which resulted in the formation of celloidosomes with needle-like and cubic-like geometries which follows the original morphology of the calcium carbonate microcrystals. Our methods for fabrication of celloidosomes may found applications in the development of novel symbiotic bio-structures, artificial multicellular organisms and in tissue engineering. The unusual structure of celloidosomes resembles the primitive forms of multicellular species, like Volvox, and other algae and could be regarded as

  9. Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models.

    Science.gov (United States)

    Kuklin, Nelly A; Pancari, Gregory D; Tobery, Timothy W; Cope, Leslie; Jackson, Jesse; Gill, Charles; Overbye, Karen; Francis, Kevin P; Yu, Jun; Montgomery, Donna; Anderson, Annaliesa S; McClements, William; Jansen, Kathrin U

    2003-09-01

    Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.

  10. Antimicrobial Resistance of Staphylococcal Strains Isolated from Various Pathological Products

    Directory of Open Access Journals (Sweden)

    Laura-Mihaela SIMON

    2010-12-01

    Full Text Available Background: The optimal choice of antimicrobial therapy is an important problem in hospital environment in which the selection of resistant and virulent strains easy occurs. S. aureus and especially MRSA(methicillin-resistant S. aureus creates difficulties in both treatment and prevention of nosocomial infections. Aim: The purpose of this study is to determine the sensitivity and the resistance to chemotherapy of staphylococci strains isolated from various pathological products. Material and Method: We identified Staphylococccus species after morphological appearance, culture properties, the production of coagulase, hemolisines and the enzyme activity. The susceptibility tests were performed on Mueller-Hinton medium according to CLSI (Clinical and Laboratory Standards Institute. Results: The strains were: MSSA (methicillin-susceptible S. aureus (74%, MRSA (8%, MLS B (macrolides, lincosamides and type B streptogramines resistance (12% and MRSA and MLS B (6%. MRSA strains were more frequently isolated from sputum. MRSA associated with the MLS B strains were more frequently isolated from pus. MLS B strains were more frequently isolated from sputum and throat secretions. All S. aureus strains were susceptible to vancomycin and teicoplanin. Conclusions: All staphylococcal infections require resistance testing before treatment. MLS B shows a high prevalence among strains of S. aureus. The association between MLS B and MRSA remains a major problem in Romania.

  11. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

    Directory of Open Access Journals (Sweden)

    Darrin V. Bann

    2012-06-01

    Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

  12. Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

    Science.gov (United States)

    Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

    2016-01-01

    Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

  13. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

    2017-02-01

    Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

  14. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Science.gov (United States)

    Khan, Naveed Ahmed; Iqbal, Junaid

    2015-01-01

    In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

  15. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Directory of Open Access Journals (Sweden)

    Naveed Ahmed Khan

    2015-01-01

    Full Text Available In the face of harsh conditions and given a choice, a cell may (i undergo programmed cell death, (ii transform into a cancer cell, or (iii enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes.

  16. Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications

    Directory of Open Access Journals (Sweden)

    Qi Tang

    2016-04-01

    Full Text Available THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells.

  17. High-content live cell imaging with RNA probes: advancements in high-throughput antimalarial drug discovery

    Directory of Open Access Journals (Sweden)

    Cervantes Serena

    2009-06-01

    Full Text Available Abstract Background Malaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes. Results After screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells. Conclusion This simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.

  18. Clinical and Molecular Epidemiology of Staphylococcal Toxic Shock Syndrome in the United Kingdom

    Science.gov (United States)

    Sharma, Hema; Smith, Debra; Turner, Claire E.; Game, Laurence; Pichon, Bruno; Hope, Russell; Hill, Robert; Kearns, Angela

    2018-01-01

    Staphylococcal toxic shock syndrome (TSS) was originally described in menstruating women and linked to TSS toxin 1 (TSST-1)–producing Staphylococcus aureus. Using UK national surveillance data, we ascertained clinical, molecular and superantigenic characteristics of TSS cases. Average annual TSS incidence was 0.07/100,000 population. Patients with nonmenstrual TSS were younger than those with menstrual TSS but had the same mortality rate. Children <16 years of age accounted for 39% of TSS cases, most caused by burns and skin and soft tissue infections. Nonmenstrual TSS is now more common than menstrual TSS in the UK, although both types are strongly associated with the tst+ clonal complex (CC) 30 methicillin-sensitive S. aureus lineage, which accounted for 49.4% of all TSS and produced more TSST-1 and superantigen bioactivity than did tst+ CC30 methicillin-resistant S. aureus strains. Better understanding of this MSSA lineage and infections in children could focus interventions to prevent TSS in the future. PMID:29350159

  19. Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

    Science.gov (United States)

    Strong, Averey D; Daniels, Richard L

    2017-08-02

    The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

  20. Fluorescent water-Soluble Probes Based on Ammonium Cation Peg Substituted Perylenepisimides: Synthesis, Photophysical Properties, and Live Cell Images

    Science.gov (United States)

    Yang, Wei; Cai, Jiaxuan; Zhang, Shuchen; Yi, Xuegang; Gao, Baoxiang

    2018-01-01

    To synthesize perylenbisimides (PBI) fluorescent probes that will improve the water-soluble ability and the cytocompatibility, the synthesis and properties of fluorescent water-soluble probes based on dendritic ammonium cation polyethylene glycol (PEG) substituted perylenebisimides(GPDIs) are presented. As we expected, with increased ammonium cation PEG, the aggregation of the PBI in an aqueous solution is completely suppressed by the hydrophilic ammonium cation PEG groups. And the fluorescence quantum yield increases from 25% for GPDI-1 to 62% for GPDI-2. When incubated with Hela cells for 48 h, the viabilities are 71% (for GPDI-1) and 76% (for GPDI-2). Live cell imaging shows that these probes are efficiently internalized by HeLa cells. The study of the photophysical properties indicated increasing the ammonium cation PEG generation can increase the fluorescence quantum yield. Live cell imaging shows that with the ammonium cation PEG chains of perylenebisimides has high biocompatibility. The exceptionally low cytotoxicity is ascribed to the ammonium cation PEG chains, which protect the dyes from nonspecifically interacting with the extracellular proteins. Live cell imaging shows that ammonium cations PEG chains can promote the internalization of these probes.