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Sample records for albicans protein microarray

  1. Protein microarrays for systems biology

    Institute of Scientific and Technical Information of China (English)

    Lina Yang; Shujuan Guo; Yang Li; Shumin Zhou; Shengce Tao

    2011-01-01

    Systems biology holds the key for understanding biological systems on a system level. It eventually holds the key for the treatment and cure of complex diseases such as cancer,diabetes, obesity, mental disorders, and many others. The '-omics' technologies, such as genomics, transcriptomics,proteomics, and metabonomics, are among the major driving forces of systems biology. Featured as highthroughput, miniaturized, and capable of parallel analysis,protein microarrays have already become an important technology platform for systems biology, In this review, we will focus on the system level or global analysis of biological systems using protein microarrays. Four major types of protein microarrays will be discussed: proteome microarrays, antibody microarrays, reverse-phase protein arrays,and lectin microarrays. We will also discuss the challenges and future directions of protein microarray technologies and their applications for systems biology. We strongly believe that protein microarrays will soon become an indispensable and invaluable tool for systems biology.

  2. Recent advances of protein microarrays

    OpenAIRE

    Hultschig, Claus; Kreutzberger, Jürgen; Seitz, Harald; Konthur, Zoltán; Büssow, Konrad; Lehrach, Hans

    2006-01-01

    Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss ...

  3. Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    ZENG Yue-bin; QIAN Yuan-shu; MA Lian; GU Hong-ni

    2007-01-01

    Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1),genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump

  4. Diagnostic and analytical applications of protein microarrays

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Christensen, C.B.V.

    2005-01-01

    DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the post...

  5. DNA microarray analysis of fluconazole resistance in a laboratory Candida albicans strain

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Several mechanisms are responsible for the acquired fluconazole (FLC) resistance in Candida albicans. In this study, we developed a FLC-resistant C. albicans strain through serial cultures of a FLC-susceptible C. albicans strain with inhibitory concentrations of FLC. Complimen-tary DNA microarray analysis and real-time reverse tran-scription-polymerase chain reaction were used to investi-gate gene expression changes during the acquisition of azole resistance in the susceptible parental strain and the resis-tant daughter strain. The differentially expressed genes rep-resented functions as diverse as transporters (e.g. CDRI, PDR17), ergosterol biosynthesis (e.g. ERG2, ERG9), sterol metabolism (e.g. ARE2, IPF6464), energy metabolism (e.g. ADH3, AOX2) and transcription factors (e.g. FCR1, ECM22). Functional analysis revealed that energy-depen-dent efflux activity of membrane transporters increased and that ergosterol content decreased with the accumulation of sterol intermediates in the resistant strain as compared with the susceptible strain. We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hy-peractivity of Tac1 could be the reason for overexpression of CDR1, CDR2, and PDR17 in the resistant strain.Furthermore, a single amino acid difference (DI9E) in ERG3 that led to the inactivation of Erg3 could account for both sterol precursor accumulation and the changes in the ex-pression of ergosterol biosynthesis genes in this resistant strain. These findings expand the understanding of poten-tial novel molecular targets of FLC resistance in clinical C.albicans isolates.

  6. Expression of surface hydrophobic proteins by Candida albicans in vivo.

    OpenAIRE

    Glee, P M; Sundstrom, P; Hazen, K C

    1995-01-01

    Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The po...

  7. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  8. Linking microarray reporters with protein functions

    Directory of Open Access Journals (Sweden)

    Gaj Stan

    2007-09-01

    Full Text Available Abstract Background The analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. Pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways. Results This paper proposes a novel method that aims to improve microarray reporter annotation by BLASTing the original reporter sequences against a species-specific EMBL subset, that was derived from and crosslinked back to the highly curated UniProt database. The resulting alignments were filtered using high quality alignment criteria and further compared with the outcome of a more traditional approach, where reporter sequences were BLASTed against EnsEMBL followed by locating the corresponding protein (UniProt entry for the high quality hits. Combining the results of both methods resulted in successful annotation of > 58% of all reporter sequences with UniProt IDs on two commercial array platforms, increasing the amount of Incyte reporters that could be coupled to Gene Ontology terms from 32.7% to 58.3% and to a local GenMAPP pathway from 9.6% to 16.7%. For Agilent, 35.3% of the total reporters are now linked towards GO nodes and 7.1% on local pathways. Conclusion Our methods increased the annotation quality of microarray reporter sequences and allowed us to visualize more reporters using pathway visualization tools. Even in cases where the original reporter annotation showed the correct description the new identifiers often allowed improved pathway and Gene Ontology linking. These methods are freely available at http://www.bigcat.unimaas.nl/public/publications/Gaj_Annotation/.

  9. Heat-shock protein 90 in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Researches on Candidal heat-shock protein 90 (HSP90) in recent years are summarized.Candida albicans is a commensal pathogen in human and animals.In immunocompromised individuals it behaves as an opportunist pathogen,giving rise to superficial or systemic infections.Systemic candidosis is a common cause of death among immunocompromised and debilitated patients,in which the mortality is as high as 70%.HSP90 is now recognized as an immunodominant antigen in C.albicans and plays a key role in systemic candidosis as a molecular chaperone.The 47-ku peptide is the breakdown product of HSP90.Patients who has recovered from systemic candidosis produce high titre of antibodies to 47-ku antigen,whereas the fatal cases have little antibody or falling titres.The three commonest epitopes of candidal HSP90 have been mapped,epitopes C,B and H.Epitopes C and H are immunogenic.The antibody probes of both epitopes may be developed into a new serological test agents for systemic candidosis due to rather high specificity and sensitivity.The recent results establish HSP90 as an ATP-dependent chaperone that is involved in the folding of cell regulatory proteins and in the refolding of stress-denatured polypeptides.Some researches on fungal HSP90 and the treatment of patients with candidosis are reviewed as well.

  10. Protein microarray: sensitive and effective immunodetection for drug residues

    Directory of Open Access Journals (Sweden)

    Zer Cindy

    2010-02-01

    Full Text Available Abstract Background Veterinary drugs such as clenbuterol (CL and sulfamethazine (SM2 are low molecular weight ( Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g than the ci-ELISA (0.1 ng/g for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

  11. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    International Nuclear Information System (INIS)

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

  12. A Protein Microarray ELISA for Screening Biological Fluids

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Woodbury, Ronald L.; Zangar, Richard C.

    2004-02-01

    Protein microarrays permit the simultaneous measurement of many proteins in a small sample volume and therefore provide an attractive approach for the quantitative measurement of proteins in biological fluids, including serum. This chapter describes a microarray ELISA assay. Capture antibodies are immobilized onto a glass surface, the covalently attached antibodies bind a specific antigen from a sample overlaying the array. A second, biotinylated antibody that recognizes the same antigen as the first antibody but at a different epitope is then used for detection. Detection is based upon an enzymatic signal enhancement method known as tyramide signal amplification (TSA). By coupling a microarray-ELISA format with the signal amplification of tyramide deposition, the assay sensitivity is as low as sub-pg/ml.

  13. Label and Label-Free Detection Techniques for Protein Microarrays

    Directory of Open Access Journals (Sweden)

    Amir Syahir

    2015-04-01

    Full Text Available Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano‑biological events.

  14. Nanotechnology in the Fabrication of Protein Microarrays.

    Science.gov (United States)

    Fuentes, Manuel; Díez, Paula; Casado-Vela, Juan

    2016-01-01

    Protein biochips are the heart of many medical and bioanalytical applications. Increasing interest of protein biochip fabrication has been focused on surface activation and subsequent functionalization strategies for the immobilization of these molecules.

  15. Producing reverse phase protein microarrays from formalin-fixed tissues.

    Science.gov (United States)

    Wolff, Claudia; Schott, Christina; Malinowsky, Katharina; Berg, Daniela; Becker, Karl-Friedrich

    2011-01-01

    In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

  16. A microarray immunoassay for simultaneous detection of proteins and bacteria

    Science.gov (United States)

    Delehanty, James B.; Ligler, Frances S.

    2002-01-01

    We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

  17. Enhancing the quality metric of protein microarray image

    Institute of Scientific and Technical Information of China (English)

    王立强; 倪旭翔; 陆祖康; 郑旭峰; 李映笙

    2004-01-01

    The novel method of improving the quality metric of protein microarray image presented in this paper reduces impulse noise by using an adaptive median filter that employs the switching scheme based on local statistics characters; and achieves the impulse detection by using the difference between the standard deviation of the pixels within the filter window and the current pixel of concern. It also uses a top-hat filter to correct the background variation. In order to decrease time consumption, the top-hat filter core is cross structure. The experimental results showed that, for a protein microarray image contaminated by impulse noise and with slow background variation, the new method can significantly increase the signal-to-noise ratio, correct the trends in the background, and enhance the flatness of the background and the consistency of the signal intensity.

  18. Protein Microarrays for Quantitative Detection of PAI-1 in Serum

    Institute of Scientific and Technical Information of China (English)

    Xu Ma; Qing-yun Zhang

    2012-01-01

    Objective:Plasminogen activator inhibitor-1 (PAl-1),one crucial component of the plasminogen activator system,is a major player in the pathogenesis of many vascular diseases as well as in cancer.High levels of PAI-1 in breast cancer tissue are associated with poor prognosis.The aim of this study is to evaluate rigorously the potential of serum PAl-1 concentration functioning as a general screening test in diagnostic or prognostic assays.Methods:A protein-microarray-based sandwich fluorescence immunoassay (FIA) was developed to detect PAl-1 in serum.Several conditions of this microarray-based FIA were optimized to establish an efficacious method.Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray.Results:The median serum PAl-1 level of breast cancer patients was higher than that of healthy women (109.7 ng/ml vs.63.4 ng/ml).Analysis of covariance revealed that PAl-1 levels of the two groups were significantly different (P<0.001) when controlling for an age effect on PAl-1 levels.However,PAl-1 values in TNM stage Ⅰ-Ⅳ patients respectively were not significantly different from each other.Conclusion:This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAl-1.

  19. Functional protein microarray as molecular decathlete: a versatile player in clinical proteomics.

    Science.gov (United States)

    Zhu, Heng; Cox, Eric; Qian, Jiang

    2012-12-01

    Functional protein microarrays were developed as a high-throughput tool to overcome the limitations of DNA microarrays and to provide a versatile platform for protein functional analyses. Recent years have witnessed tremendous growth in the use of protein microarrays, particularly functional protein microarrays, to address important questions in the field of clinical proteomics. In this review, we will summarize some of the most innovative and exciting recent applications of protein microarrays in clinical proteomics, including biomarker identification, pathogen-host interactions, and cancer biology. PMID:23027439

  20. Quantum Dots-based Reverse Phase Protein Microarray

    Energy Technology Data Exchange (ETDEWEB)

    Shingyoji, Masato; Gerion, Daniele; Pinkel, Dan; Gray, Joe W.; Chen, Fanqing

    2005-07-15

    CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP) catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R{sup 2} = 0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.

  1. Straightforward protein immobilization on Sylgard 184 PDMS microarray surface.

    Science.gov (United States)

    Heyries, Kevin A; Marquette, Christophe A; Blum, Loïc J

    2007-04-10

    In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.

  2. Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

    Science.gov (United States)

    Coleman, David Andrew

    2009-01-01

    The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

  3. Effects of salivary protein flow and indigenous microorganisms on initial colonization of Candida albicans in an in vivo model

    Directory of Open Access Journals (Sweden)

    Kanaguchi Norihiko

    2012-08-01

    Full Text Available Abstract Background Candida albicans is a dimorphic fungus that is part of the commensal microbial flora of the oral cavity. When the host immune defenses are impaired or when the normal microbial flora is disturbed, C. albicans triggers recurrent infections of the oral mucosa and tongue. Recently, we produced NOD/SCID.e2f1-/- mice that show hyposalivation, decrease of salivary protein flow, lack IgA and IgG in saliva, and have decreased NK cells. Our objective was to characterize C. albicans infection and biofilm formation in mice. Methods NOD/SCID.e2f1-/- mice were used as an animal model for C. albicans infection. C. albicans yeast and hyphal forms solutions were introduced in the oral cavity after disinfection by Chlorhexidine. Results The numbers of C. albicans colonized and decreased in a time-dependent manner in NOD/SCID.e2f1+/+ after inoculation. However, the colonization levels were higher in NOD/SCID.e2f1+/+ than NOD/SCID.e2f1-/- mice. In the mice fed 1% sucrose water before inoculation, C. albicans sample was highly contaminated by indigenous microorganisms in the oral cavity; and was not in the mice fed no sucrose water. The colonization of C. albicans was not influenced by the contamination of indigenous microorganisms. The hyphal form of C. albicans restricted the restoration of indigenous microorganisms. The decreased saliva in NOD/SCID.e2f1-/- did not increase the colonization of C. albicans in comparison to NOD/SCID.e2f1+/+ mice. We suggest that the receptor in saliva to C. albicans may not be sufficiently provided in the oral cavity of NOD/SCID.e2f1-/- mice. Conclusion The saliva protein flow may be very important for C. albicans initial colonization, where the indigenous microorganisms do not affect colonization in the oral cavity.

  4. Robust protein microarray image segmentation using improved seeded region growing algorithm

    Institute of Scientific and Technical Information of China (English)

    Liqiang Wang(王立强); Xuxiang Ni(倪旭翔); Zukang Lu(陆祖康)

    2003-01-01

    Protein microarray technology has recently emerged as a powerful tool for biomedical research. Before automatic analysis the protein microarray images, protein spots in the images must be determined appropriately by spot segmentation algorithm. In this paper, an improved seeded region growing (ISRG)algorithm for protein microarray segmentation is presented, the seeds are obtained by finding the positions of the printed spots, and the protein spot regions are grown through these seeds. The experiment results show that the presented algorithm is accurate for adaptive shape segmentation and robust for protein microarray images contaminated by noise.

  5. Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Janet F Staab

    Full Text Available Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG substrate and adhesin, Hyphal wall protein 1 (Hwp1, is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1, with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2, to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2. Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.

  6. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    Directory of Open Access Journals (Sweden)

    Adriana FIORINI

    2016-01-01

    Full Text Available Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC and sub-minimal inhibitory concentration (sub-MIC of the butanolic extract (BUTE of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1, amino acid metabolism (ILV5, PDC11 and protein synthesis (ASC1 pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides, it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

  7. [The cell-free protein synthesis-based protein microarray technology].

    Science.gov (United States)

    Lu, Linli; Lin, Bicheng

    2010-12-01

    The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay. PMID:21375003

  8. A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans.

    Science.gov (United States)

    Chen, Jiangye; Chen, Jing; Lane, Shelley; Liu, Haoping

    2002-12-01

    Candida albicans had been thought to lack a mating process until the recent discovery of a mating type-like locus and mating between MTLa and MTL(alpha) strains. To elucidate the molecular mechanisms that regulate mating in C. albicans, we examined the function of Cph1 and its upstream mitogen-activated protein (MAP) kinase pathway in mating, as they are homologues of the pheromone-responsive MAP kinase pathway in Saccharomyces cerevisiae. We found that overexpressing CPH1 in MTLa, but not in MTLa/alpha strains, induced the transcription of orthologues of S. cerevisiae pheromone-induced genes and also increased mating efficiency. Furthermore, cph1 and hst7 mutants were completely defective in mating, and cst20 and cek1 mutants showed reduced mating efficiency, as in S. cerevisiae. The partial mating defect in cek1 results from the presence of a functionally redundant MAP kinase, Cek2. CEK2 complemented the mating defect of a fus3 kss1 mutant of S. cerevisiae and was expressed only in MTLa or MTL(alpha), but not in MTLa/alpha cell types. Moreover, a cek1 cek2 double mutant was completely defective in mating. Our data suggest that the conserved MAP kinase pathway regulates mating in C. albicans. We also observed that C. albicans mating efficiency was greatly affected by medium composition, indicating the potential involvement of nutrient-sensing pathways in mating in addition to the MAP kinase pathway. PMID:12453219

  9. Rhodamine B doped silica nanoparticle labels for protein microarray detection

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule. The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays, in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanoparticles were sequentially conjugated. The calibration curve is linear over the range from 800 fg to 500 pg and the limit of detection is 100 fg, which is 8 times lower than that of streptavidin-labeled Cy3 fluorescent dyes. The dyedoped SiO2 nanoparticles show potentials for the protein array detection.

  10. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  11. Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans.

    OpenAIRE

    Hazen, K C; Hazen, B W

    1992-01-01

    Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distribu...

  12. Total Protein Profile and Drug Resistance in Candida albicans Isolated from Clinical Samples

    Directory of Open Access Journals (Sweden)

    Kamal Uddin Zaidi

    2016-01-01

    Full Text Available This study was done to assess the antifungal susceptibility of clinical isolates of Candida albicans and to evaluate its total protein profile based on morphological difference on drug resistance. Hundred and twenty clinical isolates of C. albicans from various clinical specimens were tested for susceptibility against four antifungal agents, namely, fluconazole, itraconazole, amphotericin B, and ketoconazole. A significant increase of drug resistance in clinical isolates of C. albicans was observed. The study showed 50% fluconazole and itraconazole resistance at 32 μg mL−1 with a MIC50 and MIC90 values at 34 and 47 and 36 and 49 μg mL−1, respectively. All isolates were sensitive to amphotericin B and ketoconazole. The SDS-PAGE protein profile showed a prevalent band of ~52.5 kDa, indicating overexpression of gene in 72% strains with fluconazole resistance. Since the opportunistic infections of Candida spp. are increasing along with drug resistance, the total protein profile will help in understanding the evolutionary changes in drug resistance and also to characterize them.

  13. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

    Science.gov (United States)

    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

  14. Co-occurence of filamentation defects and impaired biofilms in Candida albicans protein kinase mutants.

    Science.gov (United States)

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa.

  15. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

    Directory of Open Access Journals (Sweden)

    Constantin F Urban

    2009-10-01

    Full Text Available Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs. NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  16. Expanding the substantial interactome of NEMO using protein microarrays.

    Directory of Open Access Journals (Sweden)

    Beau J Fenner

    Full Text Available Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

  17. Expanding the substantial interactome of NEMO using protein microarrays.

    LENUS (Irish Health Repository)

    Fenner, Beau J

    2010-01-01

    Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

  18. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    Wei Gong; Kun He; Mike Covington; S.R Dinesh-Kumar; Michael Snyder; Stacey L.Harmer; Yu-Xian Zhu; Xing Wang Deng

    2008-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to constructprotein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and proteinprotein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

  19. Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

    Directory of Open Access Journals (Sweden)

    Boriollo Marcelo Fabiano Gomes

    2003-01-01

    Full Text Available The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters were analyzed, according to their homogeneous (socioeconomic category and/or same school and heterogeneous (distinct socioeconomic categories and/or schools characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0 among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low. Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a

  20. Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Jordan, Rachael P C; Williams, David W; Moran, Gary P; Coleman, David C; Sullivan, Derek J

    2014-04-01

    Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P < 0.001). However, when the yeast cells were grown at 37°C, no significant difference between the adhesion of C. dubliniensis genotype 1 and C. albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P < 0.001). Using surface plasmon resonance analysis, C. dubliniensis isolates were found to adhere in significantly greater numbers than C. albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

  1. Calling biomarkers in milk using a protein microarray on your smartphone

    NARCIS (Netherlands)

    Ludwig, S.K.J.; Tokarski, Christian; Lang, Stefan N.; Ginkel, Van L.A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, M.W.F.

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay

  2. Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

    International Nuclear Information System (INIS)

    The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto

  3. Morphogenesis of Candida albicans and Cytoplasmic Proteins Associated with Differences in Morphology, Strain, or Temperature

    OpenAIRE

    1981-01-01

    The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, whic...

  4. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

    OpenAIRE

    Maddi, Abhiram; Bowman, Shaun M.; Free, Stephen J.

    2009-01-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and 6 secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and 9 secreted proteins. Most ...

  5. Detection of protein microarrays by oblique-incidence reflectivity difference technique

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Biological microarrays with different proteins and different protein concentrations are detected without external labeling by an oblique-incidence reflectivity difference (OIRD) technique. The initial experiment results reveal that the intensities of OIRD signals can distinguish the different proteins and concentrations of protein. The OIRD technique promises feasible applications to life sciences for label-free and high-throughput detection.

  6. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

    OpenAIRE

    Ludwig, S.K.J.; Tokarski, Christian; Stefan N Lang; Ginkel, van, L.A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, M. W. F.

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV l...

  7. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    Science.gov (United States)

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  8. Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

    Science.gov (United States)

    Barbey, Raphael; Kauffmann, Ekkehard; Ehrat, Markus; Klok, Harm-Anton

    2010-12-13

    Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray

  9. Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

    Science.gov (United States)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins.

  10. An overview of innovations and industrial solutions in Protein Microarray Technology.

    Science.gov (United States)

    Gupta, Shabarni; Manubhai, K P; Kulkarni, Vishwesh; Srivastava, Sanjeeva

    2016-04-01

    The complexity involving protein array technology reflects in the fact that instrumentation and data analysis are subject to change depending on the biological question, technical compatibility of instruments and software used in each experiment. Industry has played a pivotal role in establishing standards for future deliberations in sustenance of these technologies in the form of protein array chips, arrayers, scanning devices, and data analysis software. This has enhanced the outreach of protein microarray technology to researchers across the globe. These have encouraged a surge in the adaptation of "nonclassical" approaches such as DNA-based protein arrays, micro-contact printing, label-free protein detection, and algorithms for data analysis. This review provides a unique overview of these industrial solutions available for protein microarray based studies. It aims at assessing the developments in various commercial platforms, thus providing a holistic overview of various modalities, options, and compatibility; summarizing the journey of this powerful high-throughput technology. PMID:27089056

  11. A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

    Directory of Open Access Journals (Sweden)

    Helene Andersson-Svahn

    2011-11-01

    Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  12. Detection of microarray protein biomolecules by oblique-incidence reflectivity difference technique without labelling agents

    Institute of Scientific and Technical Information of China (English)

    Zhang Hong-Yan; Lu Heng; Li Wei; Liang Ru-Qiang; Jin Kui-Juan; Zhou Yue-Liang; Ruan Kang-Cheng; Yang Guo-Zhen

    2008-01-01

    This paper reports that the detection to the protein in microarray format is carried out by oblique-incidence reflectivity difference (OI-RD) analysis without any labelling agents. The OI-RD intensities not only depend on the protein structure, but also vary with the protein concentration. The results indicate that this method should have potential application in detection of biochemical processes. The high throughout and in situ detection can be achieved by this method with further improving of the experimental system.

  13. Functional regions of Candida albicans hyphal cell wall protein Als3 that determine interaction with the oral bacterium Streptococcus gordonii.

    Science.gov (United States)

    Bamford, Caroline V; Nobbs, Angela H; Barbour, Michele E; Lamont, Richard J; Jenkinson, Howard F

    2015-01-01

    The opportunistic pathogen Candida albicans colonizes the oral cavity and gastrointestinal tract. Adherence to host cells, extracellular matrix and salivary glycoproteins that coat oral surfaces, including prostheses, is an important prerequisite for colonization. In addition, interactions of C. albicans with commensal oral streptococci are suggested to promote retention and persistence of fungal cells in mixed-species communities. The hyphal filament specific cell wall protein Als3, a member of the Als protein family, is a major determinant in C. albicans adherence. Here, we utilized site-specific in-frame deletions within Als3 expressed on the surface of heterologous Saccharomyces cerevisiae to determine regions involved in interactions of Als3 with Streptococcus gordonii. N-terminal region amino acid residue deletions Δ166-225, Δ218-285, Δ270-305 and Δ277-286 were each effective in inhibiting binding of Strep. gordonii to Als3. In addition, these deletions differentially affected biofilm formation, hydrophobicity, and adherence to silicone and human tissue proteins. Deletion of the central repeat domain (Δ434-830) did not significantly affect interaction of Als3 with Strep. gordonii SspB protein, but affected other adherence properties and biofilm formation. Deletion of the amyloid-forming region (Δ325-331) did not affect interaction of Als3 with Strep. gordonii SspB adhesin, suggesting this interaction was amyloid-independent. These findings highlighted the essential function of the N-terminal domain of Als3 in mediating the interaction of C. albicans with S. gordonii, and suggested that amyloid formation is not essential for the inter-kingdom interaction.

  14. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    Science.gov (United States)

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  15. Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

    Science.gov (United States)

    Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

    2016-01-01

    A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

  16. Structure of Protein Geranylgeranyltransferase-I from the Human Pathogen Candida albicans Complexed with a Lipid Substrate

    Energy Technology Data Exchange (ETDEWEB)

    Hast, Michael A.; Beese, Lorena S. (Duke)

    2008-11-21

    Protein geranylgeranyltransferase-I (GGTase-I) catalyzes the transfer of a 20-carbon isoprenoid lipid to the sulfur of a cysteine residue located near the C terminus of numerous cellular proteins, including members of the Rho superfamily of small GTPases and other essential signal transduction proteins. In humans, GGTase-I and the homologous protein farnesyltransferase (FTase) are targets of anticancer therapeutics because of the role small GTPases play in oncogenesis. Protein prenyltransferases are also essential for many fungal and protozoan pathogens that infect humans, and have therefore become important targets for treating infectious diseases. Candida albicans, a causative agent of systemic fungal infections in immunocompromised individuals, is one pathogen for which protein prenylation is essential for survival. Here we present the crystal structure of GGTase-I from C. albicans (CaGGTase-I) in complex with its cognate lipid substrate, geranylgeranylpyrophosphate. This structure provides a high-resolution picture of a non-mammalian protein prenyltransferase. There are significant variations between species in critical areas of the active site, including the isoprenoid-binding pocket, as well as the putative product exit groove. These differences indicate the regions where specific protein prenyltransferase inhibitors with antifungal activity can be designed.

  17. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    Science.gov (United States)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Li, Wei; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Ruan, Kangcheng; Yang, Guozhen

    2010-09-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml - 1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody-antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays.

  18. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    International Nuclear Information System (INIS)

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml−1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody–antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays

  19. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes.

  20. Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins

    OpenAIRE

    Schoenberg Fejzo, Marlena; Slamon, Dennis J.

    2001-01-01

    Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified thi...

  1. Microarray and Proteomic Analysis of Brassinosteroid- and Gibberellin-Regulated Gene and Protein Expression in Rice

    Institute of Scientific and Technical Information of China (English)

    Guangxiao Yang; Setsuko Komatsu

    2004-01-01

    Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze expression changes of genes and proteins at genome scale. In this review, we summarize rice functional genomic research by using microarray and proteomic approaches and our recent research results focusing on the comparison of cDNA microarray and proteomic analyses of BR- and GA-regulated gene and protein expression in rice. We believe our findings have important implications for understanding the mechanism by which BR and GA regulate the growth and development of rice.

  2. A Chemiluminescent Protein Microarray Method for Determining the Seroglycoid Fucosylation Index.

    Science.gov (United States)

    Zhang, Aiying; Skog, Sven; Wang, Shengqi; Ke, Yang; Zhang, Yonghong; Li, Kang; He, Ellen; Li, Ning

    2016-01-01

    The Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) is widely used to screen for hepatocellular carcinoma (HCC) in Japan and China. We developed a chemiluminescent protein microarray for determining the AFP-L3/AFP index (the ratio of AFP-L3 to total AFP, AFP-L3%) by fixing AFP-specific antibodies and Lens culinaris lectin on aldehyde-coated glass slides. Serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA) to validate the microarray. AFP-L3 was detected using Hotgen Biotech glycosyl capture spin column pretreatment technology and ELISA. When the AFP cut-off value was set to 20 ng/ml, the protein microarray displayed 89.74% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 87.17% sensitivity and 100% specificity. When the AFP-L3% cut-off value was set to 0.1, the protein microarray displayed 56.41% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 53.84% sensitivity and 100% specificity. The ROC curve for the HCC diagnosis showed that the AFP area under the ROC curve (AUC = 0.996; 95% CI: 0.986-1.005) was much higher than that of AFP-L3 (AUC = 0.857; 95% CI: 0.769-0.94) and AFP-L3% (AUC = 0.827; CI: 0.730-0.924). The microarray assay used in this study is a highly sensitive, accurate, and efficient assay for the determination of the AFP-L3%. PMID:27528397

  3. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    Science.gov (United States)

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  4. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  5. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone.

    Directory of Open Access Journals (Sweden)

    Susann K J Ludwig

    Full Text Available Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1. Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this 'protein microarray on a smartphone'-concept for on-site testing, e.g., in food safety, environment and health monitoring.

  6. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone.

    Science.gov (United States)

    Ludwig, Susann K J; Tokarski, Christian; Lang, Stefan N; van Ginkel, Leendert A; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W F

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this 'protein microarray on a smartphone'-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

  7. Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation.

    Science.gov (United States)

    Pelech, Steven; Jelinkova, Lucie; Susor, Andrej; Zhang, Hong; Shi, Xiaoqing; Pavlok, Antonin; Kubelka, Michal; Kovarova, Hana

    2008-07-01

    Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

  8. Genes transactivated by hepatitis C virus core protein, a microarray assay

    Institute of Scientific and Technical Information of China (English)

    Min Liu; Shu-Lin Zhang; Jun Cheng; Yan Liu; Lin Wang; Qing Shao; Jian Zhang; Shu-Mei Lin

    2005-01-01

    AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection.METHODS: Reverse transcribed cDNA was subjected tomicroarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods.RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene namedHCTP4 was cloned with molecular biological method in combination with bioinformatics method.CONCLUSION: HCV core is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.

  9. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    Science.gov (United States)

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  10. Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development

    OpenAIRE

    Cretich M.; Reddington A.; Monroe M.; Bagnati M.; Damin F.; Sola L.; Unlu M.S.; Chiari M.

    2011-01-01

    A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fl...

  11. Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer

    Directory of Open Access Journals (Sweden)

    Richardson Andrea L

    2011-10-01

    Full Text Available Abstract Background Na+/I- symporter (NIS-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Methods Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. Results and Discussion NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Conclusions Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

  12. Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

    Science.gov (United States)

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-01-01

    Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB. PMID:24145242

  13. Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

    OpenAIRE

    Lake April D; Phillips Keenan C; Henderson David C; Zárate Xristo; Galbraith David W

    2010-01-01

    Abstract Background Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia co...

  14. High performance protein microarrays based on glycidyl methacrylate-modified polyethylene terephthalate plastic substrate.

    Science.gov (United States)

    Liu, Yingshuai; Li, Chang Ming; Hu, Weihua; Lu, Zhisong

    2009-01-15

    There is a great challenge to immobilize high density of probe molecules for high performance protein microarrays, and this is achieved in this work by using polyethylene terephthalate (PET) plastic substrate onto which glycidyl methacrylate (GMA) photopolymer is grafted under mild conditions to introduce high density of epoxy groups for covalent immobilization of proteins. The poly(GMA)-grafted PET (PGMA-PET) surface was characterized with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy. For high density of protein immobilization and good quality of microspots, experiments were conducted to optimize the printing buffer, and an optimal buffer was found out to be PBS with 10% glycerol+0.003% triton X-100. According to the studies of loading capacity and immobilization kinetics, the optimal protein probe concentration and incubation time for the efficient immobilization are 200 microg mL(-1) and 8h, respectively. The performance of the PGMA-PET-based protein microarrays is evaluated with sandwich immunoassay using rat IgG and anti-rat IgG as model proteins, demonstrating a limit of detection (LOD) of 10 pg mL(-1) and a dynamic range of five orders of magnitude which are better than or very comparable with the reported or commercially available immunoassays, while providing a high-throughput approach. The work renders a simple and economic method to manufacture high performance protein microarrays and is expected to have great potentials in broad applications related to clinic diagnosis, drug discovery and proteomic research. PMID:19064107

  15. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

    OpenAIRE

    Palacín Gómez, Aranzazu; Gomez Casado, Cristina; Rivas, Luis; Aguirre, Jacobo; Tordesillas Villuendas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; de Frutos, Consolación; García Álvarez-Eire, Genoveva; Fernández, Francisco; Gamboa, P. M.; Muñoz, Rosa; Sánchez-Monge Laguna de Rins, Rosa

    2012-01-01

    The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant...

  16. Mycobacterium Tuberculosis Proteome Microarray for Global Studies of Protein Function and Immunogenicity

    Directory of Open Access Journals (Sweden)

    Jiaoyu Deng

    2014-12-01

    Full Text Available Poor understanding of the basic biology of Mycobacterium tuberculosis (MTB, the etiological agent of tuberculosis, hampers development of much-needed drugs, vaccines, and diagnostic tests. Better experimental tools are needed to expedite investigations of this pathogen at the systems level. Here, we present a functional MTB proteome microarray covering most of the proteome and an ORFome library. We demonstrate the broad applicability of the microarray by investigating global protein-protein interactions, small-molecule-protein binding, and serum biomarker discovery, identifying 59 PknG-interacting proteins, 30 bis-(3′-5′-cyclic dimeric guanosine monophosphate (c-di-GMP binding proteins, and 14 MTB proteins that together differentiate between tuberculosis (TB patients with active disease and recovered individuals. Results suggest that the MTB rhamnose pathway is likely regulated by both the serine/threonine kinase PknG and c-di-GMP. This resource has the potential to generate a greater understanding of key biological processes in the pathogenesis of tuberculosis, possibly leading to more effective therapies for the treatment of this ancient disease.

  17. Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

    Science.gov (United States)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

  18. Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

    Energy Technology Data Exchange (ETDEWEB)

    Daly, Don S.; Anderson, Kevin K.; White, Amanda M.; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2008-07-14

    Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensity that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting

  19. Reliability of a Tissue Microarray in Detecting Thyroid Transcription Factor-1 Protein in Lung Carcinomas

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Bai; Hong Shen

    2007-01-01

    OBJECTIVE To compare the expression of the thyroid transcription factor-1 (TTF-1) in human normal adult type Ⅱ alveolar epithelial cells,embryonic pneumocytes and cancer cells of lung carcinoma and metastatic lymph nodes using a tissue microarray (TMA) along with paired conventional full sections.and to jnvestigate the reliability of tissue microarrays in detecting protein expression in lung carcinoma.METHODS A lung carcinoma TMA including 765 cores was constructed.TTF-1 protein expression in both TMA and paired conventional full sections were detected by yhe immunohistochemical SP method using a monoclonal antibody to TTF-1.A PU (Positive Unit) of TTF-1 protein was assessed quantitatively by the Leica Q500MC image analysis system with results from the paired conventional full sections as controls.RESULTS There was no signifcance between TMA and paired conven tional full sections in TTF-1 expression in difierent nuclei of the lung tissue.CONCLUSION TTF-1 protein expression in lung carcinoma detected by TMA was highly concordanl with that of paired full sections.TMA is a reliable method in detecting protein expression.

  20. The adaptive metabolic response involves specific protein glutathionylation during the filamentation process in the pathogen Candida albicans.

    Science.gov (United States)

    Gergondey, R; Garcia, C; Serre, V; Camadro, J M; Auchère, F

    2016-07-01

    Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685). We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen. PMID:27083931

  1. 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.

  2. Design of a combinatorial dna microarray for protein-dnainteraction studies

    Energy Technology Data Exchange (ETDEWEB)

    Mintseris, Julian; Eisen, Michael B.

    2006-07-07

    Background: Discovery of precise specificity oftranscription factors is an important step on the way to understandingthe complex mechanisms of gene regulation in eukaryotes. Recently,doublestranded protein-binding microarrays were developed as apotentially scalable approach to tackle transcription factor binding siteidentification. Results: Here we present an algorithmic approach toexperimental design of a microarray that allows for testing fullspecificity of a transcription factor binding to all possible DNA bindingsites of a given length, with optimally efficient use of the array. Thisdesign is universal, works for any factor that binds a sequence motif andis not species-specific. Furthermore, simulation results show that dataproduced with the designed arrays is easier to analyze and would resultin more precise identification of binding sites. Conclusion: In thisstudy, we present a design of a double stranded DNA microarray forprotein-DNA interaction studies and show that our algorithm allowsoptimally efficient use of the arrays for this purpose. We believe such adesign will prove useful for transcription factor binding siteidentification and other biological problems.

  3. Protein microarrays for the identification of praja1 e3 ubiquitin ligase substrates.

    Science.gov (United States)

    Loch, Christian M; Eddins, Michael J; Strickler, James E

    2011-06-01

    Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3's profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3's, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2's to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme's function.

  4. The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans.

    Science.gov (United States)

    Donohue, Dagmara S; Ielasi, Francesco S; Goossens, Katty V Y; Willaert, Ronnie G

    2011-06-01

    The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

  5. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans.

    Science.gov (United States)

    Guan, Guobo; Wang, Haitao; Liang, Weihong; Cao, Chengjun; Tao, Li; Naseem, Shamoon; Konopka, James B; Wang, Yue; Huang, Guanghua

    2015-08-01

    The fungus Candida albicans is both a pathogen and a commensal in humans. The ability to utilize different carbon sources available in diverse host niches is vital for both commensalism and pathogenicity. N-acetylglucosamine (GlcNAc) is an important signaling molecule as well as a carbon source in C. albicans. Here, we report the discovery of a novel gene MCU1 essential for GlcNAc utilization. Mcu1 is located in mitochondria and associated with multiple energy- and metabolism-related proteins including Por1, Atp1, Pet9, and Mdh1. Consistently, inactivating Por1 impaired GlcNAc utilization as well. Deletion of MCU1 also caused defects in utilizing non-fermentable carbon sources and amino acids. Furthermore, MCU1 is required for filamentation in several inducing conditions and virulence in a mouse systemic infection model. We also deleted TGL99 and GUP1, two genes adjacent to MCU1, and found that the gup1/gup1 mutant exhibited mild defects in the utilization of several carbon sources including GlcNAc, maltose, galactose, amino acids, and ethanol. Our results indicate that MCU1 exists in a cluster of genes involved in the metabolism of carbon sources. Given its importance in metabolism and lack of a homolog in humans, Mcu1 could be a potential target for developing antifungal agents.

  6. Microarrays, Integrated Analytical Systems

    Science.gov (United States)

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  7. Improving the sensitivity of protein microarray by evanescent-field-induced fluorescence

    Institute of Scientific and Technical Information of China (English)

    WANG Li-qiang; LU Zu-kang

    2005-01-01

    To improve the sensitivity of protein microarray, a prism surface replaces the surface of the common microscope slide.The protein targets arrayed on the surface are hybridized and labelled by fluorescent probes. Evanescent excitation occurs when the convergent laser reaches the surface, and a photomultiplier tube detects the emitted fluorescent signal. A two-dimensional actuator scans the whole surface to achieve planar laser excitation and fluorescence collection. The penetration depth of the evanescent field into the protein targets is only some hundred nanometers and can be controlled by different incident angle of the laser beam, so the undesired background signals are reduced dramatically and the detection sensitivity is improved by a factor of 50 to 100 comparing to confocal excitation. This approach can detect low abundance analytes without signal amplification.

  8. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Delorme, Caroline; Joshi, Monika [Department of Biomedical and Molecular Sciences, Queen' s University, Kingston, ON, Canada K7L 3N6 (Canada); Allingham, John S., E-mail: allinghj@queensu.ca [Department of Biomedical and Molecular Sciences, Queen' s University, Kingston, ON, Canada K7L 3N6 (Canada)

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  9. Exploring host-pathogen interactions through genome wide protein microarray analysis.

    Science.gov (United States)

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-06-15

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

  10. Exploring host-pathogen interactions through genome wide protein microarray analysis

    Science.gov (United States)

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F.; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J.; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-01-01

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis. PMID:27302108

  11. Exploring host-pathogen interactions through genome wide protein microarray analysis.

    Science.gov (United States)

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-01-01

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis. PMID:27302108

  12. Mutation of G234 amino acid residue in candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport.

    Science.gov (United States)

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-Lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  13. Multicolor surface plasmon resonance imaging of ink jet-printed protein microarrays.

    Science.gov (United States)

    Singh, Bipin K; Hillier, Andrew C

    2007-07-15

    We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.

  14. A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases.

    Directory of Open Access Journals (Sweden)

    Jia-Xu Chen

    Full Text Available BACKGROUND: Food-borne helminthiases (FBHs have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA. The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI: 95.3-98.7% to 100.0% (95% CI: 100.0% in the protein microarray and from 97.7% (95% CI: 96.2-99.2% to 100.0% (95% CI: 100.0% in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3% to 92.1% (95% CI: 83.5-100.0% in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6% to 92.1% (95% CI: 83.5-100.0%. Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. CONCLUSIONS/SIGNIFICANCE: The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

  15. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    Science.gov (United States)

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

  16. Microarray-based method for screening of immunogenic proteins from bacteria

    Directory of Open Access Journals (Sweden)

    Hoppe Sebastian

    2012-03-01

    Full Text Available Abstract Background Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures. Results Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168. Conclusions The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and

  17. Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays.

    Science.gov (United States)

    Malinowsky, K; Wolff, C; Ergin, B; Berg, D; Becker, K F

    2010-11-01

    The current transition in cancer therapy from general treatment approaches, based mainly on chemotherapy and radiotherapy, to more directed approaches that aim to inhibit specific molecular targets has brought about new challenges for pathology. In the past, classical assignment of pathology consisted of tumor diagnosis and staging for further therapy decisions; nowadays, pathologists are asked to predict possible therapeutic results by detecting and quantifying therapeutic targets in tumors such as the human epidermal growth factor receptor 2 (HER2). The best approach to analyze such molecular targets is to provide a tumor-specific protein expression profile prior to therapy. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow fast, precise, cheap, and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling quantitative measurement of proteins. Recently, methods for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues have become available. In this article, we demonstrate how the use of RPPA to analyze signaling pathways from FFPE tissues may improve quantification of therapeutic targets and diagnostic markers in the near future.

  18. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  19. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    Science.gov (United States)

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  20. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Karen Elizabeth Kempsell

    2015-08-01

    Full Text Available A commercial Bacillus anthracis (Anthrax whole genome protein microarray has been used to identify immunogenic Anthrax proteins using sera from groups of donors with (a confirmed B. anthracis naturally acquired cutaneous infection, (b confirmed B. anthracis intravenous drug use-acquired infection (c occupational exposure in a wool-sorters factory (d humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups.Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However a number of other chromosomally-located and plasmid encoded open reading frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and are not currently found in the UK licensed Anthrax Vaccine (AVP. These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis ‘infectome’. These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  1. Candidate Genes for Testicular Cancer Evaluated by In Situ Protein Expression Analyses on Tissue Microarrays

    Directory of Open Access Journals (Sweden)

    Rolf I. Skotheim

    2003-09-01

    Full Text Available By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intratubular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT.

  2. Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

    Science.gov (United States)

    Han, Jin-Hee; Li, Jian; Wang, Bo; Lee, Seong-Kyun; Nyunt, Myat Htut; Na, Sunghun; Park, Jeong-Hyun; Han, Eun-Taek

    2015-08-01

    Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

  3. EFFECTS OF SYSTEMIC FLUCONAZOLE THERAPY ON IN VITRO ADHESION OF CANDIDA ALBICANS TO BUCCAL EPITHELIAL CELLS AND CHANGES OF THE CELL SURFACE PROTEINS OF THE EPITHELIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    吴绍熙; 郭宁如; 侯幼红

    1996-01-01

    This paper presented the effects of systemic fluconazole therapy via intravenous (IV) and oral (PO) administrations on the adhesion of Candida albicans (C. albicans) to the huccal epithelial ceils (BEC) from five treated patients with three candidosis, one mucornlycosis and one sporotrichosis and at the same time,an analysis of the cell surface proteins involving candidal adherent receptor in the BEC of the patients in the course of 7 days were exposed to 3H-leucine radiolabaled C. atbicans for in vitro eandidal adherent assay,and the BEC from first intake day and the last intake day of the patients were extracted by dithiothreitol(DTT)-iodoacetamide treatment for SDS-PAGE. These results indicate that the systemic iluconazole therapy resuks in the inhibitory effect of candldal adhesion to BEC of treated patients to prevent them from oral candidosis for a prolonged time, which is based on the absent surface protein (35KDa) of the BEC.

  4. Snf7p, a Component of the ESCRT-III Protein Complex, Is an Upstream Member of the RIM101 Pathway in Candida albicans

    OpenAIRE

    Kullas, Amy L.; Li, Mingchun; Davis, Dana A.

    2004-01-01

    The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pat...

  5. Discovery and validation of an INflammatory PROtein-driven GAstric cancer Signature (INPROGAS) using antibody microarray-based oncoproteomics

    OpenAIRE

    Puig-Costa, Manuel; Codina-Cazador, Antonio; Cortés-Pastoret, Elisabet; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Flaquer, Sílvia; Llopis-Puigmarti, Francesca; Pujol-Amado, Eulalia; Corominas-Faja, Bruna; Cuyàs, Elisabet; Ortiz, Rosa; Lopez-Bonet, Eugeni; Queralt, Bernardo; Guardeño, Raquel; Martin-Castillo, Begoña

    2014-01-01

    This study aimed to improve gastric cancer (GC) diagnosis by identifying and validating an INflammatory PROtein-driven GAstric cancer Signature (hereafter INPROGAS) using low-cost affinity proteomics. The detection of 120 cytokines, 43 angiogenic factors, 41 growth factors, 40 inflammatory factors and 10 metalloproteinases was performed using commercially available human antibody microarray-based arrays. We identified 21 inflammation-related proteins (INPROGAS) with significant differences in...

  6. Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial

    OpenAIRE

    Iresjö, Britt‐Marie; Engström, Cecilia; Lundholm, Kent

    2016-01-01

    Abstract Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short‐term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty...

  7. Analyzing the basic principles of tissue microarray data measuring the cooperative phenomena of marker proteins in invasive breast cancer

    OpenAIRE

    Korsching, Eberhard; Buerger, Horst; Boecker, Florian; Packeisen, Jens; Agelopoulos, Konstantin; Poos, Kathrin; Nadler, Walter

    2013-01-01

    Background: The analysis of a protein-expression pattern from tissue microarray (TMA) data will not immediately give an answer on synergistic or antagonistic effects between the observed proteins. But contrary to apparent first impression, it is possible to reveal those cooperative phenomena from TMA data. The data is (1) preserving a lot of the original physiological information content and (2) because of minor variances between the tumor samples, contains several related slightly different ...

  8. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  9. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs using microarray in a multicenter study.

    Directory of Open Access Journals (Sweden)

    Arantxa Palacín

    Full Text Available The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

  10. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

    Science.gov (United States)

    Palacín, Arantxa; Gómez-Casado, Cristina; Rivas, Luis A; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; de Frutos, Consolación; Alvarez-Eire, Genoveva García; Fernández, Francisco J; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Sirvent, Sofía; Torres, María J; Varela-Losada, Susana; Rodríguez, Rosalía; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

    2012-01-01

    The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens. PMID:23272072

  11. The Candida genome database incorporates multiple Candida species: multispecies search and analysis tools with curated gene and protein information for Candida albicans and Candida glabrata.

    Science.gov (United States)

    Inglis, Diane O; Arnaud, Martha B; Binkley, Jonathan; Shah, Prachi; Skrzypek, Marek S; Wymore, Farrell; Binkley, Gail; Miyasato, Stuart R; Simison, Matt; Sherlock, Gavin

    2012-01-01

    The Candida Genome Database (CGD, http://www.candidagenome.org/) is an internet-based resource that provides centralized access to genomic sequence data and manually curated functional information about genes and proteins of the fungal pathogen Candida albicans and other Candida species. As the scope of Candida research, and the number of sequenced strains and related species, has grown in recent years, the need for expanded genomic resources has also grown. To answer this need, CGD has expanded beyond storing data solely for C. albicans, now integrating data from multiple species. Herein we describe the incorporation of this multispecies information, which includes curated gene information and the reference sequence for C. glabrata, as well as orthology relationships that interconnect Locus Summary pages, allowing easy navigation between genes of C. albicans and C. glabrata. These orthology relationships are also used to predict GO annotations of their products. We have also added protein information pages that display domains, structural information and physicochemical properties; bibliographic pages highlighting important topic areas in Candida biology; and a laboratory strain lineage page that describes the lineage of commonly used laboratory strains. All of these data are freely available at http://www.candidagenome.org/. We welcome feedback from the research community at candida-curator@lists.stanford.edu.

  12. Detection of hybridization of protein microarrays using an oblique-incidence reflectivity difference method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Mouse-Immunoglobulin G(mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference(OI-RD) method.Two detection signals,consisting of an imaginary part(Im{Δp-Δs}) and a real part(Re{Δp-Δs}) of OI-RD,were obtained simultaneously.The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans.In particular,we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates.The detection sensitivity of OI-RD can reach 14 fg.These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.

  13. Boosting AthaMap Database Content with Data from Protein Binding Microarrays.

    Science.gov (United States)

    Hehl, Reinhard; Norval, Leo; Romanov, Artyom; Bülow, Lorenz

    2016-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) and small RNA target sites for the whole Arabidopsis thaliana genome. With the advent of protein binding microarrays (PBM), the number of known TFBS for A. thaliana transcription factors (TFs) has increased dramatically. Using 113 positional weight matrices (PWMs) generated from a single PBM study and representing a total number of 68 different TFs, the number of predicted TFBS in AthaMap was now increased by about 3.8 × 10(7) to 4.9 × 10(7). The number of TFs with PWM-predicted TFBS annotated in AthaMap has increased to 126, representing a total of 29 TF families and newly including ARF, AT-Hook, YABBY, LOB/AS2 and SRS. Furthermore, links from all Arabidopsis TFs and genes to the newly established Arabidopsis Information Portal (AIP) have been implemented. With this qualitative and quantitative update, the improved AthaMap increases its value as a resource for the analysis of A. thaliana gene expression regulation at www.athamap.de. PMID:26542109

  14. Detection of hybridization of protein microarrays using an oblique-incidence reflectivity difference method

    Science.gov (United States)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Lu, Huibin; Zhou, Yueliang; Jin, Kui-Juan; Yang, Guozhen; Li, Wei; Ruan, Kangcheng

    2010-07-01

    Mouse-Immunoglobulin G (mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference (OI-RD) method. Two detection signals, consisting of an imaginary part (Im{Δp-Δs}) and a real part (Re{Δp-Δs}) of OI-RD, were obtained simultaneously. The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans. In particular, we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates. The detection sensitivity of OI-RD can reach 14 fg. These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.

  15. The conserved dual phosphorylation sites of the Candida albicans Hog1 protein are crucial for white-opaque switching, mating, and pheromone-stimulated cell adhesion.

    Science.gov (United States)

    Chang, Wen-Han; Liang, Shen-Huan; Deng, Fu-Sheng; Lin, Ching-Hsuan

    2016-08-01

    Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion. PMID:27118797

  16. Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.

    Science.gov (United States)

    Klenkar, Goran; Valiokas, Ramûnas; Lundström, Ingemar; Tinazli, Ali; Tampé, Robert; Piehler, Jacob; Liedberg, Bo

    2006-06-01

    The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.

  17. A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

    Science.gov (United States)

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Deghou, Samy; Ceschia, Stefano; Tischer, Christian; Kugler, Karl G; Bork, Peer; Ellenberg, Jan; Gavin, Anne-Claude

    2016-06-01

    Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales. PMID:27149326

  18. Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

    Science.gov (United States)

    Yun, Bogeon; Lee, HeeJung; Jayaraja, Sabarirajan; Suram, Saritha; Murphy, Robert C; Leslie, Christina C

    2016-03-25

    In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

  19. A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

    Science.gov (United States)

    Loch, Christian M; Strickler, James E

    2012-11-01

    Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

  20. Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial.

    Science.gov (United States)

    Iresjö, Britt-Marie; Engström, Cecilia; Lundholm, Kent

    2016-06-01

    Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short-term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty-two patients (11/group) scheduled for upper GI surgery due to malignant or benign disease received a continuous peripheral all-in-one TPN infusion (30 kcal/kg/day, 0.16 gN/kg/day) or saline infusion for 12 h prior operation. Biopsies from the rectus abdominis muscle were taken at the start of operation for isolation of muscle RNA RNA expression microarray analyses were performed with Agilent Sureprint G3, 8 × 60K arrays using one-color labeling. 447 mRNAs were differently expressed between study and control patients (P muscle mRNA alterations during overnight standard TPN infusions at constant rate altered mRNAs associated with mTOR signaling; increased initiation of protein translation; and suppressed autophagy/lysosomal degradation of proteins. This indicates that overnight preoperative parenteral nutrition is effective to promote muscle protein metabolism. PMID:27273879

  1. Microarray analysis of ncRNA expression patterns in Caenorhabditis elegans after RNAi against snoRNA associated proteins

    Directory of Open Access Journals (Sweden)

    Skogerbø Geir

    2008-06-01

    Full Text Available Abstract Background Short non-coding RNAs (ncRNAs perform their cellular functions in ribonucleoprotein (RNP complexes, which are also essential for maintaining the stability of the ncRNAs. Depletion of individual protein components of non-coding ribonucleoprotein (ncRNP particles by RNA interference (RNAi may therefore affect expression levels of the corresponding ncRNA, and depletion of candidate associated proteins may constitute an alternative strategy when investigating ncRNA-protein interactions and ncRNA functions. Therefore, we carried out a pilot study in which the effects of RNAi against protein components of small nucleolar RNPs (snoRNPs in Caenorhabditis elegans were observed on an ncRNA microarray. Results RNAi against individual C. elegans protein components of snoRNPs produced strongly reduced mRNA levels and distinct phenotypes for all targeted proteins. For each type of snoRNP, individual depletion of at least three of the four protein components produced significant (P ≦ 1.2 × 10-5 reductions in the expression levels of the corresponding small nucleolar RNAs (snoRNAs, whereas the expression levels of other ncRNAs were largely unaffected. The effects of depletion of individual proteins were in accordance with snoRNP structure analyses obtained in other species for all but two of the eight targeted proteins. Variations in snoRNA size, sequence and secondary structure characteristics were not systematically reflected in the affinity for individual protein component of snoRNPs. The data supported the classification of nearly all annotated snoRNAs and suggested the presence of several novel snoRNAs among unclassified short ncRNA transcripts. A number of transcripts containing canonical Sm binding element sequences (Sm Y RNAs also showed reduced expression after depletion of protein components of C/D box snoRNPs, whereas the expression of some stem-bulge RNAs (sbRNAs was increased after depletion of the same proteins. Conclusion

  2. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    Science.gov (United States)

    Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza. PMID:26938453

  3. Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: a comparative proteomics and microarray study.

    Science.gov (United States)

    Yang, Kyeong Eun; Kwon, Joseph; Rhim, Ji-Heon; Choi, Jong Soon; Kim, Seung Il; Lee, Seung-Hoon; Park, Junsoo; Jang, Ik-Soon

    2011-07-01

    The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.

  4. Discovery and validation of an INflammatory PROtein-driven GAstric cancer Signature (INPROGAS) using antibody microarray-based oncoproteomics

    Science.gov (United States)

    Puig-Costa, Manuel; Codina-Cazador, Antonio; Cortés-Pastoret, Elisabet; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Flaquer, Sílvia; Llopis-Puigmarti, Francesca; Pujol-Amado, Eulalia; Corominas-Faja, Bruna; Cuyàs, Elisabet; Ortiz, Rosa; Lopez-Bonet, Eugeni; Queralt, Bernardo; Guardeño, Raquel; Martin-Castillo, Begoña; Roig, Josep; Joven, Jorge; Menendez, Javier A.

    2014-01-01

    This study aimed to improve gastric cancer (GC) diagnosis by identifying and validating an INflammatory PROtein-driven GAstric cancer Signature (hereafter INPROGAS) using low-cost affinity proteomics. The detection of 120 cytokines, 43 angiogenic factors, 41 growth factors, 40 inflammatory factors and 10 metalloproteinases was performed using commercially available human antibody microarray-based arrays. We identified 21 inflammation-related proteins (INPROGAS) with significant differences in expression between GC tissues and normal gastric mucosa in a discovery cohort of matched pairs (n=10) of tumor/normal gastric tissues. Ingenuity pathway analysis confirmed the “inflammatory response”, “cellular movement” and “immune cell trafficking” as the most overrepresented biofunctions within INPROGAS. Using an expanded independent validation cohort (n = 22), INPROGAS classified gastric samples as “GC” or “non-GC” with a sensitivity of 82% (95% CI 59-94) and a specificity of 73% (95% CI 49-89). The positive predictive value and negative predictive value in this validation cohort were 75% (95% CI 53-90) and 80% (95% CI 56-94), respectively. The positive predictive value and negative predictive value in this validation cohort were 75% (95% CI 53-90) and 80% (95% CI 56-94), respectively. Antibody microarray analyses of the GC-associated inflammatory proteome identified a 21-protein INPROGAS that accurately discriminated GC from noncancerous gastric mucosa. PMID:24722433

  5. Development of a Novel Protein Microarray Method for Serotyping Salmonella enterica Strains

    OpenAIRE

    Cai, H.Y.; Lu, L; Muckle, C A; Prescott, J F; Chen, S.

    2005-01-01

    An antibody microarray assay was developed for Salmonella serotyping based on the Kauffmann-White scheme. A model (8 by 15) array was constructed using 35 antibodies for identification of 20 common Salmonella serovars and evaluated using 117 target and 73 nontarget Salmonella strains. The assay allowed complete serovar identification of 86 target strains and partial identification of 30 target strains and allowed exclusion of the 73 nontarget strains from the target serovars.

  6. Expression of the G protein-coupled estrogen receptor (GPER in endometriosis: a tissue microarray study

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    Samartzis Nicolas

    2012-04-01

    Full Text Available Abstract Background The G protein-coupled estrogen receptor (GPER is thought to be involved in non-genomic estrogen responses as well as processes such as cell proliferation and migration. In this study, we analyzed GPER expression patterns from endometriosis samples and normal endometrial tissue samples and compared these expression profiles to those of the classical sex hormone receptors. Methods A tissue microarray, which included 74 samples from different types of endometriosis (27 ovarian, 19 peritoneal and 28 deep-infiltrating and 30 samples from normal endometrial tissue, was used to compare the expression levels of the GPER, estrogen receptor (ER-alpha, ER-beta and progesterone receptor (PR. The immunoreactive score (IRS was calculated separately for epithelium and stroma as the product of the staining intensity and the percentage of positive cells. The expression levels of the hormonal receptors were dichotomized into low (IRS  =6 expression groups. Results The mean epithelial IRS (+/−standard deviation, range of cytoplasmic GPER expression was 1.2 (+/−1.7, 0–4 in normal endometrium and 5.1 (+/−3.5, 0–12 in endometriosis (p p = 0.71, of ER-alpha 10.6 (+/−2.4, 3–12 and 9.8 (+/−3.0, 2–12; p = 0.26, of ER-beta 2.4 (+/−2.2; 0–8 and 5.6 (+/−2.6; 0–10; p p p p = 0.001, of ER-beta 1.8 (+/−2.0; 0–8 and 5.4 (+/−2.5; 0–10; p p���= 0.044, respectively. Cytoplasmic GPER expression was not detectable in the stroma of endometrium and endometriosis. The observed frequency of high epithelial cytoplasmic GPER expression levels was 50% (n = 30/60 in the endometriosis and none (0/30 in the normal endometrium samples (p p = 0.01, as compared to peritoneal (9/18, 50% or deep-infiltrating endometriotic lesions (7/22, 31.8%. The frequency of high stromal nuclear GPER expression levels was 100% (n = 74/74 in endometriosis and 76.7% (n = 23/30 in normal endometrium (p

  7. Ethanolic extract of Passiflora edulis Sims leaves inhibits protein glycation and restores the oxidative burst in diabetic rat macrophages after Candida albicans exposure

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    Carolina Fernandes Ribas Martins

    2015-12-01

    Full Text Available abstract This study was conducted to evaluate the effects of the ethanolic extract of Passiflora edulis leaves on blood glucose, protein glycation, NADPH oxidase activity and macrophage phagocytic capacity after Candida albicans exposure in diabetic rats. The Passiflora edulis Sims leaves were dried to 40°C, powdered, extracted by maceration in 70% ethanol, evaporated under reduced pressure and lyophilised. The biochemical tests performed were total phenolic content (TP as determined by the Folin-Ciocalteu assay, trapping potential DPPH assay and total iron-reducing potential. Diabetes was induced by alloxan injection. Protein glycation was determined by AGE and fructosamine serum concentrations. Extract-treated diabetic animals demonstrated lower fructosamine concentrations compared with the diabetic group. Our results suggest that ethanolic Passiflora edulis Sims leaf extraction may have beneficial effects on diabetes and may improve glycaemic control in diabetic rats.

  8. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray

    Energy Technology Data Exchange (ETDEWEB)

    Zhijian, Chen [Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, Zhejiang (China); Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang (China); Xiaoxue, Li [Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang (China); Wei, Zheng [Zhejiang International Travel Healthcare Center, 230 Zhonghezhong Road, Hangzhou 310003 (China); Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin [Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang (China); Jiliang, He, E-mail: he_jiliang@hotmail.com [Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang (China)

    2013-03-29

    Highlights: ► Protein microarray shows the differential expression of 27 proteins induced by RFR. ► RPA32 related to DNA repair is down-regulated in Western blot. ► p73 related to cell genome stability and apoptosis is up-regulated in Western blot. -- Abstract: In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P < 0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P < 0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P < 0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.

  9. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

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    He Zu-gen

    2006-12-01

    Full Text Available Abstract Background The development of esophageal squamous cell carcinoma (ESCC progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS and carcinoma in situ (CIS, subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

  10. Aptamer Microarrays

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    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  11. Candida albicans infection leads to barrier breakdown and a MAPK/NF-κB mediated stress response in the intestinal epithelial cell line C2BBe1.

    Science.gov (United States)

    Böhringer, Michael; Pohlers, Susann; Schulze, Sylvie; Albrecht-Eckardt, Daniela; Piegsa, Judith; Weber, Michael; Martin, Ronny; Hünniger, Kerstin; Linde, Jörg; Guthke, Reinhard; Kurzai, Oliver

    2016-07-01

    Intestinal epithelial cells (IEC) form a tight barrier to the gut lumen. Paracellular permeability of the intestinal barrier is regulated by tight junction proteins and can be modulated by microorganisms and other stimuli. The polymorphic fungus Candida albicans, a frequent commensal of the human mucosa, has the capacity of traversing this barrier and establishing systemic disease within the host. Infection of polarized C2BBe1 IEC with wild-type C. albicans led to a transient increase of transepithelial electric resistance (TEER) before subsequent barrier disruption, accompanied by a strong decline of junctional protein levels and substantial, but considerably delayed cytotoxicity. Time-resolved microarray-based transcriptome analysis of C. albicans challenged IEC revealed a prominent role of NF-κB and MAPK signalling pathways in the response to infection. Hence, we inferred a gene regulatory network based on differentially expressed NF-κB and MAPK pathway components and their predicted transcriptional targets. The network model predicted activation of GDF15 by NF-κB was experimentally validated. Furthermore, inhibition of NF-κB activation in C. albicans infected C2BBe1 cells led to enhanced cytotoxicity in the epithelial cells. Taken together our study identifies NF-κB activation as an important protective signalling pathway in the response of epithelial cells to C. albicans. PMID:26752615

  12. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  13. Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms.

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    Chessa, S; Chiatti, F; Ceriotti, G; Caroli, A; Consolandi, C; Pagnacco, G; Castiglioni, B

    2007-01-01

    The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.

  14. Pattern recognition in pulmonary tuberculosis defined by high content peptide microarray chip analysis representing 61 proteins from M. tuberculosis.

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    Simani Gaseitsiwe

    Full Text Available BACKGROUND: Serum antibody-based target identification has been used to identify tumor-associated antigens (TAAs for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M. tuberculosis (MTB infection for diagnosis or TB vaccine development in clinically well defined populations. METHOD: We constructed a high-content peptide microarray with 61 M. tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/-. FINDINGS: Three distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals and are highly predictive of the division into TB+ and TB-, other targets were exclusively recognized in all patients with TB (e.g. sigmaF but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35 but no in TB+ patients. The segregation between TB+ and TB- does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope 'hotspots' at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background. CONCLUSIONS: A uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize

  15. Identification of a putative DEAD-box RNA helicase and a zinc-finger protein in Candida albicans by functional complementation of the S. cerevisiae rok1 mutation.

    Science.gov (United States)

    Kim, W I; Lee, W B; Song, K; Kim, J

    2000-03-30

    We identified two novel genes, CHR1 and CSR1, of the fungal pathogen Candida albicans, by functional complementation of the Saccharomyces cerevisiae rok1 mutation. The Rok1 protein is a member of the DEAD protein family of ATP-dependent RNA helicases. ROK1 is required for cell cycle progression and also for rRNA processing. The CHR1 gene product of 578 amino acids is highly homologous to the Rok1 protein (54% identity) and is considered to be a putative DEAD-box RNA helicase. We predict that the CSR1 gene encodes a 73 kDa protein of 612 amino acids with five zinc-finger motifs at the C-terminal region. CHR1 or CSR1 on a high-copy number plasmid showed a slow-growth phenotype in a condition where the ROK1 expression is turned on from the GAL1 promoter. This result is consistent with the lethality caused by the ROK1 overexpression. We conclude that CHR1 encodes a functional homologue of Rok1 protein and CSR1 is a heterologous suppressor of the rok1 mutation. PMID:10705369

  16. Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

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    Lake April D

    2010-06-01

    Full Text Available Abstract Background Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. Results Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. Conclusion The results are consistent with the hypothesis that the wheat germ extract enhances the protein

  17. Microarray Analysis in Glioblastomas

    Science.gov (United States)

    Bhawe, Kaumudi M.; Aghi, Manish K.

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930–2942, 2012)To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013)To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59–70, 2013; Verhaak et al., Cancer Cell 17(1):98–110, 2010) While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  18. HER2/neu (c-erbB-2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen;

    2009-01-01

    Published studies have reported widely variable incidence of HER2/neu (c-erbB-2) protein expression and HER2/neu (c-erbB-2) gene amplification in cervical carcinoma. We examined tissue microarrays (TMAs) constructed from 814 formaldehyde-fixed paraffin-embedded archival specimens of cervical intr...

  19. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Yansheng Liu

    Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  20. Endoftalmite por Candida albicans Candida albicans endophthalmitis

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    Pedro Duraes Serracarbassa

    2003-10-01

    Full Text Available O autor descreve os aspectos epidemiológicos, histopatológicos e clínicos da endoftalmite endógena por Candida albicans. Apresenta ainda novos métodos diagnósticos e opções terapêuticas utilizadas no tratamento das infecções fúngicas intra-oculares, por meio de revisão bibliográfica.The author describes epidemiological, histopathological and clinical aspects of endogenous Candida albicans endophthalmitis. He also presents new diagnostic methods and therapeutical options to treat intraocular fungal infections, based on literature review.

  1. Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays

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    Evans David

    2008-01-01

    Full Text Available Abstract Background The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. Results We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. Conclusion The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

  2. Measurement of Small Molecule Binding Kinetics on a Protein Microarray by Plasmonic-Based Electrochemical Impedance Imaging

    Science.gov (United States)

    2015-01-01

    We report on a quantitative study of small molecule binding kinetics on protein microarrays with plasmonic-based electrochemical impedance microscopy (P-EIM). P-EIM measures electrical impedance optically with high spatial resolution by converting a surface charge change to a surface plasmon resonance (SPR) image intensity change, and the signal is not scaled to the mass of the analyte. Using P-EIM, we measured binding kinetics and affinity between small molecule drugs (imatinib and SB202190) and their target proteins (kinases Abl1 and p38-α). The measured affinity values are consistent with reported values measured by an indirect competitive binding assay. We also found that SB202190 has weak bindings to ABL1 with KD > 10 μM, which is not reported in the literature. Furthermore, we found that P-EIM is less prone to nonspecific binding, a long-standing issue in SPR. Our results show that P-EIM is a novel method for high-throughput measurement of small molecule binding kinetics and affinity, which is critical to the understanding of small molecules in biological systems and discovery of small molecule drugs. PMID:25153794

  3. Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms.

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    Vitor Cabral

    2014-12-01

    Full Text Available Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power

  4. [THE POSSIBILITIES OF APPLICATION OF TECHNOLOGY PROTEIN MICROARRAY (MICROCHIPS) FOR ANALYSIS OF PROTEIN COMPOSITION OF BLOOD SERUM].

    Science.gov (United States)

    Gumanova, N G; Klimushina, M V; Metelskaya, V A; Boitsov, S A

    2015-10-01

    The microchip technology represents convenient and relatively economic tool of analyzing specific biomarkers with the purpose to diagnose diseases, to evaluate effectiveness of therapy and to investigate signaling pathways. To analyze protein composition of blood serum certain types of finished microchips which were not applied previously on the territory of Russia. The detection from 2% to 5% out of matrix of chips depending on their variety was managed without preliminary depletion of serum (removal of proteins of major fractions). Hence, partial protein composition of blood serum can be analyzed with microchips even without preliminary removal of proteins of major fractions.

  5. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

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    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  6. A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

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    Kumar Pat

    2007-11-01

    Full Text Available Abstract Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1, matrix metalloproteinase 11 (MMP11 and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

  7. Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer

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    Clifford John

    2009-01-01

    Full Text Available Abstract Background Eukaryotic initiation factor 4E (eIF4E is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC, vascular endothelial growth factor (VEGF and Tousled-like kinase 1B (TLK1B. However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products. Results Breast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu. Conclusion We conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

  8. Microarray analysis of the effect of Streptococcus equi subsp. zooepidemicus M-like protein in infecting porcine pulmonary alveolar macrophage.

    Directory of Open Access Journals (Sweden)

    Zhe Ma

    Full Text Available Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, which belongs to Lancefield group C streptococci, is an important pathogen of domesticated species, causing septicemia, meningitis and mammitis. M-like protein (SzP is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. To increase our knowledge of the mechanism of SzP in infection, we profiled the response of porcine pulmonary alveolar macrophage (PAM to infection with S. zooepidemicus ATCC35246 wild strain (WD and SzP-knockout strain (KO using the Roche NimbleGen Porcine Genome Expression Array. We found SzP contributed to differential expression of 446 genes, with upregulation of 134 genes and downregulation of 312 genes. Gene Ontology category and KEGG pathway were analyzed for relationships among differentially expressed genes. These genes were represented in a variety of functional categories, including genes involved in immune response, regulation of chemokine production, signal transduction and regulation of apoptosis. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data will contribute to understanding of SzP mediated mechanisms of S. zooepidemicus pathogenesis.

  9. Microarray analysis applied to the study of milk protein loci in cattle

    Directory of Open Access Journals (Sweden)

    G. Pagnacco

    2010-01-01

    Full Text Available Milk proteins still arise much interest because of the proved effects of the most common milk genetic polymorphisms on quantitative and qualitative milk production, as well as on milk technological properties. The role of αs1-casein (CSN1S1, β-casein (CSN2, k-casein (CSN3 and β-lactoglobulin (LGB polymorphisms in the genetic improvement of milk production was already demonstrated in cattle, as reviewed by Di Stasio and Mariani (2000 and Martin et al. (2002.

  10. Identification of Novel Protein-Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a small number of clones will dominate the selection output, making it difficult to comprehensively identify potentially important interactions due to low representation in the selection output. We have developed a novel method to address this problem. By analyzing selection outputs using high-density human exon microarrays, the full potential of selection output diversity can be revealed in one experiment. FACS-based selection using yeast surface displayed human cDNA libraries combined with exon microarray analysis of the selection outputs is a powerful way of rapidly identifying protein fragments with affinity for any soluble ligand that can be fluorescently detected, including small biological molecules and drugs. In this report we present protocols for exon microarray-based analysis of yeast surface display human cDNA library selection outputs. PMID:26060075

  11. Label-Free and High-Throughput Detection of Protein Microarrays by Oblique-Incidence Reflectivity Difference Method

    Science.gov (United States)

    Wang, Xu; Lu, Heng; Wen, Juan; Yuan, Kun; LÜ, Hui-Bin; Jin, Kui-Juan; Zhou, Yue-Liang; Yang, Guo-Zhen

    2010-10-01

    We label-free detected the biological process of preparing a microarray that includes 400 spots of mouse immunoglobulin G (IgG) as well as the specific hybridization between mouse IgG and goat anti-mouse IgG by an oblique-incidence reflectivity difference (OI-RD) method. The detection results after each process including printing, washing, blocking, and hybridization, demonstrate that the OI-RD method can trace the preparation process of a microarray and detect the specific hybridization between antigens and antibodies. OI-RD is a promising method for label-free and high-throughput detection of biological microarrays.

  12. Label-Free and High-Throughput Detection of Protein Microarrays by Oblique-Incidence Reflectivity Difference Method

    International Nuclear Information System (INIS)

    We label-free detected the biological process of preparing a microarray that includes 400 spots of mouse immunoglobulin G (IgG) as well as the specific hybridization between mouse IgG and goat anti-mouse IgG by an oblique-incidence reflectivity difference (OI-RD) method. The detection results after each process including printing, washing, blocking, and hybridization, demonstrate that the OI-RD method can trace the preparation process of a microarray and detect the specific hybridization between antigens and antibodies. OI-RD is a promising method for label-free and high-throughput detection of biological microarrays

  13. Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner

    Science.gov (United States)

    Landry, James P.; Proudian, Andrew P.; Malovichko, Galina; Zhu, Xiangdong

    2013-02-01

    Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.

  14. Baicalin prevents Candida albicans infections via increasing its apoptosis rate

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shulong; Fu, Yingyuan, E-mail: yingyuanfu@126.com; Wu, Xiuzhen; Zhou, Zhixing; Xu, Jing; Zeng, Xiaoping; Kuang, Nanzhen; Zeng, Yurong

    2014-08-15

    Highlights: • Baicalin increases the ratio of the G0/G1 stages and C. albicans apoptosis. • Baicalin decreases the proliferation index of C. albicans. • Baicalin inhibits the biosynthesis of DNA, RNA and protein in C. albicans. • Baicalin depresses Succinate Dehydrogenase and Ca{sup 2+}–Mg{sup 2+} ATPase in C. albicans. • Baicalin increases the endocytic free Ca{sup 2+} concentration in C. albicans. - Abstract: Background: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. Methodology and principal findings: We detected the baicalin inhibition effects on three isotope-labeled precursors of {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca{sup 2+}–Mg{sup 2+} ATPase, cytosolic Ca{sup 2+} concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca{sup 2+} concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. Innovation and significance: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase, increasing

  15. Baicalin prevents Candida albicans infections via increasing its apoptosis rate

    International Nuclear Information System (INIS)

    Highlights: • Baicalin increases the ratio of the G0/G1 stages and C. albicans apoptosis. • Baicalin decreases the proliferation index of C. albicans. • Baicalin inhibits the biosynthesis of DNA, RNA and protein in C. albicans. • Baicalin depresses Succinate Dehydrogenase and Ca2+–Mg2+ ATPase in C. albicans. • Baicalin increases the endocytic free Ca2+ concentration in C. albicans. - Abstract: Background: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. Methodology and principal findings: We detected the baicalin inhibition effects on three isotope-labeled precursors of 3H-UdR, 3H-TdR and 3H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca2+–Mg2+ ATPase, cytosolic Ca2+ concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited 3H-UdR, 3H-TdR and 3H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca2+–Mg2+ ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca2+ concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. Innovation and significance: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca2+–Mg2+ ATPase, increasing cytosolic Ca2+ content and damaging the ultrastructure of C. albicans

  16. Chromosome Microarray.

    Science.gov (United States)

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  17. Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

    OpenAIRE

    Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

    2010-01-01

    We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is...

  18. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    International Nuclear Information System (INIS)

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues. -- Highlights: • We examined the effects of Bt proteins on gene expression of Folsomia candida. • Eleven transcripts were up-regulated by Bt proteins (Cry1Ab and Cry1Ac). • Only three of the eleven transcripts were annotated. • The responses of 11 transcripts were tested on both Cry1Ab and Cry1Ac. • These transcripts did not respond to the Bt proteins in Bt-rice residues. -- Eleven potential molecular biomarkers of Folsomia candida to Cry1Ab and Cry1Ac were screened by microarray and qPCR analysis

  19. Is Candida albicans a trigger in the onset of coeliac disease?

    NARCIS (Netherlands)

    Nieuwenhuizen, W.F.; Pieters, R.H.H.; Knippels, L.M.J.; Jansen, M.C.J.F.; Koppelman, S.J.

    2003-01-01

    Coeliac disease is a T-cell-mediated autoimmune disease of the small intestine that is induced by ingestion of gluten proteins from wheat, barley, or rye. We postulate that Candida albicans is a trigger in the onset of coeliac disease. The virulence factor of C albicans - hyphal wall protein 1 (HWP1

  20. Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP-transgenic mouse as a model

    Directory of Open Access Journals (Sweden)

    Grossman Gail

    2005-12-01

    Full Text Available Abstract Background Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens. Methods Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction. Results Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips were up-regulated and 198 genes (1.59% were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1. Conclusion Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional

  1. Short peptides allowing preferential detection of Candida albicans hyphae.

    Science.gov (United States)

    Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

    2015-09-01

    Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

  2. Sap6, a secreted aspartyl proteinase, participates in maintenance the cell surface integrity of Candida albicans

    OpenAIRE

    Buu, Leh-Miauh; Chen, Yee-Chun

    2013-01-01

    Background The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases. R...

  3. Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis.

    Science.gov (United States)

    Tati, Swetha; Davidow, Peter; McCall, Andrew; Hwang-Wong, Elizabeth; Rojas, Isolde G; Cormack, Brendan; Edgerton, Mira

    2016-03-01

    Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

  4. Orally administered lactoperoxidase increases expression of the FK506 binding protein 5 gene in epithelial cells of the small intestine of mice: a DNA microarray study.

    Science.gov (United States)

    Wakabayashi, Hiroyuki; Miyauchi, Hirofumi; Shin, Kouichirou; Yamauchi, Koji; Matsumoto, Ichiro; Abe, Keiko; Takase, Mitsunori

    2007-09-01

    Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

  5. Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data

    Science.gov (United States)

    Raju, Hemalatha B.; Tsinoremas, Nicholas F.; Capobianco, Enrico

    2016-01-01

    Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein–protein

  6. Imaging morphogenesis of Candida albicans during infection in a live animal

    Science.gov (United States)

    Mitra, Soumya; Dolan, Kristy; Foster, Thomas H.; Wellington, Melanie

    2010-01-01

    Candida albicans is an opportunistic human fungal pathogen that requires an intact host immune response to prevent disease. Thus, studying host-pathogen interactions is critical to understanding and preventing this disease. We report a new model infection system in which ongoing C. albicans infections can be imaged at high spatial resolution in the ears of living mice. Intradermal inoculation into mouse ears with a C. albicans strain expressing green fluorescent protein results in systemic C. albicans infection that can be imaged in vivo using confocal microscopy. We observed filamentous growth of the organism in vivo as well as formation of microabscesses. This model system will allow us to gain significant new information about C. albicans pathogenesis through studies of host-C. albicans interactions in the native environment.

  7. Development of a high-throughput Candida albicans biofilm chip.

    Directory of Open Access Journals (Sweden)

    Anand Srinivasan

    Full Text Available We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B. Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.

  8. Candida albicans commensalism in the gastrointestinal tract.

    Science.gov (United States)

    Neville, B Anne; d'Enfert, Christophe; Bougnoux, Marie-Elisabeth

    2015-11-01

    Candida albicans is a polymorphic yeast species that often forms part of the commensal gastrointestinal mycobiota of healthy humans. It is also an important opportunistic pathogen. A tripartite interaction involving C. albicans, the resident microbiota and host immunity maintains C. albicans in its commensal form. The influence of each of these factors on C. albicans carriage is considered herein, with particular focus on the mycobiota and the approaches used to study it, models of gastrointestinal colonization by C. albicans, the C. albicans genes and phenotypes that are necessary for commensalism and the host factors that influence C. albicans carriage.

  9. Microarray-based detection and expression analysis of extracellular matrix proteins in drug‑resistant ovarian cancer cell lines.

    Science.gov (United States)

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Zabel, Maciej

    2014-11-01

    Ovarian cancer is the most lethal gynecological malignancy. Multiple drug resistance (MDR) development leads to resistance of cancer cells to chemotherapy. Microarray methods can provide information regarding new candidate genes that can play a role in resistance to cytostatic drugs. Extracellular matrix (ECM) can influence drug resistance by inhibiting the penetration of the drug into cancer tissue as well as increased apoptosis resistance. In the present study, we report changes in the ECM and related gene expression pattern in methotrexate-, cisplatin-, doxorubicin-, vincristine-, topotecan- and paclitaxel-resistant variants of the W1 ovarian cancer cell line. The resistant variants of the W1 cell line were generated by stepwise selection of cells with an increasing concentration of the indicated drugs. Affymetrix GeneChip® Human Genome U219 Array Strips were used for hybridizations. Independent t-tests were used to determinate the statistical significance of results. Genes whose expression levels were higher than the assumed threshold (upregulated, >5-fold and downregulated, 20-fold. These genes were: ITGB1BP3, COL3A1, COL5A2, COL15A1, TGFBI, DCN, LUM, MATN2, POSTN and EGFL6. The expression of seven genes decreased very significantly: ITGA1, COL1A2, LAMA2, GPC3, KRT23, VIT and HMCN1. The expression pattern of ECM and related genes provided the preliminary view into the role of ECM components in cytostatic drug resistance of cancer cells. The exact role of the investigated genes in drug resistance requires further investigation.

  10. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac).

    Science.gov (United States)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue; Roelofs, Dick; Chen, Fajun; Zhu-Salzman, Keyan; Liang, Yuyong; Sun, Yucheng; Ge, Feng

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues.

  11. The Stanford Tissue Microarray Database.

    Science.gov (United States)

    Marinelli, Robert J; Montgomery, Kelli; Liu, Chih Long; Shah, Nigam H; Prapong, Wijan; Nitzberg, Michael; Zachariah, Zachariah K; Sherlock, Gavin J; Natkunam, Yasodha; West, Robert B; van de Rijn, Matt; Brown, Patrick O; Ball, Catherine A

    2008-01-01

    The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license. PMID:17989087

  12. Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Yun Fei BAI; Qin Yu GE; Tong Xiang LI; Jin Ke WANG; Quan Jun LIU; Zu Hong LU

    2005-01-01

    The double-stranded DNA (dsDNA) probe contains two different protein binding sites.One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme.The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

  13. Skin Immunity to Candida albicans.

    Science.gov (United States)

    Kashem, Sakeen W; Kaplan, Daniel H

    2016-07-01

    Candida albicans is a dimorphic commensal fungus that colonizes healthy human skin, mucosa, and the reproductive tract. C. albicans is also a predominantly opportunistic fungal pathogen, leading to disease manifestations such as disseminated candidiasis and chronic mucocutaneous candidiasis (CMC). The differing host susceptibilities for the sites of C. albicans infection have revealed tissue compartmentalization with tailoring of immune responses based on the site of infection. Furthermore, extensive studies of host genetics in rare cases of CMC have identified conserved genetic pathways involved in immune recognition and the response to the extracellular pathogen. We focus here on human and mouse skin as a site of C. albicans infection, and we review established and newly discovered insights into the cellular pathways that promote cutaneous antifungal immunity. PMID:27178391

  14. Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer

    International Nuclear Information System (INIS)

    Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was

  15. Allium sativum (garlic) inhibits lipid synthesis by Candida albicans.

    OpenAIRE

    Adetumbi, M; Javor, G T; Lau, B H

    1986-01-01

    The effect of aqueous garlic extract on the macromolecular synthesis of Candida albicans was studied. Protein and nucleic acid syntheses were inhibited to the same extent as growth, but lipid synthesis was completely arrested. Blockage of lipid synthesis is likely an important component of the anticandidal activity of garlic.

  16. Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis.

    Science.gov (United States)

    Wolff, Claudia; Malinowsky, Katharina; Berg, Daniela; Schragner, Kerstin; Schuster, Tibor; Walch, Axel; Bronger, Holger; Höfler, Heinz; Becker, Karl-Friedrich

    2011-01-01

    The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

  17. Nocvel potential targets and related genes of transcription factor Caplp in Candida albicans 1

    Institute of Scientific and Technical Information of China (English)

    YahWANG; Yong-bingCAO; Xin-mingJIA; De-junWANG; ZhengXU; HuiSHEN; KangYING; Wan-shengCHEN; Yuan-yingJIANG

    2005-01-01

    AIM Capl p, encoded by CAP1 in Candida albicans, is highly homologous to Saccharomyces cerevisiae transcription factor Yapl p. It has been associated with tolerance to oxidative stress and resistance to a variety of toxicants previously. We used homemade microarray to reveal Capl p related genes in a broad spectrum as well as to lucubrate the functions of Capl p. METHODS Microarray analysis was used to identify differentially expressed genes between CAP1 deletion strain CJD21 and its parental strain CAI4. CAP1 over-expression strain was constructed to confirm the relationship between CAP1 and some differentially expressedgenes. Bioinformatics was applied to reveal promoters with Capl p binding site as well as the clusters of differentially expressed genes. RT-PCR and drug efflux analysis were used to lucubrate the functions of Caplp in Candida albicans.

  18. DNA Microarray Technique

    Directory of Open Access Journals (Sweden)

    Thakare SP

    2012-11-01

    Full Text Available DNA Microarray is the emerging technique in Biotechnology. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. It also includes screening and diagnostic applications. The DNA microarray hybridization applications include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs, and short tandem repeats (STRs. In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes.

  19. Morphological and physiological changes induced by contact-dependent interaction between Candida albicans and Fusobacterium nucleatum

    Science.gov (United States)

    Bor, Batbileg; Cen, Lujia; Agnello, Melissa; Shi, Wenyuan; He, Xuesong

    2016-01-01

    Candida albicans and Fusobacterium nucleatum are well-studied oral commensal microbes with pathogenic potential that are involved in various oral polymicrobial infectious diseases. Recently, we demonstrated that F. nucleatum ATCC 23726 coaggregates with C. albicans SN152, a process mainly mediated by fusobacterial membrane protein RadD and Candida cell wall protein Flo9. The aim of this study was to investigate the potential biological impact of this inter-kingdom interaction. We found that F. nucleatum ATCC 23726 inhibits growth and hyphal morphogenesis of C. albicans SN152 in a contact-dependent manner. Further analysis revealed that the inhibition of Candida hyphal morphogenesis is mediated via RadD and Flo9 protein pair. Using a murine macrophage cell line, we showed that the F. nucleatum-induced inhibition of Candida hyphal morphogenesis promotes C. albicans survival and negatively impacts the macrophage-killing capability of C. albicans. Furthermore, the yeast form of C. albicans repressed F. nucleatum-induced MCP-1 and TNFα production in macrophages. Our study suggests that the interaction between C. albicans and F. nucleatum leads to a mutual attenuation of virulence, which may function to promote a long-term commensal lifestyle within the oral cavity. This finding has significant implications for our understanding of inter-kingdom interaction and may impact clinical treatment strategies. PMID:27295972

  20. Streptococcus gordonii glucosyltransferase promotes biofilm interactions with Candida albicans

    Directory of Open Access Journals (Sweden)

    Austin Ricker

    2014-01-01

    Full Text Available Background: Candida albicans co-aggregates with Streptococcus gordonii to form biofilms and their interactions in mucosal biofilms may lead to pathogenic synergy. Although the functions of glucosyltransferases (Gtf of Mutans streptococci have been well characterized, the biological roles of these enzymes in commensal oral streptococci, such as S. gordonii, in oral biofilm communities are less clear. Objective: The objective of this work was to explore the role of GtfG, the single Gtf enzyme of S. gordonii, in biofilm interactions with C. albicans. Design: Biofilms were grown under salivary flow in flow cells in vitro, or under static conditions in 96 well plates. A panel of isogenic S. gordonii CH1 gtfG mutants and complemented strains were co-inoculated with C. albicans strain SC5314 to form mixed biofilms. Biofilm accretion and binding interactions between the two organisms were tested. Biofilms were quantified using confocal microscopy or the crystal violet assay. Results: The presence of GtfG enhanced dual biofilm accretion, and sucrose supplementation further augmented dual biofilm formation, pointing to a role of newly synthesized glucans. GtfG also promoted binding to C. albicans preformed biofilms. Soluble α-1,6-glucans played a role in these interactions since: 1 a strain producing only soluble glucans (CH107 formed robust dual biofilms under conditions of salivary flow; and 2 the dual biofilm was susceptible to enzymatic breakdown by dextranase which specifically degrades soluble α-1,6-glucans. Conclusion: Our work identified a novel molecular mechanism for C. albicans and S. gordonii biofilm interactions, mediated by GtfG. This protein promotes early biofilm binding of S. gordonii to C. albicans which leads to increased accretion of streptococcal cells in mixed biofilms. We also showed that soluble glucans, with α-1,6-linkages, promoted inter-generic adhesive interactions.

  1. O-mannosylation in Candida albicans enables development of interkingdom biofilm communities.

    Science.gov (United States)

    Dutton, Lindsay C; Nobbs, Angela H; Jepson, Katy; Jepson, Mark A; Vickerman, M Margaret; Aqeel Alawfi, Sami; Munro, Carol A; Lamont, Richard J; Jenkinson, Howard F

    2014-04-15

    Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. IMPORTANCE In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus. We find that

  2. Impact of protein supplementation and exercise in preventing changes in gene expression profiling in woman muscles after long-term bedrest as revealed by microarray analysis.

    Science.gov (United States)

    Chopard, Angele; Lecunff, Martine; Danger, Richard; Teusan, Raluca; Jasmin, Bernard J.; Marini, Jean-Francois; Leger, Jean

    Long duration space flights have a dramatic impact on human physiology and under such a condition, skeletal muscles are known to be one of the most affected systems. A thorough understanding of the basic mechanisms leading to muscle impairment under microgravity, which causes significant loss of muscle mass as well as structural disorders, is necessary for the development of efficient space flight countermeasures. This study was conducted under the aegis of the European Space Agency (ESA), the National Aeronautics and Space Administration of the USA (NASA), the Canadian Space Agency (CSA), and the French "Centre National d'Etudes Spatiales" (CNES). It gave us the opportunity to investigate for the first time the effects of prolonged disuse (long-term bedrest, LTBR) on the transcriptome of different muscle types in healthy women (control, n=8), as well as the potential beneficial impact of protein supplementation (nutrition, n=8) and a combined resistance and aerobic exercise training program (exercise, n=8). Pre- (LTBR -8) and post- (LTBR +59) biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles from each subject. Skeletal muscle gene expression profiles were obtained using a custom made microarray containing 6681 muscle-relevant genes. 555 differentiallyexpressed and statistically-significant genes were identified in control group following 60 days of LTBR, including 348 specific for SOL, 83 specific for VL, and 124 common for the two types of muscle (p<0.05). After LTBR, both muscle types exhibited a consistent decrease in pathways involved in fatty acid oxidation, ATP synthesis, and oxidative phosphorylation (p<0.05). However, the postural SOL muscle exhibited a higher level of changes with mRNA encoding proteins involved in protein synthesis and activation of protein degradation (mainly ubiquitinproteasome components) (p<0.05). Major changes in muscle function, such as those involved in calcium signaling and muscle structure including

  3. Candida albicans skin abscess Abscesso de pele por Candida albicans

    Directory of Open Access Journals (Sweden)

    Felipe Francisco Tuon

    2006-10-01

    Full Text Available Subcutaneous candidal abscess is a very rare infection even in immunocompromised patients. Some cases are reported when breakdown in the skin occurs, as bacterial cellulites or abscess, iatrogenic procedures, trauma and parenteral substance abuse. We describe a case of Candida albicans subcutaneous abscess without fungemia, which can be associated with central venous catheter.Abscesso subcutâneo por Candida é infecção muito rara mesmo em pacientes imunocomprometidos. Alguns casos são relatados quando ocorre dano na pele, como celulite bacteriana ou abscesso, procedimentos iatrogênicos, trauma e abuso de substância parenteral. Relatamos caso de abscesso subcutâneo por Candida albicans sem fungemia, que pode estar associado com cateter venoso central.

  4. Histone deacetylase-mediated morphological transition in Candida albicans.

    Science.gov (United States)

    Kim, Jueun; Lee, Ji-Eun; Lee, Jung-Shin

    2015-12-01

    Candida albicans is the most common opportunistic fungal pathogen, which switches its morphology from single-cell yeast to filament through the various signaling pathways responding to diverse environmental cues. Various transcriptional factors such as Nrg1, Efg1, Brg1, Ssn6, and Tup1 are the key components of these signaling pathways. Since C. albicans can regulate its transcriptional gene expressions using common eukaryotic regulatory systems, its morphological transition by these signaling pathways could be linked to the epigenetic regulation by chromatin structure modifiers. Histone proteins, which are critical components of eukaryotic chromatin structure, can regulate the eukaryotic chromatin structure through their own modifications such as acetylation, methylation, phosphorylation and ubiquitylation. Recent studies revealed that various histone modifications, especially histone acetylation and deacetylation, participate in morphological transition of C. albicans collaborating with well-known transcription factors in the signaling pathways. Here, we review recent studies about chromatin-mediated morphological transition of C. albicans focusing on the interaction between transcription factors in the signaling pathways and histone deacetylases.

  5. An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis

    OpenAIRE

    Chen, Changbin; Pande, Kalyan; French, Sarah D.; Tuch, Brian B.; Noble, Suzanne M.

    2011-01-01

    The mammalian gastrointestinal tract and bloodstream are highly disparate biological niches that differ in concentrations of nutrients such as iron. However, some commensal-pathogenic microorganisms, such as the yeast Candida albicans, thrive in both environments. We report the evolution of a transcription circuit in C. albicans that controls iron uptake and determines its fitness in both niches. Our analysis of DNA-binding proteins that regulate iron uptake by this organism suggests the evol...

  6. Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans.

    LENUS (Irish Health Repository)

    Jackson, Andrew P

    2009-12-01

    Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.

  7. Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

    Directory of Open Access Journals (Sweden)

    Romain Vauchelles

    Full Text Available The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP together with fluorescence loss in photobleaching (FLIP studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

  8. Tissue Microarrays for Analysis of Expression Patterns

    OpenAIRE

    Lindskog Bergström, Cecilia

    2013-01-01

    Proteins are essential building blocks in every living cell, and since the complete human genome was sequenced in 2004, researchers have attempted to map the human proteome, which is the functional representation of the genome. One such initiative is the Human Protein Atlas programme (HPA), which generates monospecific antibodies towards all human proteins and uses these for high-throughput tissue profiling on tissue microarrays (TMAs). The results are publically available at the website www....

  9. Development and application of protein microarray technology for serodiagnosis of Salmonella typhi and paratyphi%伤寒、副伤寒分型诊断蛋白质芯片法的建立和应用

    Institute of Scientific and Technical Information of China (English)

    彭杰雄; 林连成; 邓兆享; 林文浩; 裴春丽

    2012-01-01

    目的 探讨应用蛋白质芯片技术诊断伤寒、副伤寒的可行性.方法 利用蛋白质芯片方法,将伤寒抗原(To、Th)和副伤寒抗原(Ta、Tb、Tc)及伤寒Vi荚膜多糖抗原集成到已经活化处理的玻璃芯片上,使其保持蛋白质活性和立体结构不变.对86例经血培养确认的已知标本和100例健康体检标本进行检测,并与肥达氏反应作比较.结果 蛋白质芯片法检测结果与血培养结果符合率高,灵敏度为97.6%,特异度为98.0%,而肥达氏反应灵敏度为60.4%,特异度98.0%,两方法之间差异有统计学意义(P<0.01).结论 蛋白质芯片法具有高通量、可并行检测伤寒、副伤寒多种抗体的优势,为伤寒、副伤寒的诊断提供了有效手段.%Objective To evaluate a new protein microarray method for the diagnosis of typhoid and paratyphoid fever. Methods Salmonella typhi antigens To and Th,Salmonella paratyphi antigens Ta,Tb and Tc,and Vi capsular polysaccharide of Salmonella typhi were spotted on glass slides, which were modified with specific active groups. A total of 86 cases of known samples,confirmed by blood culture,and 100 cases of samples from healthy subjects were detected by constructed method, and the detection results were compared with those of Widal test. Results There was a high consistence between protein microarray and blood culture. The sensitivity and specificity of protein microarray were respectively 97. 6% and 98. 0% ,higher than 60. A% and 98. 0% of Widal test (Pprotein microarray technology might with more advantages than Widal test and blood culture,and it be with much potentials to apply in clinical diagnosis.

  10. Chemiluminescence microarrays in analytical chemistry: a critical review.

    Science.gov (United States)

    Seidel, Michael; Niessner, Reinhard

    2014-09-01

    Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

  11. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

    Science.gov (United States)

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon; Nile, Christopher

    2015-08-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

  12. Genetic organization and mRNA expression of enolase genes of Candida albicans.

    Science.gov (United States)

    Postlethwait, P; Sundstrom, P

    1995-04-01

    In previous work, we cloned a Candida albicans cDNA for the glycolytic enzyme enolase and found a single, abundant enolase transcript on Northern (RNA) blots and a single protein on immunoblots, using antiserum raised against a recombinant enolase fusion protein. Because C. albicans enolase is abundantly produced during infection and elicits strong host immune responses, the mechanisms regulating enolase production are important for understanding the growth of C. albicans in vivo. To obtain more information on enolase gene expression by C. albicans, we used the enolase cDNA clone to investigate the genetic organization of enolase genes and the steady-state levels of enolase mRNA under several growth conditions. Gene disruption techniques in combination with Southern blot analyses of genomic DNA showed the presence of two enolase gene loci that could be distinguished by the locations of ClaI and Mn/I sites in their 3' flanking regions. Enolase steady-state mRNA levels were greatest during the middle phase of the logarithmic growth curve and were low during stationary phase. Minimal differences in enolase mRNA levels between yeast cells and hyphae were found. Propagation of C. albicans in glucose did not cause increased enolase mRNA levels compared with growth in a nonfermentable carbon source (pyruvate). It was concluded that two gene loci exist for C. albicans enolase and that enolase mRNA is constitutively produced at high levels during active metabolism. PMID:7896700

  13. Global transcriptome sequencing identifies chlamydospore specific markers in Candida albicans and Candida dubliniensis.

    Directory of Open Access Journals (Sweden)

    Katja Palige

    Full Text Available Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2 which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens.

  14. Global Transcriptome Sequencing Identifies Chlamydospore Specific Markers in Candida albicans and Candida dubliniensis

    LENUS (Irish Health Repository)

    Palige, Katja

    2013-04-15

    Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens.

  15. Identification of Drosophila gene products required for phagocytosis of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Shannon L Stroschein-Stevenson

    2006-01-01

    Full Text Available Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr, shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps, and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli. Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis.

  16. Specific induction of fibronectin binding activity by hemoglobin in Candida albicans grown in defined media.

    Science.gov (United States)

    Yan, S; Nègre, E; Cashel, J A; Guo, N; Lyman, C A; Walsh, T J; Roberts, D D

    1996-08-01

    Fibronectin (FN) is a major component of host extracellular matrix that may play an important role in the initiation and dissemination of Candida albicans infections. Expression of FN binding requires growth of C albicans blastoconidia in complex medium, and the regulation of FN receptor expression is poorly understood. We now demonstrate that hemoglobin is a potent and specific inducer of FN receptor expression and describe a defined medium supplemented with hemoglobin that greatly and stably enhances the binding activity of C. albicans for soluble FN. Enhancement of FN binding by hemoglobin in strain 44807 was concentration dependent and was maximal at 0.1% hemoglobin with 20- to 80-fold enhancement. The hemoglobin-induced FN binding to C. albicans was saturable, with a Kd of 2.7 X 10(-8) M. Enhancement required growth of C. albicans in hemoglobin-containing medium, since simply exposing blastoconidia to hemoglobin in a nongrowing status did not enhance binding. Induction was reversible following removal of hemoglobin from the growth medium and not associated with germination. Inorganic or protein-bound iron was not sufficient for the induction, since other iron-containing proteins or inorganic iron salts were inactive. Growth in the simple medium yeast nitrogen base supplemented with hemoglobin increased cell adhesion to immobilized FN and to cultured monolayers of bovine corneal endothelial cells. These data suggest that hemoglobin may be an important regulator of FN binding activity in C. albicans and thus may play a role in its pathogenesis. PMID:8757815

  17. A comprehensive Candida albicans PeptideAtlas build enables deep proteome coverage.

    Science.gov (United States)

    Vialas, Vital; Sun, Zhi; Reales-Calderón, Jose A; Hernáez, María L; Casas, Vanessa; Carrascal, Montserrat; Abián, Joaquín; Monteoliva, Lucía; Deutsch, Eric W; Moritz, Robert L; Gil, Concha

    2016-01-10

    To provide new and expanded proteome documentation of the opportunistically pathogen Candida albicans, we have developed new protein extraction and analysis routines to provide a new, extended and enhanced version of the C. albicans PeptideAtlas. Two new datasets, resulting from experiments consisting of exhaustive subcellular fractionations and different growing conditions, plus two additional datasets from previous experiments on the surface and the secreted proteomes, have been incorporated to increase the coverage of the proteome. High resolution precursor mass spectrometry (MS) and ion trap tandem MS spectra were analyzed with three different search engines using a database containing allele-specific sequences. This approach, novel for a large-scale C. albicans proteomics project, was combined with the post-processing and filtering implemented in the Trans Proteomic Pipeline consistently used in the PeptideAtlas project and resulted in 49,372 additional peptides (3-fold increase) and 1630 more proteins (1.6-fold increase) identified in the new C. albicans PeptideAtlas with respect to the previous build. A total of 71,310 peptides and 4174 canonical (minimal non-redundant set) proteins (4115 if one protein per pair of alleles is considered) were identified representing 66% of the 6218 proteins in the predicted proteome. This makes the new PeptideAtlas build the most comprehensive C. albicans proteomics resource available and the only large-scale one with detections of individual alleles.

  18. Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium.

    Science.gov (United States)

    Moffa, Eduardo B; Mussi, Maria C M; Xiao, Yizhi; Garrido, Saulo S; Machado, Maria A A M; Giampaolo, Eunice T; Siqueira, Walter L

    2015-01-01

    Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37°C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37°C at 5% CO2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.

  19. Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics

    DEFF Research Database (Denmark)

    Reales-Calderón, Jose Antonio; Sylvester, Marc; Strijbis, Karin;

    2013-01-01

    Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We expose...

  20. Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions.

    Science.gov (United States)

    Santos, Jéssica Diane dos; Piva, Elisabete; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata and Candida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (10(7) cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a single C. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

  1. Design of a covalently bonded glycosphingolipid microarray

    DEFF Research Database (Denmark)

    Arigi, Emma; Blixt, Klas Ola; Buschard, Karsten;

    2012-01-01

    , a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G(M2)). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays......, the major classes of plant and fungal GSLs. In this work, a prototype "universal" GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release...

  2. Up-to-Date Applications of Microarrays and Their Way to Commercialization

    Directory of Open Access Journals (Sweden)

    Sarah Schumacher

    2015-04-01

    Full Text Available This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

  3. Up-to-Date Applications of Microarrays and Their Way to Commercialization.

    Science.gov (United States)

    Schumacher, Sarah; Muekusch, Sandra; Seitz, Harald

    2015-04-23

    This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

  4. Combining Affymetrix microarray results

    Directory of Open Access Journals (Sweden)

    Doerge RW

    2005-03-01

    Full Text Available Abstract Background As the use of microarray technology becomes more prevalent it is not unusual to find several laboratories employing the same microarray technology to identify genes related to the same condition in the same species. Although the experimental specifics are similar, typically a different list of statistically significant genes result from each data analysis. Results We propose a statistically-based meta-analytic approach to microarray analysis for the purpose of systematically combining results from the different laboratories. This approach provides a more precise view of genes that are significantly related to the condition of interest while simultaneously allowing for differences between laboratories. Of particular interest is the widely used Affymetrix oligonucleotide array, the results of which are naturally suited to a meta-analysis. A simulation model based on the Affymetrix platform is developed to examine the adaptive nature of the meta-analytic approach and to illustrate the usefulness of such an approach in combining microarray results across laboratories. The approach is then applied to real data involving a mouse model for multiple sclerosis. Conclusion The quantitative estimates from the meta-analysis model tend to be closer to the "true" degree of differential expression than any single lab. Meta-analytic methods can systematically combine Affymetrix results from different laboratories to gain a clearer understanding of genes' relationships to specific conditions of interest.

  5. Microarray technology and its applications

    CERN Document Server

    Müller, UR

    2006-01-01

    It presents detailed overviews of the different techniques of fabricating microarrays, of the chemistries and preparative steps involved, of the different types of microarrays, and of the instrumentation and optical issues involved.

  6. Innate Immunity and Saliva in Candida albicans-mediated Oral Diseases.

    Science.gov (United States)

    Salvatori, O; Puri, S; Tati, S; Edgerton, M

    2016-04-01

    The oral cavity is a unique niche where Candida albicans infections occur in immunocompetent as well as immunosuppressed individuals. Here we critically review the significance of human innate immune response in preventing oral candidiasis. One important line of defense against oropharyngeal candidiasis is the oral microbiota that prevents infection by competing for space and nutrients as well as by secreting antagonistic molecules and triggering local inflammatory responses. C. albicans is able to induce mucosal defenses through activation of immune cells and production of cytokines. Also, saliva contains various proteins that affect C. albicans growth positively by promoting mucosal adherence and negatively through immune exclusion and direct fungicidal activity. We further discuss the role of saliva in unifying host innate immune defenses against C. albicans as a communicating medium and how C. albicans overgrowth in the oral cavity may be a result of aberrations ranging from microbial dysbiosis and salivary dysfunction to epithelial damage. Last we underscore select oral diseases in which C. albicans is a contributory microorganism in immune-competent individuals.

  7. Expression of centromere protein F (CENP-F associated with higher FDG uptake on PET/CT, detected by cDNA microarray, predicts high-risk patients with primary breast cancer

    Directory of Open Access Journals (Sweden)

    Ishida Jiro

    2008-12-01

    Full Text Available Abstract Background Higher standardized uptake value (SUV detected by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT correlates with proliferation of primary breast cancer. The purpose of this study is to identify specific molecules upregulated in primary breast cancers with a high SUV and to examine their clinical significance. Methods We compared mRNA expression profiles between 14 tumors with low SUVs and 24 tumors with high SUVs by cDNA microarray. We identified centromere protein F (CENP-F and CDC6 were upregulated in tumors with high SUVs. RT-PCR and immunohistochemical analyses were performed to validate these data. Clinical implication of CENP-F and CDC6 was examined for 253 archival breast cancers by the tissue microarray. Results The relative ratios of CENP-F and CDC6 expression levels to β-actin were confirmed to be significantly higher in high SUV tumors than in low SUV tumors (p = 0.027 and 0.025, respectively by RT-PCR. In immunohistochemical analysis of 47 node-negative tumors, the CENP-F expression was significantly higher in the high SUV tumors (74% than the low SUV tumors (45% (p = 0.04, but membranous and cytoplasmic CDC6 expressions did not significantly differ between both groups (p = 0.9 each. By the tissue microarray, CENP-F (HR = 2.94 as well as tumor size (HR = 4.49, nodal positivity (HR = 4.1, and Ki67 (HR = 2.05 showed independent impact on the patients' prognosis. Conclusion High CENP-F expression, correlated with high SUV, was the prognostic indicators of primary breast cancer. Tumoral SUV levels may serve as a pretherapeutic indicator of aggressiveness of breast cancer.

  8. Mechanisms of the antifungal action of marine metagenome-derived peptide, MMGP1, against Candida albicans.

    Directory of Open Access Journals (Sweden)

    Muthuirulan Pushpanathan

    Full Text Available BACKGROUND: Development of resistant variants to existing antifungal drugs continues to be the serious problem in Candida albicans-induced fungal pathogenesis, which has a considerable impact on animal and human health. Identification and characterization of newer drugs against C. albicans is, therefore, essential. MMGP1 is a direct cell-penetrating peptide recently identified from marine metagenome, which was found to possess potent antifungal activity against C. albicans. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of antifungal action of MMGP1 against C. albicans. Agarose gel shift assay found the peptide to be having a remarkable DNA-binding ability. The modification of the absorption spectra and fluorescence quenching of the tryptophyl residue correspond to the stacking between indole ring and nucleotide bases. The formation of peptide-DNA complexes was confirmed by fluorescence quenching of SYTO 9 probe. The interaction of peptide with plasmid DNA afforded protection of DNA from enzymatic degradation by DNase I. In vitro transcription of mouse β-actin gene in the presence of peptide led to a decrease in the level of mRNA synthesis. The C. albicans treated with MMGP1 showed strong inhibition of biosynthetic incorporation of uridine analog 5-ethynyluridine (EU into nascent RNA, suggesting the peptide's role in the inhibition of macromolecular synthesis. Furthermore, the peptide also induces endogenous accumulation of reactive oxygen species (ROS in C. albicans. MMGP1 supplemented with glutathione showed an increased viability of C. albicans cells. The hyper-produced ROS by MMGP1 leads to increased levels of protein carbonyls and thiobarbituric acid reactive substances and it also causes dissipation of mitochondrial membrane potential and DNA fragmentation in C. albicans cells. CONCLUSION: And Significance: Therefore, the antifungal activity of MMGP1 could be attributed to its binding with DNA, causing

  9. Iron restriction-induced adaptations in the wall proteome of Candida albicans

    NARCIS (Netherlands)

    A.G. Sorgo; S. Brul; C.G. de Koster; L.J. de Koning; F.M. Klis

    2013-01-01

    The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized GPI-proteins, it can exploit iron from host hemoglobin, ferritin, and transferrin. Culturing C. al

  10. A Human-Curated Annotation of the Candida albicans Genome.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

  11. Navigating public microarray databases.

    Science.gov (United States)

    Penkett, Christopher J; Bähler, Jürg

    2004-01-01

    With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources. PMID:18629145

  12. Coaggregation of Candida albicans, Actinomyces naeslundii and Streptococcus mutans is Candida albicans strain dependent.

    Science.gov (United States)

    Arzmi, Mohd Hafiz; Dashper, Stuart; Catmull, Deanne; Cirillo, Nicola; Reynolds, Eric C; McCullough, Michael

    2015-08-01

    Microbial interactions are necessarily associated with the development of polymicrobial oral biofilms. The objective of this study was to determine the coaggregation of eight strains of Candida albicans with Actinomyces naeslundii and Streptococcus mutans. In autoaggregation assays, C. albicans strains were grown in RPMI-1640 and artificial saliva medium (ASM) whereas bacteria were grown in heart infusion broth. C. albicans, A. naeslundii and S. mutans were suspended to give 10(6), 10(7) and 10(8) cells mL(-1) respectively, in coaggregation buffer followed by a 1 h incubation. The absorbance difference at 620 nm (ΔAbs) between 0 h and 1 h was recorded. To study coaggregation, the same protocol was used, except combinations of microorganisms were incubated together. The mean ΔAbs% of autoaggregation of the majority of RPMI-1640-grown C. albicans was higher than in ASM grown. Coaggregation of C. albicans with A. naeslundii and/or S. mutans was variable among C. albicans strains. Scanning electron microscopy images showed that A. naeslundii and S. mutans coaggregated with C. albicans in dual- and triculture. In conclusion, the coaggregation of C. albicans, A. naeslundii and S. mutans is C. albicans strain dependent. PMID:26054855

  13. Carbohydrate Microarrays in Plant Science

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik; Pedersen, H.L.; Vidal-Melgosa, S.;

    2012-01-01

    Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important...... industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high...... for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities....

  14. Top-down characterization data on the speciation of the Candida albicans immunome in candidemia.

    Science.gov (United States)

    Pitarch, Aida; Nombela, César; Gil, Concha

    2016-03-01

    The characterization of pathogen-specific antigenic proteins at the protein species level is crucial in the development and molecular optimization of novel immunodiagnostics, vaccines or immunotherapeutics for infectious diseases. The major requirements to achieve this molecular level are to obtain 100% sequence coverage and identify all post-translational modifications of each antigenic protein species. In this article, we show nearly complete sequence information for five discrete antigenic species of Candida albicans Tdh3 (glyceraldehyde-3-phosphate dehydrogenase), which have been reported to be differentially recognized both among candidemia patients and between candidemia and control patients. A comprehensive description of the top-down immunoproteomic strategy used for seroprofiling at the C. albicans protein species level in candidemia as well as for the chemical characterization of this immunogenic protein (based on high-resolution 2-DE, Western blotting, peptide mass fingerprinting, tandem mass spectrometry and de novo peptide sequencing) is also provided. The top-down characterization data on the speciation of the C. albicans immunome in candidemia presented here are related to our research article entitled "Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia" (Pitarch et al., J. Proteomics, 2015, http://dx.doi.org/10.1016/j.jprot.2015.10.022). PMID:26862568

  15. Top-down characterization data on the speciation of the Candida albicans immunome in candidemia

    Directory of Open Access Journals (Sweden)

    Aida Pitarch

    2016-03-01

    Full Text Available The characterization of pathogen-specific antigenic proteins at the protein species level is crucial in the development and molecular optimization of novel immunodiagnostics, vaccines or immunotherapeutics for infectious diseases. The major requirements to achieve this molecular level are to obtain 100% sequence coverage and identify all post-translational modifications of each antigenic protein species. In this article, we show nearly complete sequence information for five discrete antigenic species of Candida albicans Tdh3 (glyceraldehyde-3-phosphate dehydrogenase, which have been reported to be differentially recognized both among candidemia patients and between candidemia and control patients. A comprehensive description of the top-down immunoproteomic strategy used for seroprofiling at the C. albicans protein species level in candidemia as well as for the chemical characterization of this immunogenic protein (based on high-resolution 2-DE, Western blotting, peptide mass fingerprinting, tandem mass spectrometry and de novo peptide sequencing is also provided. The top-down characterization data on the speciation of the C. albicans immunome in candidemia presented here are related to our research article entitled “Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia” (Pitarch et al., J. Proteomics, 2015, http://dx.doi.org/10.1016/j.jprot.2015.10.022.

  16. In vitro modification of Candida albicans invasiveness.

    Science.gov (United States)

    Fontenla de Petrino, S E; de Jorrat, M E; Sirena, A; Valdez, J C; Mesón, O

    1986-05-01

    Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability. The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities. The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr. Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.

  17. Adaptive immune responses to Candida albicans infection.

    Science.gov (United States)

    Richardson, Jonathan P; Moyes, David L

    2015-01-01

    Fungal infections are becoming increasingly prevalent in the human population and contribute to morbidity and mortality in healthy and immunocompromised individuals respectively. Candida albicans is the most commonly encountered fungal pathogen of humans, and is frequently found on the mucosal surfaces of the body. Host defense against C. albicans is dependent upon a finely tuned implementation of innate and adaptive immune responses, enabling the host to neutralise the invading fungus. Central to this protection are the adaptive Th1 and Th17 cellular responses, which are considered paramount to successful immune defense against C. albicans infections, and enable tissue homeostasis to be maintained in the presence of colonising fungi. This review will highlight the recent advances in our understanding of adaptive immunity to Candida albicans infections.

  18. The ABCs of Candida albicans Multidrug Transporter Cdr1.

    Science.gov (United States)

    Prasad, Rajendra; Banerjee, Atanu; Khandelwal, Nitesh Kumar; Dhamgaye, Sanjiveeni

    2015-12-01

    In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms. PMID:26407965

  19. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

    OpenAIRE

    Netea, M.G.; Gow, N.A.; Munro, C.A.; S. Bates; Collins, C; Ferwerda, G.; HOBSON, R. P.; Bertram, G; Hughes, H.B.; Jansen, T.; Jacobs, L; Buurman, E.T.; Gijzen, K.; Williams, D. L.; Torensma, R.

    2006-01-01

    The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of β-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by ...

  20. CalPFl4030 negatively modulates intracellular ATP levels during the development of azole resistance in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    Xin-ming JIA; Ying WANG; Jun-dong ZHANG; Hong-yue TAN; Yuan-yingJIANG; Jun GU

    2011-01-01

    Aim:Widespread and repeated use of azoles, particularly fiuconazole, has led to the rapid development of azole resistance in Candida albicans.We investigated the role of CalPF14030 during the development of azole resistance in C albicans.Methods:The expression of CalPF14030 was measured by quantitative RT-PCR, and CalPF14030 was disrupted by the hisG-URA3-hisG(URA-blaster)method.The sensitivity of C albicans to azoles was examined using a spot assay, and the intracellular ATP concentrations were measured by a luminometer.Results:CalPF14030 expression in C albicans was up-regulated by Ca2+ in a calcineurin-dependent manner, and the protein was overexpressed during the stepwise acquisition of azole resistance.However,disruption or ectopic overexpression of CalPFl4030 did not affect the sensitivity of C albicans to azoles.Finally,we demonstrated that disruption of CalPFll4030 significantly increased intracellular ATP levels.and overexpression significantly decreased intracellular ATP levels jn C albicans.Conclusion:CalPF14030 may negatively modulate intracellular ATP levels during the development of azole resistance in C albicans.

  1. Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

    Directory of Open Access Journals (Sweden)

    Manish Biyani

    2015-07-01

    Full Text Available Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density, ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.

  2. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses.

    Science.gov (United States)

    Abd El-Rehim, Dalia M; Ball, Graham; Pinder, Sarah E; Rakha, Emad; Paish, Claire; Robertson, John F R; Macmillan, Douglas; Blamey, Roger W; Ellis, Ian O

    2005-09-01

    Recent studies on gene molecular profiling using cDNA microarray in a relatively small series of breast cancer have identified biologically distinct groups with apparent clinical and prognostic relevance. The validation of such new taxonomies should be confirmed on larger series of cases prior to acceptance in clinical practice. The development of tissue microarray (TMA) technology provides methodology for high-throughput concomitant analyses of multiple proteins on large numbers of archival tumour samples. In our study, we have used immunohistochemistry techniques applied to TMA preparations of 1,076 cases of invasive breast cancer to study the combined protein expression profiles of a large panel of well-characterized commercially available biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and gene products known to be altered in some forms of breast cancer. Using hierarchical clustering methodology, 5 groups with distinct patterns of protein expression were identified. A sixth group of only 4 cases was also identified but deemed too small for further detailed assessment. Further analysis of these clusters was performed using multiple layer perceptron (MLP)-artificial neural network (ANN) with a back propagation algorithm to identify key biomarkers driving the membership of each group. We have identified 2 large groups by their expression of luminal epithelial cell phenotypic characteristics, hormone receptors positivity, absence of basal epithelial phenotype characteristics and lack of c-erbB-2 protein overexpression. Two additional groups were characterized by high c-erbB-2 positivity and negative or weak hormone receptors expression but showed differences in MUC1 and E-cadherin expression. The final group was characterized by strong basal epithelial characteristics, p53 positivity, absent hormone receptors and weak to low luminal epithelial cytokeratin expression. In addition, we have identified significant

  3. Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2006-12-07

    Protein arrays are ideal tools for the rapid analysis of whole proteomes as well as for the development of reliable and cheap biosensors. The objective of this proposal is to develop a new ligand assisted ligation method based in the naturally occurring protein trans-splicing process. This method has been used for the generation of spatially addressable arrays of multiple protein components by standard micro-lithographic techniques. Key to our approach is the use of the protein trans-splicing process. This naturally occurring process allows the development of a truly generic and highly efficient method for the covalent attachment of proteins through its C-terminus to any solid support. This technology has been used for the creation of protein chips containing several virulence factors from the human pathogen Y. pestis.

  4. Proteolytic activity and cytokine up-regulation by non-albicans Candida albicans.

    Science.gov (United States)

    Nawaz, Ali; Pärnänen, Pirjo; Kari, Kirsti; Meurman, Jukka H

    2015-05-01

    Mouth is an important source of infections and oral infections such as Candida infections increase the risk of mortality. Our purpose was to investigate differences in proteolytic activity of non-albicans Candida albicans (non-albicans Candida) between clinical isolates and laboratory samples. The second aim was to assess the concentration of pro- and anti-inflammatory cytokine levels IL-1β, IL-10, and TNF-α in saliva of patients with the non-albicans Candida and Candida-negative saliva samples. Clinical yeast samples from our laboratory were used for analyses. Candida strains were grown in YPG at 37 °C for 24 h in water bath with shaking. The activity of Candida proteinases of cell and cell-free fractions were analyzed by MDPF-gelatin zymography. The levels of IL-1β, IL-10, and TNF-α were measured from saliva with ELISA. The study showed differences in the proteolytic activity among the non-albicans Candida strains. C. tropicalis had higher proteolytic activity when compared to the other strains. Significant difference was found in salivary IL-1β levels between the non-albicans Candida and control strains (P albicans Candida strains. The increased IL-1β concentration may be one of the host response components associated with non-albicans Candida infection.

  5. Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species.

    Science.gov (United States)

    Whibley, Natasha; Gaffen, Sarah L

    2015-11-01

    The fungal genus Candida encompasses numerous species that inhabit a variety of hosts, either as commensal microbes and/or pathogens. Candida species are a major cause of fungal infections, yet to date there are no vaccines against Candida or indeed any other fungal pathogen. Our knowledge of immunity to Candida mainly comes from studies on Candida albicans, the most frequent species associated with disease. However, non-albicans Candida (NAC) species also cause disease and their prevalence is increasing. Although research into immunity to NAC species is still at an early stage, it is becoming apparent that immunity to C. albicans differs in important ways from non-albicans species, with important implications for treatment, therapy and predicted demographic susceptibility. This review will discuss the current understanding of immunity to NAC species in the context of immunity to C. albicans, and highlight as-yet unanswered questions.

  6. Antifungal activity, kinetics and molecular mechanism of action of garlic oil against Candida albicans.

    Science.gov (United States)

    Li, Wen-Ru; Shi, Qing-Shan; Dai, Huan-Qin; Liang, Qing; Xie, Xiao-Bao; Huang, Xiao-Mo; Zhao, Guang-Ze; Zhang, Li-Xin

    2016-01-01

    The antifungal activity, kinetics, and molecular mechanism of action of garlic oil against Candida albicans were investigated in this study using multiple methods. Using the poisoned food technique, we determined that the minimum inhibitory concentration of garlic oil was 0.35 μg/mL. Observation by transmission electron microscopy indicated that garlic oil could penetrate the cellular membrane of C. albicans as well as the membranes of organelles such as the mitochondria, resulting in organelle destruction and ultimately cell death. RNA sequencing analysis showed that garlic oil induced differential expression of critical genes including those involved in oxidation-reduction processes, pathogenesis, and cellular response to drugs and starvation. Moreover, the differentially expressed genes were mainly clustered in 19 KEGG pathways, representing vital cellular processes such as oxidative phosphorylation, the spliceosome, the cell cycle, and protein processing in the endoplasmic reticulum. In addition, four upregulated proteins selected after two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis were identified with high probability by mass spectrometry as putative cytoplasmic adenylate kinase, pyruvate decarboxylase, hexokinase, and heat shock proteins. This is suggestive of a C. albicans stress responses to garlic oil treatment. On the other hand, a large number of proteins were downregulated, leading to significant disruption of the normal metabolism and physical functions of C. albicans. PMID:26948845

  7. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    DEFF Research Database (Denmark)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue;

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida...

  8. Transcriptomics Analysis of Candida albicans Treated with Huanglian Jiedu Decoction Using RNA-seq

    Directory of Open Access Journals (Sweden)

    Qianqian Yang

    2016-01-01

    Full Text Available Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often life-threatening. Resistance of C. albicans to antifungal agents and limited antifungal agents has potentially serious implications for management of infections. As a famous multiherb prescription in China, Huanglian Jiedu Decoction (HLJJD, Orengedokuto in Japan is efficient against Trichophyton mentagrophytes and C. albicans. But the antifungal mechanism of HLJDD remains unclear. In this study, by using RNA-seq technique, we performed a transcriptomics analysis of gene expression changes for C. albicans under the treatment of HLJDD. A total of 6057 predicted protein-encoding genes were identified. By gene expression analysis, we obtained a total of 735 differentially expressed genes (DEGs, including 700 upregulated genes and 35 downregulated genes. Genes encoding multidrug transporters such as ABC transporter and MFS transporter were identified to be significantly upregulated. Meanwhile, by pathway enrichment analysis, we identified 26 significant pathways, in which pathways of DNA replication and transporter activity were mainly involved. These results might provide insights for the inhibition mechanism of HLJDD against C. albicans.

  9. Compressive Sensing DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Richard G. Baraniuk

    2009-01-01

    Full Text Available Compressive sensing microarrays (CSMs are DNA-based sensors that operate using group testing and compressive sensing (CS principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed.

  10. Wild-type Drosophila melanogaster as a model host to analyze nitrogen source dependent virulence of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Monica M Davis

    Full Text Available The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5 activates two transcription factors (Stp1 and Stp2 to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively. Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.

  11. DNA Microarray-Based Diagnostics.

    Science.gov (United States)

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  12. DNA Microarray-Based Diagnostics.

    Science.gov (United States)

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  13. The parasexual lifestyle of Candida albicans.

    Science.gov (United States)

    Bennett, Richard J

    2015-12-01

    Candida albicans is both a prevalent human commensal and the most commonly encountered human fungal pathogen. This lifestyle is dependent on the ability of the fungus to undergo rapid genetic and epigenetic changes, often in response to specific environmental cues. A parasexual cycle in C. albicans has been defined that includes several unique properties when compared to the related model yeast, Saccharomyces cerevisiae. Novel features include strict regulation of mating via a phenotypic switch, enhanced conjugation within a sexual biofilm, and a program of concerted chromosome loss in place of a conventional meiosis. It is expected that several of these adaptations co-evolved with the ability of C. albicans to colonize the mammalian host.

  14. Candida albicans escapes from mouse neutrophils

    DEFF Research Database (Denmark)

    Ermert, David; Niemiec, Maria J; Röhm, Marc;

    2013-01-01

    is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed......, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans...

  15. Design, construction, characterization, and application of a hyperspectral microarray scanner.

    Science.gov (United States)

    Sinclair, Michael B; Timlin, Jerilyn A; Haaland, David M; Werner-Washburne, Margaret

    2004-04-01

    We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research. The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 microm and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray. This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments. Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate. We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media.

  16. Cell-Based Microarrays for In Vitro Toxicology

    Science.gov (United States)

    Wegener, Joachim

    2015-07-01

    DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

  17. Bruton's Tyrosine Kinase (BTK and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

    Directory of Open Access Journals (Sweden)

    Karin Strijbis

    Full Text Available Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

  18. Candida albicans adhesion to composite resin materials.

    Science.gov (United States)

    Bürgers, Ralf; Schneider-Brachert, Wulf; Rosentritt, Martin; Handel, Gerhard; Hahnel, Sebastian

    2009-09-01

    The adhesion of Candida albicans to dental restorative materials in the human oral cavity may promote the occurrence of oral candidosis. This study aimed to compare the susceptibility of 14 commonly used composite resin materials (two compomers, one ormocer, one novel silorane, and ten conventional hybrid composites) to adhere Candida albicans. Differences in the amount of adhering fungi should be related to surface roughness, hydrophobicity, and the type of matrix. Cylindrical specimens of each material were made according to the manufacturers' instructions. Surface roughness R (a) was assessed by perthometer measurements and the degree of hydrophobicity by computerized contact angle analysis. Specimens were incubated with a reference strain of C. albicans (DMSZ 1386), and adhering fungi were quantified by using a bioluminometric assay in combination with an automated plate reader. Statistical differences were analyzed by the Kruskal-Wallis test and Mann-Whitney U test. Spearman's rank correlation coefficients were calculated to assess correlations. Median R (a) of the tested composite resin materials ranged between 0.04 and 0.23 microm, median contact angles between 69.2 degrees and 86.9 degrees . The two compomers and the ormocer showed lower luminescence intensities indicating less adhesion of fungi than all tested conventional hybrid composites. No conclusive correlation was found between surface roughness, hydrophobicity, and the amount of adhering C. albicans.

  19. The Role of Mms22p in DNA Damage Response in Candida albicans.

    Science.gov (United States)

    Yan, Lan; Xiong, Juan; Lu, Hui; Lv, Quan-zhen; Ma, Qian-yao; Côte, Pierre; Whiteway, Malcolm; Jiang, Yuan-ying

    2015-12-01

    To ensure correct DNA replication, eukaryotes have signaling pathways that respond to replication-associated DNA damage and trigger repair. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, a complex of proteins, including the cullin protein Rtt101p and two adapter proteins Mms22p and Mms1p, is important for proper response to replication stress. We have investigated this system in Candida albicans. In this pathogen, Mms22p is important for recovery from DNA replication damage induced by agents including methylmethane sulfonate, camptothecin, and ionizing radiation. Although no clear ortholog of Mms1p has been identified in C. albicans, loss of either Mms22p or Rtt101p generates similar damage sensitivity, consistent with a common function. In S. cerevisiae, the Mrc1p-Csm3p-Tof1p complex stabilizes stalled replication forks and activates a replication checkpoint and interacts with Mms22p. A similar complex in S. pombe, consisting of the Tof1p and Csm3p orthologs Swi1p and Swi3p, along with the fission yeast Mrc1p, genetically also interacts with Mms22p. Intriguingly in C. albicans only Mrc1p and Csm3p appear involved in damage repair, and Mms22p is required for responding to DNA damage agents in MRC1 or CSM3 conditional mutants. In C. albicans, although the loss of RAD57 greatly impairs response in the pathogen to many DNA-damaging agents, lethality due to camptothecin damage requires concomitant loss of Rad57p and Mms22p, suggesting that Mms22p is only essential for homologous recombination induced by camptothecin. These results establish that although C. albicans uses conserved cellular modules to respond to DNA damage and replication blocks, the specific details of these modules differ significantly from the S. cerevisiae model.

  20. Microarray Scanner for Fluorescence Detection

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel pseudo confocal microarray scanner is introduced, in which one dimension scanning is performed by a galvanometer optical scanner and a telecentric objective, another dimension scanning is performed by a stepping motor.

  1. Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

    Directory of Open Access Journals (Sweden)

    Peng Huiru

    2011-04-01

    Full Text Available Abstract Background Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat. Results In this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress. Conclusion Our results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future.

  2. Hubungan Kadar Glukosa Darah dengan Pertumbuhan Candida Albicans pada Penderita Diabetes Mellitus

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    Sri Hernawati

    2015-10-01

    Full Text Available Diabetes mellitus is comma only hereditary metabolic disorder. The signs were hyperglycemic and glucosuric with or without acute or chronic clinically symptoms. It was cause effectively insulin deficiency. The primary was carbohydrate metabolism disorder which followed lipid and protein metabolism disorders. The increase of boold. Glucose conentration could increase salivary glucose concentration. Glucose was a good media for the growth of microorganism, for example: candida albicans. The most frequently infection on oral mucous diabetes mellitus patients was candidacies. The purpose of the study was to determine the relation of blood glucose concentration and C. albicans growth on oral cavity diabetes mellitus patients. The subject consisted of 8 non regulated diabetes mellitus, 8 regulated diabetes mellitus, and 8 normal patients, respectively. The assessment of blood glucose concentration used Bio-Rad Diastat Halmoglobine A1c method. The growth of C. albicans was determined using swab on oral mucous. The result of swab was into culated on sabaurond agar, than gram stining and glucose test was done. Data was analyzed using spearman test. The result indicated that the growth of C. albicans was eughen on non regulated diabetes mellitus than regulated diabetes mellitus. It's also on regulated diabetes mellitus that normal patient.

  3. Comparison of the hemolytic activity between C. albicans and non-albicans Candida species.

    Science.gov (United States)

    Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2013-01-01

    The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37 ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

  4. The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

    OpenAIRE

    Lamb, David C.; Kelly, Diane E.; White, Theodore C.; Kelly, Steven L.

    2000-01-01

    The cytochrome P450 sterol 14α-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum ...

  5. Development of DNA probes for Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  6. Development of DNA probes for Candida albicans

    International Nuclear Information System (INIS)

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis

  7. Phage displaying epitope of Candida albicans HSP90 and serodiagnosis

    Institute of Scientific and Technical Information of China (English)

    杨琼; 王丽; 卢大宁; 邢沈阳; 尹东; 朱筱娟

    2004-01-01

    @@ Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused an increase in the frequency of life-threatening systemic candidiasis.1 Studies by Matthews et al2 indicated HSP90 fragments are major targets for the immune system in infection due to C. albicans, and anti-epitope LKVIRK of HSP90 antibody is a serological marker for diagnosis of invasive candidiasis. Cloning and sequencing HSP90 antigen revealed that the linear epitope LKVIRK, localized near the C-terminus of the 47 kDa protein which circulates in the sera of patients with invasive candidiasis, as a heat-stable breakdown product of large more heat-labile antigen HSP90.2 In this study, epitope LKVIRK was displayed on the surface of phage fd to develop a new serological test for systemic candidiasis.

  8. White-opaque switching in Candida albicans

    OpenAIRE

    Lohse, Matthew B.; Johnson, Alexander D.

    2009-01-01

    The human commensal yeast Candida albicans undergoes an epigenetic switch between two distinct types of cells, referred to as white and opaque. These two cell types differ in many respects, including their cell and colony morphologies, their metabolic states, their mating behaviors, their preferred niches in the host, and their interactions with the host immune system. Each of the two cell types is heritable for many generations and switching between them appears stochastic; however, environm...

  9. Tetracycline Effects on Candida Albicans Virulence Factors

    OpenAIRE

    Logan McCool; Hanh Mai; Michael Essmann; Bryan Larsen

    2008-01-01

    Object. To determine if tetracycline, previously reported to increase the probability of developing symptomatic vaginal yeast infections, has a direct effect on Candida albicans growth or induction of virulent phenotypes. Method. In vitro, clinical isolates of yeast were cultivated with sublethal concentrations of tetracycline and yeast cell counts, hyphal formation, drug efflux pump activity, biofilm production, and hemolysin production were determined by previously reported methods. Resul...

  10. Humoral immunity links Candida albicans infection and celiac disease.

    Directory of Open Access Journals (Sweden)

    Marion Corouge

    Full Text Available The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI may be a triggering factor for Celiac disease (CeD onset. We investigated cross-immune reactivity between CeD and CI.Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC. IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1.CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004 and anti-gliadin (p=0.002 and p=0.0009 antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039. During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides.Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

  11. The fungus Candida albicans tolerates ambiguity at multiple codons

    Directory of Open Access Journals (Sweden)

    João Salvador Simões

    2016-03-01

    Full Text Available The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and other warm-blooded animals. It occurs in a broad range of body sites and has high capacity to survive and proliferate in adverse environments with drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients and temperature. Its biology is unique due to flexible reassignment of the leucine CUG codon to serine and synthesis of statistical proteins. Under standard growth conditions 3% of leucine and 97% of serine are incorporated at CUG sites on a proteome wide scale, but leucine incorporation fluctuates in response to environmental stressors and can be artificially increased up to 98%. In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons. Expression of these tRNAs had minor effects on fitness, but growth of the mistranslating strains at different temperatures, in medium with different pH and nutrients composition was often enhanced relatively to the wild type (WT strain, supporting our previous data on adaptive roles of CUG ambiguity in variable growth conditions. Parallel evolution of the recombinant strains (100 generations followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP and one SNP in the deneddylase (JAB1 gene in all strains. Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.

  12. Humoral Immunity Links Candida albicans Infection and Celiac Disease

    Science.gov (United States)

    Fradin, Chantal; Salleron, Julia; Damiens, Sébastien; Moragues, Maria Dolores; Souplet, Vianney; Jouault, Thierry; Robert, Raymond; Dubucquoi, Sylvain; Sendid, Boualem; Colombel, Jean Fréderic; Poulain, Daniel

    2015-01-01

    Objective The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. Methods Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. Results CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides. Conclusions Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals. PMID:25793717

  13. Triclosan antagonizes fluconazole activity against Candida albicans.

    LENUS (Irish Health Repository)

    Higgins, J

    2012-01-01

    Triclosan is a broad-spectrum antimicrobial compound commonly used in oral hygiene products. Investigation of its activity against Candida albicans showed that triclosan was fungicidal at concentrations of 16 mg\\/L. However, at subinhibitory concentrations (0.5-2 mg\\/L), triclosan antagonized the activity of fluconazole. Although triclosan induced CDR1 expression in C. albicans, antagonism was still observed in cdr1Δ and cdr2Δ strains. Triclosan did not affect fluconazole uptake or alter total membrane sterol content, but did induce the expression of FAS1 and FAS2, indicating that its mode of action may involve inhibition of fatty acid synthesis, as it does in prokaryotes. However, FAS2 mutants did not exhibit increased susceptibility to triclosan, and overexpression of both FAS1 and FAS2 alleles did not alter triclosan susceptibility. Unexpectedly, the antagonistic effect was specific for C. albicans under hypha-inducing conditions and was absent in the non-filamentous efg1Δ strain. This antagonism may be due to the membranotropic activity of triclosan and the unique composition of hyphal membranes.

  14. Candida albicans versus Candida dubliniensis: Why Is C. albicans More Pathogenic?

    LENUS (Irish Health Repository)

    Moran, Gary P

    2012-01-01

    Candida albicans and Candida dubliniensis are highly related pathogenic yeast species. However, C. albicans is far more prevalent in human infection and has been shown to be more pathogenic in a wide range of infection models. Comparison of the genomes of the two species has revealed that they are very similar although there are some significant differences, largely due to the expansion of virulence-related gene families (e.g., ALS and SAP) in C. albicans, and increased levels of pseudogenisation in C. dubliniensis. Comparative global gene expression analyses have also been used to investigate differences in the ability of the two species to tolerate environmental stress and to produce hyphae, two traits that are likely to play a role in the lower virulence of C. dubliniensis. Taken together, these data suggest that C. dubliniensis is in the process of undergoing reductive evolution and may have become adapted for growth in a specialized anatomic niche.

  15. Game and player: C. albicans biofilm lifestyle and extracellular DNA

    OpenAIRE

    Martins, Margarida Isabel Barros Coelho; Uppuluri, Priya; Thomas, Derek P.; Cleary, Ian A.; Henriques, Mariana; Lopez-Ribot, José L.; Oliveira, Rosário

    2010-01-01

    DNA is as a structural component of bacterial biofilms extracellular matrix (ECM). Although evidences have shown that DNA may play a role in C. albicans biofilms, further studies are required to understand the contribution of extracellular DNA (eDNA) in C. albicans biofilm lifestyle. Herein we aimed to determine the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I and exogenous DNA treatments on biofilm formation and biofilm cells susceptibility to antifungals. First, ...

  16. Identification of two germ-tube-specific cell wall antigens of Candida albicans.

    OpenAIRE

    Ponton, J; J. M. Jones

    1986-01-01

    Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antig...

  17. Interactions Between Candida albicans and Host Interações entre Candida albicans e Hospedeiro

    Directory of Open Access Journals (Sweden)

    Tatiane De Rossi

    2011-06-01

    Full Text Available Candida albicans can cause grave infections in patients who are immunocompromised by diseases, by surgery, or by immunesupresive therapy. The high levels of morbidity and mortality resulting from those infections in hospitalized patients show that C. albicans became a prominent human pathogen. Although the host immune system is the major factor balancing the transition from commensalisms to pathogenicity, several virulence attributes expressed by C. albicans, such as adhesion factors, phenotypic switching, dimorphic behavior, and secretion of hydrolytic enzymes, might contribute to the persistence of colonization as well as the development of symptomatic episodes. Host defense against candidiasis relies mainly on the ingestion and elimination of C. albicans by phagocytic cells, which present receptors Toll-like 4, dectin–1 associated to receptors Toll-like2 and mannose receptors. The cytokine IL-10 (IL-10 produced by phagocytes has a crucial role on susceptibility of host fungal infection, whereas IL-10 produced by regulatory T cells is mainly responsible by commensalisms. In contrast, productions of tumour necrosis factor - α (TNF-α, interleukin–1 β (lL-1 β, (IL-6 and (Il-12 provided protective cell–mediated immunity. The interferon-γ produced by natural killer and TH1 cells stimulates migration of phagocytes and major efficacy on destruction of fungi. In epithelial cells from mucosas the NOD-like receptors and defensins-β cytoplasmatic prevent the translocation of C. albicans from microbiota to tissues, which are modulated by IL-1 β, Il-17 and Il-22 cytokines. to pathogenicity, several virulence attributes expressed by C. albicans, such as adhesion factors, phenotypic switching, dimorphic behavior, and secretion of hydrolytic enzymes, might contribute to the persistence of colonization as well as the development of symptomatic episodes. Host defense against candidiasis relies mainly on the ingestion and elimination of C. albicans

  18. Intestinal colonization with Candida albicans and mucosal immunity

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Bai; Xian-Hua Liu; Qing-Ying Tong

    2004-01-01

    AIM: To observe the relationship between intestinal lumen colonization with Candida albicans and mucosal secretory IgA (sIgA).METHODS: A total of 82 specific-pathogen-free mice were divided randomly into control and colonization groups. After Candida albicans were inoculated into specific-pathogenfree mice, the number of Candida albicans adhering to cecum and mucosal membrane was counted. The lymphocyte proliferation in Peyer's patch and in lamina propria was shown by BrdU incorporation, while mucosal sIgA (surface membrane) isotype switch in Peyer's patch was investigated. IgA plasma cells in lamina propria were observed by immunohistochemical staining. Specific IgA antibodies to Candida albicans were measured with ELISA.RESULTS: From d 3 to d 14 after Candida albicans gavaging to mice, the number of Candida albicans colonizing in lumen and adhering to mucosal membrane was sharply reduced.Candida albicans translocation to mesenteric lymph nodes occurred at early time points following gavage administration and disappeared at later time points. Meanwhile, the content of specific IgA was increased obviously. Proliferation and differentiation of lymphocytes in lamina propria were also increased.CONCLUSION: Lymphocytes in lamina propria play an important role in intestinal mucosal immunity of specificpathogen-free mice when they are first inoculated with Candida albicans. The decreasing number of Candida albicans in intestine is related to the increased level of specific IgA antibodies in the intestinal mucus.

  19. Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Jiyoti Verma-Gaur

    2015-10-01

    Full Text Available The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

  20. Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects.

    Science.gov (United States)

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; van Houdt, Michel; Verkuijlen, Paul J J H; Herbst, Martin; Liese, Johannes G; van de Veerdonk, Frank L; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2014-07-24

    Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease. PMID:24948657

  1. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

    NARCIS (Netherlands)

    Netea, M.G.; Gow, N.A.; Munro, C.A.; Bates, S.; Collins, C.; Ferwerda, G.; Hobson, R.P.; Bertram, G.; Hughes, H.B.; Jansen, T.; Jacobs, L.; Buurman, E.T.; Gijzen, K.; Williams, D.L.; Torensma, R.; McKinnon, A.; MacCallum, D.M.; Odds, F.C.; Meer, J.W.M. van der; Brown, A.; Kullberg, B.J.

    2006-01-01

    The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages

  2. CEK2, a Novel MAPK from Candida albicans Complement the Mating Defect of fus3/kss1 Mutant.

    Science.gov (United States)

    Chen, Jing; Wang, Qing; Chen, Jiang-Ye

    2000-01-01

    A novel MAPK(mitogen-activated protein kinase) gene, CEK2(Candida albicans extracellular signal-regulated kinase 2), was isolated by screening the Candida albicans library based on oligonucleotide probe hybridization and degenerated PCR. The CEK2 gene is 1 119 bp in length, and coding for a 373 aa protein. The CEK2 shares 56% homology with CEK1 from Candida albicans, 55 % homology with FUS3 from S. cerevisiae. From the deduced amino acid sequence, the Cek2 protein contains a conserved ATP binding site and a Ser/Thr kinase activity signal and a conserved TEY sequence was located at L(12) region. In vitro kinase activity assay showed Cek2 could phosphorylate MBP(myelin bovine protein) but not histone H1. CEK2 gene could complement the fus3/kss1 mutant and underwent mating signal induction by a tester strain, but CEK1 gene could not complement with the fus3/kss1 mutant. CEK2 is therefore a FUS3 homolog in Candida albicans. PMID:12075459

  3. Roles of Zinc-responsive transcription factor Csr1 in filamentous growth of the pathogenic Yeast Candida albicans.

    Science.gov (United States)

    Kim, Min-Jeong; Kil, Minkwang; Jung, Jong-Hwan; Kim, Jinmi

    2008-02-01

    In the fungal pathogen Candida albicans, the yeast-to-hyphal transition occurs in response to a broad range of environmental stimuli and is considered to be a major virulence factor. To address whether the zinc homeostasis affects the growth or pathogenicity of C. albicans, we functionally characterized the zinc-finger protein Csr1 during filamentation. The deduced amino acid sequence of Csr1 showed a 49% similarity to the zinc-specific transcription factor, Zap1 of Saccharomyces cerevisiae. Sequential disruptions of CSR1 were carried out in diploid C. albicans. The csr1/csr1 mutant strain showed severe growth defects under zinc-limited growth conditions and the filamentation defect under hyphainducing media. The colony morphology and the germ-tube formation were significantly affected by the csr1 mutation. The expression of the hyphae-specific gene HWP1 was also impaired in csr1/csr1 cells. The C. albicans homologs of ZRT1 and ZRT2, which are zinc-transporter genes in S. cerevisiae, were isolated. High-copy number plasmids of these genes suppressed the filamentation defect of the csr1/csr1 mutant strain. We propose that the filamentation phenotype of C. albicans is closely associated with the zinc homeostasis in the cells and that Csr1 plays a critical role in this regulation. PMID:18309267

  4. Assessing the Application of Tissue Microarray Technology to Kidney Research

    OpenAIRE

    Zhang, Ming-Zhi; Su, Yinghao; Yao, Bing; Zheng, Wei; deCaestecker, Mark; Harris, Raymond C.

    2010-01-01

    Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and...

  5. Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

    Science.gov (United States)

    Bandara, H M H N; K Cheung, B P; Watt, R M; Jin, L J; Samaranayake, L P

    2013-02-01

    Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation. PMID:23194472

  6. Seeded Bayesian Networks: Constructing genetic networks from microarray data

    Directory of Open Access Journals (Sweden)

    Quackenbush John

    2008-07-01

    Full Text Available Abstract Background DNA microarrays and other genomics-inspired technologies provide large datasets that often include hidden patterns of correlation between genes reflecting the complex processes that underlie cellular metabolism and physiology. The challenge in analyzing large-scale expression data has been to extract biologically meaningful inferences regarding these processes – often represented as networks – in an environment where the datasets are often imperfect and biological noise can obscure the actual signal. Although many techniques have been developed in an attempt to address these issues, to date their ability to extract meaningful and predictive network relationships has been limited. Here we describe a method that draws on prior information about gene-gene interactions to infer biologically relevant pathways from microarray data. Our approach consists of using preliminary networks derived from the literature and/or protein-protein interaction data as seeds for a Bayesian network analysis of microarray results. Results Through a bootstrap analysis of gene expression data derived from a number of leukemia studies, we demonstrate that seeded Bayesian Networks have the ability to identify high-confidence gene-gene interactions which can then be validated by comparison to other sources of pathway data. Conclusion The use of network seeds greatly improves the ability of Bayesian Network analysis to learn gene interaction networks from gene expression data. We demonstrate that the use of seeds derived from the biomedical literature or high-throughput protein-protein interaction data, or the combination, provides improvement over a standard Bayesian Network analysis, allowing networks involving dynamic processes to be deduced from the static snapshots of biological systems that represent the most common source of microarray data. Software implementing these methods has been included in the widely used TM4 microarray analysis package.

  7. Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation

    OpenAIRE

    Richard, Arianne C.; Lyons, Paul A.; Peters, James E.; Biasci, Daniele; Flint, Shaun M; James C Lee; McKinney, Eoin F; Siegel, Richard M.; Smith, Kenneth GC

    2014-01-01

    Background Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray u...

  8. Microarray analysis of the developing cortex.

    Science.gov (United States)

    Semeralul, Mawahib O; Boutros, Paul C; Likhodi, Olga; Okey, Allan B; Van Tol, Hubert H M; Wong, Albert H C

    2006-12-01

    Abnormal development of the prefrontal cortex (PFC) is associated with a number of neuropsychiatric disorders that have an onset in childhood or adolescence. Although the basic laminar structure of the PFC is established in utero, extensive remodeling continues into adolescence. To map the overall pattern of changes in cortical gene transcripts during postnatal development, we made serial measurements of mRNA levels in mouse PFC using oligonucleotide microarrays. We observed changes in mRNA transcripts consistent with known postnatal morphological and biochemical events. Overall, most transcripts that changed significantly showed a progressive decrease in abundance after birth, with the majority of change between postnatal weeks 2 and 4. Genes with cell proliferative, cytoskeletal, extracellular matrix, plasma membrane lipid/transport, protein folding, and regulatory functions had decreases in mRNA levels. Quantitative PCR verified the microarray results for six selected genes: DNA methyltransferase 3A (Dnmt3a), procollagen, type III, alpha 1 (Col3a1), solute carrier family 16 (monocarboxylic acid transporters), member 1 (Slc16a1), MARCKS-like 1 (Marcksl1), nidogen 1 (Nid1) and 3-hydroxybutyrate dehydrogenase (heart, mitochondrial) (Bdh).

  9. Direct calibration of PICKY-designed microarrays

    Directory of Open Access Journals (Sweden)

    Ronald Pamela C

    2009-10-01

    Full Text Available Abstract Background Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples. Results Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu under the PICKY Download area. Conclusion PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.

  10. Melittin induces apoptotic features in Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Park, Cana [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of); Lee, Dong Gun, E-mail: dglee222@knu.ac.kr [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of)

    2010-03-26

    Melittin is a well-known antimicrobial peptide with membrane-active mechanisms. In this study, it was found that Melittin exerted its antifungal effect via apoptosis. Candida albicans exposed to Melittin showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining. Fluorescence microscopy staining with FITC-annexin V, TUNEL and DAPI further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, and DNA and nuclear fragmentation. The current study suggests that Melittin possesses an antifungal effect with another mechanism promoting apoptosis.

  11. Candida albicans osteomyelitis of the cervical spine

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Jang-Gyu; Hong, Hyun-Sook [Soonchunhyang University Bucheon Hospital, Department of Radiology, Bucheon-Si, Gyeonggi-Do (Korea); Koh, Yoon-Woo [Soonchunhyang University Bucheon Hospital, Department of Otolaryngology - Head and Neck Surgery, Bucheon-Si, Gyeonggi-Do (Korea); Kim, Hee-Kyung [Soonchunhyang University Bucheon Hospital, Department of Pathology, Bucheon-Si, Gyeonggi-Do (Korea); Park, Jung-Mi [Soonchunhyang University Bucheon Hospital, Department of Nuclear Medicine, Bucheon-Si, Gyeonggi-Do (Korea)

    2008-04-15

    Fungal osteomyelitis is a rare infection that usually develops in immunocompromised patients. Additionally, involvement of the cervical spine by Candida albicans is extremely rare; only three previous cases of Candida vertebral osteomyelitis have been reported in the literature. The diagnosis may be delayed due to nonspecific radiologic findings and a slow progression. We report the CT, MRI, bone scan, and PET-CT findings in a patient who developed Candida osteomyelitis, which was initially misdiagnosed as metastasis, at the atlas and axis following treatment for nasopharyngeal cancer. (orig.)

  12. Candida albicans osteomyelitis of the cervical spine

    International Nuclear Information System (INIS)

    Fungal osteomyelitis is a rare infection that usually develops in immunocompromised patients. Additionally, involvement of the cervical spine by Candida albicans is extremely rare; only three previous cases of Candida vertebral osteomyelitis have been reported in the literature. The diagnosis may be delayed due to nonspecific radiologic findings and a slow progression. We report the CT, MRI, bone scan, and PET-CT findings in a patient who developed Candida osteomyelitis, which was initially misdiagnosed as metastasis, at the atlas and axis following treatment for nasopharyngeal cancer. (orig.)

  13. Gymnemic acids inhibit hyphal growth and virulence in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Govindsamy Vediyappan

    Full Text Available Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine.

  14. The malfunction of peroxisome has an impact on the oxidative stress sensitivity in Candida albicans.

    Science.gov (United States)

    Chen, Yulu; Yu, Qilin; Wang, Honggang; Dong, Yijie; Jia, Chang; Zhang, Bing; Xiao, Chenpeng; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2016-10-01

    The peroxisome plays an essential role in eukaryotic cellular metabolism, including β-oxidation of fatty acids and detoxification of hydrogen peroxide. However, its functions in the important fungal pathogen, C. albicans, remain to be investigated. In this study, we identified a homologue of Saccharomyces cerevisiae peroxisomal protein Pex1 in this pathogen, and explored its functions in stress tolerance. Fluorescence observation revealed that C. albicans Pex1 was localized in the peroxisomes, and its loss led to the defect in peroxisome formation. Interestingly, the pex1Δ/Δ mutant had increased tolerance to oxidative stress, which was neither associated with the Cap1 pathway, nor related to the altered distribution of catalase. However, under oxidative stress, the pex1Δ/Δ mutant showed increased expression of autophagy-related genes, with enhanced cytoplasm-to-vacuole transport and degradation of the autophagy markers Atg8 and Lap41. Moreover, the double mutants pex1Δ/Δatg8Δ/Δ and pex1Δ/Δatg1Δ/Δ, both of which were defective in autophagy and peroxisome formation, showed remarkable attenuated tolerance to oxidative stress. These results indicated that autophagy is involved in resistance to oxidative stress in pex1Δ/Δ mutant. Taken together, this study provides evidence that the peroxisomal protein Pex1 regulates oxidative stress tolerance in an autophagy-dependent manner in C. albicans. PMID:27473887

  15. Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Rung-Yi; Huang, Siyu; Fenwick, Michael K.; Hazra, Amrita; Zhang, Yang; Rajashankar, Kanagalaghatta; Philmus, Benjamin; Kinsland, Cynthia; Sanders, Jennie Mansell; Ealick, Steven E.; Begley, Tadhg P. (Cornell); (TAM)

    2012-06-26

    In Saccharomyces cerevisiae, thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.

  16. Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria.

    Science.gov (United States)

    Cavalcanti, Yuri Wanderley; Morse, Daniel James; da Silva, Wander José; Del-Bel-Cury, Altair Antoninha; Wei, Xiaoqing; Wilson, Melanie; Milward, Paul; Lewis, Michael; Bradshaw, David; Williams, David Wynne

    2015-01-01

    This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.

  17. Sensitization of Candida albicans to terbinafine by berberine and berberrubine

    Science.gov (United States)

    LAM, PIKLING; KOK, STANTON HON LUNG; LEE, KENNETH KA HO; LAM, KIM HUNG; HAU, DESMOND KWOK PO; WONG, WAI YEUNG; BIAN, ZHAOXIANG; GAMBARI, ROBERTO; CHUI, CHUNG HIN

    2016-01-01

    Candida albicans (C. albicans) is an opportunistic fungal pathogen, particularly observed in immunocompromised patients. C. albicans accounts for 50–70% of cases of invasive candidiasis in the majority of clinical settings. Terbinafine, an allylamine antifungal drug, has been used to treat fungal infections previously. It has fungistatic activity against C. albicans. Traditional Chinese medicines can be used as complementary medicines to conventional drugs to treat a variety of ailments and diseases. Berberine is a quaternary alkaloid isolated from the traditional Chinese herb, Coptidis Rhizoma, while berberrubine is isolated from the medicinal plant Berberis vulgaris, but is also readily derived from berberine by pyrolysis. The present study demonstrates the possible complementary use of berberine and berberrubine with terbinafine against C. albicans. The experimental findings assume that the potential application of these alkaloids together with reduced dosage of the standard drug would enhance the resulting antifungal potency. PMID:27073630

  18. Disc-based microarrays: principles and analytical applications.

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    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays. PMID:26922341

  19. Convergent Regulation of Candida albicans Aft2 and Czf1 in Invasive and Opaque Filamentation.

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    Xu, Ning; Dong, Yi-Jie; Yu, Qi-Lin; Zhang, Bing; Zhang, Meng; Jia, Chang; Chen, Yu-Lu; Zhang, Biao; Xing, Lai-Jun; Li, Ming-Chun

    2015-09-01

    Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions.

  20. Microarray results: how accurate are they?

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    Mane Shrikant

    2002-08-01

    Full Text Available Abstract Background DNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially. Results In this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene. Conclusions In view of these pitfalls, data from microarray analysis need to be interpreted cautiously.

  1. Optimisation algorithms for microarray biclustering.

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    Perrin, Dimitri; Duhamel, Christophe

    2013-01-01

    In providing simultaneous information on expression profiles for thousands of genes, microarray technologies have, in recent years, been largely used to investigate mechanisms of gene expression. Clustering and classification of such data can, indeed, highlight patterns and provide insight on biological processes. A common approach is to consider genes and samples of microarray datasets as nodes in a bipartite graphs, where edges are weighted e.g. based on the expression levels. In this paper, using a previously-evaluated weighting scheme, we focus on search algorithms and evaluate, in the context of biclustering, several variations of Genetic Algorithms. We also introduce a new heuristic "Propagate", which consists in recursively evaluating neighbour solutions with one more or one less active conditions. The results obtained on three well-known datasets show that, for a given weighting scheme, optimal or near-optimal solutions can be identified. PMID:24109756

  2. How Can Microarrays Unlock Asthma?

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    Alen Faiz

    2012-01-01

    Full Text Available Asthma is a complex disease regulated by the interplay of a large number of underlying mechanisms which contribute to the overall pathology. Despite various breakthroughs identifying genes related to asthma, our understanding of the importance of the genetic background remains limited. Although current therapies for asthma are relatively effective, subpopulations of asthmatics do not respond to these regimens. By unlocking the role of these underlying mechanisms, a source of novel and more effective treatments may be identified. In the new age of high-throughput technologies, gene-expression microarrays provide a quick and effective method of identifying novel genes and pathways, which would be impossible to discover using an individual gene screening approach. In this review we follow the history of expression microarray technologies and describe their contributions to advancing our current knowledge and understanding of asthma pathology.

  3. Microarray analysis in pulmonary hypertension.

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    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea; Kwapiszewska, Grazyna

    2016-07-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  4. MBL-mediated opsonophagocytosis of Candida albicans by human neutrophils is coupled with intracellular Dectin-1-triggered ROS production.

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    Dongsheng Li

    Full Text Available Mannan-binding lectin (MBL, a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans by human neutrophils, and has the capacity to bind to CR1 (CD35 expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production.

  5. Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans.

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    Wang, Ying; Liu, Jin-Yan; Shi, Ce; Li, Wen-Jing; Zhao, Yue; Yan, Lan; Xiang, Ming-Jie

    2015-11-01

    The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.

  6. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

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    Shi Qun Zhang

    2016-05-01

    Full Text Available The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3-glucan, a crucial pathogen-associated molecular pattern (PAMP of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

  7. A genome-wide 20 K citrus microarray for gene expression analysis

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    Gadea Jose

    2008-07-01

    Full Text Available Abstract Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database 1 was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global

  8. Molecular mechanisms of primary resistance to flucytosine in Candida albicans.

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    Hope, William W; Tabernero, Lydia; Denning, David W; Anderson, Michael J

    2004-11-01

    Primary resistance in Candida albicans to flucytosine (5-FC) was investigated in 25 strains by identifying and sequencing the genes FCA1, FUR1, FCY21, and FCY22, which code for cytosine deaminase, uracil phosphoribosyltransferase (UPRT), and two purine-cytosine permeases, respectively. These proteins are involved in pyrimidine salvage and 5-FC metabolism. An association between a polymorphic nucleotide and resistance to 5-FC was found within FUR1 where the substitution of cytidylate for thymidylate at nucleotide position 301 results in the replacement of arginine with cysteine at amino acid position 101 in UPRT. Isolates that are homozygous for this mutation display increased levels of resistance to 5-FC, whereas heterozygous isolates have reduced susceptibility. Three-dimensional protein modeling of UPRT suggests that the Arg101Cys mutation disturbs the quaternary structure of the enzyme, which is postulated to compromise optimal enzyme activity. A single resistant isolate, lacking the above polymorphism in FUR1, has a homozygous polymorphism in FCA1 that results in a glycine-to-aspartate substitution at position 28 in cytosine deaminase.

  9. AI-2 of Aggregatibacter actinomycetemcomitans Inhibits Candida albicans Biofilm Formation

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    Endang W. Bachtiar

    2014-07-01

    Full Text Available Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2, synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.

  10. In vitro activity of eugenol against Candida albicans biofilms.

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    He, Miao; Du, Minquan; Fan, Mingwen; Bian, Zhuan

    2007-03-01

    Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC(50) for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections. PMID:17356790

  11. Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2

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    Gibson Joanne

    2007-10-01

    Full Text Available Abstract Background In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. Results The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt was inserted between fused lacZ and luciferase (luc genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. Conclusion This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

  12. PREVALENCE OF BIOFILM PRODUCING MDR CANDIDA ALBICANS AND NON CANDIDA ALBICANS ISOLATE FROM MEDICAL DEVICES

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    P. Rajeswari

    2012-12-01

    Full Text Available Totally 56% of occurrence was observed from 6 types of sources. Among them highest prevalence was observed from urinary catheter (68% next in line is intravenous tubes (66.66%, venflon needles (65%, and blood bags (53.33% respectively. Four types of Candida species were identified by using selective media and biochemical tests. The Candida albicans was predominant isolates in all sources especially in urinary catheter. In this study, 60.2% of non Candida albicans were observed. All isolates were subjected to antifungal stability test, 6 antifungal agents were used. Among the 6 antifungal agents Itraconazole had highly resistance activity and Fluconazole had highly sensitive activity against the isolates. The antifungal resistance of isolates were highly observed in non Candida albicans such as Candida tropicalis (83.3% and followed by Candida glabrata (74.5%. All isolates were have the ability to produce biofilm, among them 37.4% of isolates were strong biofilm producer and 100% of protease producing isolates were observed in the last part of the study.

  13. Role of SFP1 in the Regulation of Candida albicans Biofilm Formation.

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    Hsueh-Fen Chen

    Full Text Available Candida albicans is a major human fungal pathogen. One of the important features of C. albicans pathogenicity is the ability to form biofilms on mucosal surfaces and indwelling medical devices. Biofilm formation involves complex processes in C. albicans, including cell adhesion, filamentous growth, extracellular matrix secretion and cell dispersion. In this work, we characterized the role of the transcription factor Sfp1, particularly with respect to its function in the regulation of biofilm formation. The deletion of the SFP1 gene enhanced cell adhesion and biofilm formation in comparison to the wild-type strain. Interestingly, the sfp1-deleted mutant also exhibited an increase in the expression of the ALS1, ALS3 and HWP1 genes, which encode adhesin proteins. In addition, Sfp1 was demonstrated to function downstream of the Rhb1-TOR signaling pathway. Bcr1 and Efg1 are transcription factors that are critical for controlling biofilm formation, and Efg1 is also required for hyphal growth. Deleting either the BCR1 or EFG1 gene in the sfp1-null background led to reduced adhesin gene expression. As a result, the bcr1/sfp1 or efg1/sfp1 double deletion mutants exhibited dramatically reduced biofilm formation. The results indicated that Sfp1 negatively regulates the ALS1, ALS3 and HWP1 adhesin genes and that the repression of these genes is mediated by the inhibition of Bcr1 and Efg1.

  14. Modelling the regulation of thermal adaptation in Candida albicans, a major fungal pathogen of humans.

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    Michelle D Leach

    Full Text Available Eukaryotic cells have evolved mechanisms to sense and adapt to dynamic environmental changes. Adaptation to thermal insults, in particular, is essential for their survival. The major fungal pathogen of humans, Candida albicans, is obligately associated with warm-blooded animals and hence occupies thermally buffered niches. Yet during its evolution in the host it has retained a bona fide heat shock response whilst other stress responses have diverged significantly. Furthermore the heat shock response is essential for the virulence of C. albicans. With a view to understanding the relevance of this response to infection we have explored the dynamic regulation of thermal adaptation using an integrative systems biology approach. Our mathematical model of thermal regulation, which has been validated experimentally in C. albicans, describes the dynamic autoregulation of the heat shock transcription factor Hsf1 and the essential chaperone protein Hsp90. We have used this model to show that the thermal adaptation system displays perfect adaptation, that it retains a transient molecular memory, and that Hsf1 is activated during thermal transitions that mimic fever. In addition to providing explanations for the evolutionary conservation of the heat shock response in this pathogen and the relevant of this response to infection, our model provides a platform for the analysis of thermal adaptation in other eukaryotic cells.

  15. Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target.

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    Katharine S Dobb

    Full Text Available Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.

  16. Propolis Is an Efficient Fungicide and Inhibitor of Biofilm Production by Vaginal Candida albicans.

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    Capoci, Isis Regina Grenier; Bonfim-Mendonça, Patrícia de Souza; Arita, Glaucia Sayuri; Pereira, Raphaela Regina de Araújo; Consolaro, Marcia Edilaine Lopes; Bruschi, Marcos Luciano; Negri, Melyssa; Svidzinski, Terezinha Inez Estivalet

    2015-01-01

    Vulvovaginal candidiasis (VVC) is one of the most common genital infections in women. The therapeutic arsenal remains restricted, and some alternatives to VVC treatment are being studied. The present study evaluated the influence of a propolis extractive solution (PES) on biofilm production by Candida albicans isolated from patients with VVC. Susceptibility testing was used to verify the minimum inhibitory concentration (MIC) of PES, with fluconazole and nystatin as controls. The biofilm formation of 29 vaginal isolates of C. albicans and a reference strain that were exposed to PES was evaluated using crystal violet staining. Colony-forming units were evaluated, proteins and carbohydrates of the matrix biofilm were quantified, and scanning electron microscopy was performed. The MIC of PES ranged from 68.35 to 546.87 μg/mL of total phenol content in gallic acid. A concentration of 546.87 μg/mL was able to cause the death of 75.8% of the isolates. PES inhibited biofilm formation by C. albicans from VVC. Besides antifungal activity, PES appears to present important antibiofilm activity on abiotic surfaces, indicating that it may have an additional beneficial effect in the treatment of VVC. PMID:25815029

  17. Propolis Is an Efficient Fungicide and Inhibitor of Biofilm Production by Vaginal Candida albicans

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    Isis Regina Grenier Capoci

    2015-01-01

    Full Text Available Vulvovaginal candidiasis (VVC is one of the most common genital infections in women. The therapeutic arsenal remains restricted, and some alternatives to VVC treatment are being studied. The present study evaluated the influence of a propolis extractive solution (PES on biofilm production by Candida albicans isolated from patients with VVC. Susceptibility testing was used to verify the minimum inhibitory concentration (MIC of PES, with fluconazole and nystatin as controls. The biofilm formation of 29 vaginal isolates of C. albicans and a reference strain that were exposed to PES was evaluated using crystal violet staining. Colony-forming units were evaluated, proteins and carbohydrates of the matrix biofilm were quantified, and scanning electron microscopy was performed. The MIC of PES ranged from 68.35 to 546.87 μg/mL of total phenol content in gallic acid. A concentration of 546.87 μg/mL was able to cause the death of 75.8% of the isolates. PES inhibited biofilm formation by C. albicans from VVC. Besides antifungal activity, PES appears to present important antibiofilm activity on abiotic surfaces, indicating that it may have an additional beneficial effect in the treatment of VVC.

  18. Molecular concordance of concurrent Candida albicans candidemia and candiduria.

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    Huang, Po-Yen; Hung, Min-Hui; Shie, Shian-Sen; Su, Lin-Hui; Chen, Ke-Yuan; Ye, Jung-Jr; Chiang, Ping-Cheng; Leu, Hsieh-Shong; Huang, Ching-Tai

    2013-07-01

    The significance of candiduria remains unclear. We correlated Candida albicans candidemia with candiduria by molecular genotyping. 33 pairs of concurrent blood and urine C. albicans isolates from 31 adult (≥ 18 years) were genotyped with infrequent-restriction-site PCR. The molecular concordance rates of three major genotypes were 100% for I, 82% for II, and 71% for III. The molecular concordance between concurrent C. albicans candidemia and candiduria was frequent. Our findings substantiate the importance of candiduria in appropriate clinical context as the majority of our patients were from intensive care units.

  19. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

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    Deforce Dieter

    2006-08-01

    Full Text Available Abstract Background Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence gene family can be performed using quantitative PCR (qPCR. In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans biofilms, using the geNorm Visual Basic Application (VBA for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. Results The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable as follows: ACT1 (β-actin/PMA1 (adenosine triphosphatase, RIP (ubiquinol cytochrome-c reductase complex component, RPP2B (cytosolic ribosomal acidic protein P2B, LSC2 (succinyl-CoA synthetase β-subunit fragment, IMH3 (inosine-5'-monophosphate dehydrogenase fragment, CPA1 (carbamoyl-phosphate synthethase small subunit and GAPDH (glyceraldehyde-3-phosphate dehydrogenase. Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control, for 24 hours. Conclusion In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2 for normalization, when analysing differences in gene expression levels between C. albicans biofilm

  20. Control of the C. albicans cell wall damage response by transcriptional regulator Cas5.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available The fungal cell wall is vital for growth, development, and interaction of cells with their environment. The response to cell wall damage is well understood from studies in the budding yeast Saccharomyces cerevisiae, where numerous cell wall integrity (CWI genes are activated by transcription factor ScRlm1. Prior evidence suggests the hypothesis that both response and regulation may be conserved in the major fungal pathogen Candida albicans. We have tested this hypothesis by using a new C. albicans genetic resource: we have screened mutants defective in putative transcription factor genes for sensitivity to the cell wall biosynthesis inhibitor caspofungin. We find that the zinc finger protein CaCas5, which lacks a unique ortholog in S. cerevisiae, governs expression of many CWI genes. CaRlm1 has a modest role in this response. The transcriptional coactivator CaAda2 is also required for expression of many CaCas5-dependent genes, as expected if CaCas5 recruits CaAda2 to activate target gene transcription. Many caspofungin-induced C. albicans genes specify endoplasmic reticulum and secretion functions. Such genes are not induced in S. cerevisiae, but promote its growth in caspofungin. We have used a new resource to identify a key C. albicans transcriptional regulator of CWI genes and antifungal sensitivity. Our gene expression findings indicate that both divergent and conserved response genes may have significant functional roles. Our strategy may be broadly useful for identification of pathogen-specific regulatory pathways and critical response genes.

  1. Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Eduardo Lopez-Medina

    2015-08-01

    Full Text Available Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

  2. Ontology-Based Analysis of Microarray Data.

    Science.gov (United States)

    Giuseppe, Agapito; Milano, Marianna

    2016-01-01

    The importance of semantic-based methods and algorithms for the analysis and management of biological data is growing for two main reasons. From a biological side, knowledge contained in ontologies is more and more accurate and complete, from a computational side, recent algorithms are using in a valuable way such knowledge. Here we focus on semantic-based management and analysis of protein interaction networks referring to all the approaches of analysis of protein-protein interaction data that uses knowledge encoded into biological ontologies. Semantic approaches for studying high-throughput data have been largely used in the past to mine genomic and expression data. Recently, the emergence of network approaches for investigating molecular machineries has stimulated in a parallel way the introduction of semantic-based techniques for analysis and management of network data. The application of these computational approaches to the study of microarray data can broad the application scenario of them and simultaneously can help the understanding of disease development and progress.

  3. CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans*

    OpenAIRE

    Bi, Liangkuan; Gojestani, Sara; Wu, Weihui; Hsu, Yen-Michael S.; Zhu, Jiayuan; Ariizumi, Kiyoshi; Lin, Xin

    2010-01-01

    The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our dat...

  4. Construction of metastatic spinal cancer tissue microarrays

    Institute of Scientific and Technical Information of China (English)

    Yang Xinghai; Chen Huajiang; Xiao Jianru; Yuan Wen; Jia Lianshun

    2009-01-01

    Objective: To explore the construction of metastatic spinal cancer (MSC) tissue microarrays and validate its value in immunohistochemical study of MSC. Methods: Paraffin-embedded specimens from 71 MSC cases and 6 primary tumor cases were selected as donor blocks and prepared into MSC tissue microarrays by tissue array arrangement, the steps of which included location, punching, sampling, sample seeding, and re-diagnosis by hematoxylin-eosin (HE) as well as MMP-9 and MMP-14 immunohistochemical staining. Results: The MSC tissue microarrays thus constructed were intact and crackless, containing 154 complete and well arranged microarray points. None of the sectioned tissue microarrays was lost, and the results of HE staining was consistent with the primary pathologic diagnoses. Immunohistochemical staining was also good without non-specific or marginal effect. Conclusion: The MSC tissue microarrays have a high value in the immunohistochemical study of MSC.

  5. Comprehensive comparison of six microarray technologies

    OpenAIRE

    Yauk, Carole L.; Berndt, M. Lynn; Williams, Andrew; Douglas, George R

    2004-01-01

    Microarray technology is extensively used in biological research. The applied technologies vary greatly between laboratories, and outstanding questions remain regarding the degree of correlation among approaches. Recently, there has been a drive toward ensuring high-quality microarray data by the implementation of MIAME (Minimal Information About a Microarray Experiment) guidelines and an emphasis on ensuring public-availability to all datasets. However, despite its current widespread use and...

  6. MICROARRAYS AND THEIR POTENTIAL IN MEDICINE

    Institute of Scientific and Technical Information of China (English)

    Erick Ling; Jie Xu

    2003-01-01

    Advancement in microarray technology can revolutionize many aspects of medicine. Microarrays have applications in gene expression profiling, genotyping, mutation analysis, gene identification, and pharmacology. This paper provides a brief review on the use of microarrays in studies of cancer, infectious diseases, chromosome disorders, neurological/mental disorders, and drugs, along with a prospect on its great potential in diagnosis, prognosis and the treatment of human diseases.

  7. Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida albicans

    DEFF Research Database (Denmark)

    Cheon, Seon Ah; Bal, Jyotiranjan; Song, Yunkyoung;

    2012-01-01

    Lag1p and Lac1p catalyse ceramide synthesis in Saccharomyces cerevisiae. This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to S. cerevisiae where these proteins are functionally redundant, C. albicans Lag1p (CaLag1......p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of C. albicans. Deletion of CaLAG1 decreased expression of the hypha-specific HWP1 and ECE1 genes. Moreover, overexpression of CaLAG1 induced pseudohyphal....... albicans....

  8. Characterization of extracellular nucleotide metabolism in Candida albicans.

    Science.gov (United States)

    Rodrigues, Lisa; Russo-Abrahão, Thais; Cunha, Rodrigo A; Gonçalves, Teresa; Meyer-Fernandes, José Roberto

    2016-01-01

    Candida albicans is the most frequent agent of human disseminated fungal infection. Ectophosphatase and ectonucleotidase activities are known to influence the infectious potential of several microbes, including other non-albicans species of Candida. With the present work we aim to characterize these ecto-enzymatic activities in C. albicans. We found that C. albicans does not have a classical ecto-5'-nucleotidase enzyme and 5'AMP is cleaved by a phosphatase instead of exclusively by a nucleotidase that also can use 3'AMP as a substrate. Moreover, these enzymatic activities are not dependent on secreted soluble enzymes and change when the yeast cells are under infection conditions, including low pH, and higher temperature and CO2 content.

  9. Detection of Candida albicans by mass spectrometric fingerprinting.

    Science.gov (United States)

    Zehm, Sarah; Schweinitz, Simone; Würzner, Reinhard; Colvin, Hans Peter; Rieder, Josef

    2012-03-01

    Candida albicans is one of the most frequent causes of fungal infections in humans. Significant correlation between candiduria and invasive candidiasis has previously been described. The existing diagnostic methods are often time-consuming, cost-intensive and lack in sensitivity and specificity. In this study, the profile of low-molecular weight volatile compounds in the headspace of C. albicans-urine suspensions of four different fungal cell concentrations compared to nutrient media and urine without C. albicans was determined using proton-transfer reaction mass spectrometry (PTR-MS). At fungal counts of ≥1.5 × 10(5) colony forming units (CFU)/ml signals at 45, 47 and 73 atomic mass units (amu) highly significantly increased. At fungal counts of albicans-urine suspensions of different fungal cell concentrations. PTR-MS represents a promising approach to rapid, highly sensitive and non-invasive clinical diagnostics allowing qualitative and quantitative analysis.

  10. Innate immune cell response upon Candida albicans infection.

    Science.gov (United States)

    Qin, Yulin; Zhang, Lulu; Xu, Zheng; Zhang, Jinyu; Jiang, Yuan-Ying; Cao, Yongbing; Yan, Tianhua

    2016-07-01

    Candida albicans is a polymorphic fungus which is the predominant cause of superficial and deep tissue fungal infections. This microorganism has developed efficient strategies to invade the host and evade host defense systems. However, the host immune system will be prepared for defense against the microbe by recognition of receptors, activation of signal transduction pathways and cooperation of immune cells. As a consequence, C. albicans could either be eliminated by immune cells rapidly or disseminate hematogenously, leading to life-threatening systemic infections. The interplay between Candida albicans and the host is complex, requiring recognition of the invaded pathogens, activation of intricate pathways and collaboration of various immune cells. In this review, we will focus on the effects of innate immunity that emphasize the first line protection of host defense against invaded C. albicans including the basis of receptor-mediated recognition and the mechanisms of cell-mediated immunity. PMID:27078171

  11. Dental Caries in Rats Associated with Candida albicans

    OpenAIRE

    Klinke, Thomas; Guggenheim, Bernhard; Klimm, Wolfgang; Thurnheer, Thomas

    2014-01-01

    In addition to occasional opportunistic colonization of the oral mucosa, Candida albicans is frequently found in carious dentin. The yeast’s potential to induce dental caries as a consequence of its pronounced ability to produce and tolerate acids was investigated. Eighty caries-active Osborne-Mendel rats were raised on an ampicillin-supplemented diet and exposed to C. albicans and/or Streptococcus mutans, except for controls. Throughout the 28-day test period, the animals were offered the mo...

  12. Blood group glycolipids as epithelial cell receptors for Candida albicans.

    OpenAIRE

    Cameron, B J; Douglas, L J

    1996-01-01

    The role of glycosphingolipids as possible epithelial cell receptors for Candida albicans was examined by investigating the binding of biotinylated yeasts to lipids extracted from human buccal epithelial cells and separated on thin-layer chromatograms. Binding was visualized by the addition of 125I-streptavidin followed by autoradiography. Five C. albicans strains thought from earlier work to have a requirement for fucose-containing receptors all bound to the same three components in the lipi...

  13. Improved assay for surface hydrophobic avidity of Candida albicans cells.

    OpenAIRE

    Hazen, K C; LeMelle, W G

    1990-01-01

    A simple method that distinguishes among hydrophobic avidity levels of highly hydrophobic isolates of the pathogenic fungus Candida albicans is described. This method involves mixing polystyrene microspheres at different concentrations with a constant concentration of yeast cells and plotting the data in accordance with the Langmuir isotherm equation. A 10-fold difference between the C. albicans isolates with the lowest and highest avidity (KH) values was found. This method may also demonstra...

  14. Candida albicans specializations for iron homeostasis: from commensalism to virulence

    OpenAIRE

    Noble, Suzanne

    2013-01-01

    Candida albicans is a fungal commensal-pathogen that persistently associates with its mammalian hosts. Between the commensal and pathogenic lifestyles, this microorganism inhabits host niches that differ markedly in the levels of bioavailable iron. A number of recent studies have exposed C. albicans specializations for acquiring iron from specific host molecules in regions where iron is scarce, while also defending against iron-related toxicity in regions where iron occurs in surfeit. Togethe...

  15. Metabolic gene clusters encoding the enzymes of two branches of the 3-oxoadipate pathway in the pathogenic yeast Candida albicans.

    Science.gov (United States)

    Gérecová, Gabriela; Neboháčová, Martina; Zeman, Igor; Pryszcz, Leszek P; Tomáška, Ľubomír; Gabaldón, Toni; Nosek, Jozef

    2015-05-01

    The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway. PMID:25743787

  16. "Harshlighting" small blemishes on microarrays

    Directory of Open Access Journals (Sweden)

    Wittkowski Knut M

    2005-03-01

    Full Text Available Abstract Background Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs. Results We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Conclusion Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization.

  17. Expression and Detection the Enzyme Activity or the Wild and Mutation Type of CYP51 Protein of Candida albicans%白色念珠菌CYP51蛋白功能性氨基酸残基定点突变、蛋白表达及活性测定

    Institute of Scientific and Technical Information of China (English)

    陈双红; 盛春泉; 徐晓辉; 姜远英; 张万年; 何成

    2009-01-01

    The Y118A、Y118F、Y118T、S378A、S378T、H310A、H310R mutants of Candida albicans sterol 14α-demethylase (CACYP51) were constructed and heterologously expressed in D12667, the recon-structed strain with the deletion of CYP51 gene of the Y12667. With the strains obtained and microsome enzymes separated, the western blot and the ultraviolet absorption spectrophotometry were used to qualita-tive and quantitative detect the expressed protein, the GC-MS was used to detect the metabolism activity of the protein. The results showed that, the target protein expressed successfully in the reconstructed strains, with the expression level up to 25% of the total microsome proteins. The results also showed that, the wild type protein had the catalytic activity to its nature substrate. While after alteration the wild gene with Y118A、Y118F、Y118T、S378A、S378T、H310A、H31 OR by a single base substitution, the catalytic activity of protein markedly decreased respectively. So the wild type and mutation CYP51 were expressed success-fully in Saccharomyces cerevisiae and the expression products preserved the activity to metabolism their nature substrate.%实验设计了白色念珠菌CYP51蛋白功能性氨基酸残基突变体Y118A、Y118F、Y118T、S378A、S378T、H310A、H310R,并转入基因工程菌D12667中表达.用Western及紫外分光光度法定性、定量检测蛋白,用GC-MS法测定蛋白代谢活性.结果表明,成功表达目标蛋白,蛋白诱导表达量接近微粒体蛋白总量的25%.活性测定表明,表达的野生型蛋白保持其对天然底物的代谢能力;相较于野生型蛋白,突变体蛋白代谢活性不同程度改变,最多可下降1/2左右.因此,本研究中成功表达了野生型和突变型CYP51蛋白,表达的蛋白保留了对天然底物的代谢活性.

  18. Oxidative stress of photodynamic antimicrobial chemotherapy inhibits Candida albicans virulence

    Science.gov (United States)

    Kato, Ilka Tiemy; Prates, Renato Araujo; Tegos, George P.; Hamblin, Michael R.; Simões Ribeiro, Martha

    2011-03-01

    Photodynamic antimicrobial chemotherapy (PACT) is based on the principal that microorganisms will be inactivated using a light source combined to a photosensitizing agent in the presence of oxygen. Oxidative damage of cell components occurs by the action of reactive oxygen species leading to cell death for microbial species. It has been demonstrated that PACT is highly efficient in vitro against a wide range of pathogens, however, there is limited information for its in vivo potential. In addition, it has been demonstrated that sublethal photodynamic inactivation may alter the virulence determinants of microorganisms. In this study, we explored the effect of sublethal photodynamic inactivation to the virulence factors of Candida albicans. Methylene Blue (MB) was used as photosensitizer for sublethal photodynamic challenge on C. albicans associated with a diode laser irradiation (λ=660nm). The parameters of irradiation were selected in causing no reduction of viable cells. The potential effects of PACT on virulence determinants of C. albicans cells were investigated by analysis of germ tube formation and in vivo pathogenicity assays. Systemic infection was induced in mice by the injection of fungal suspension in the lateral caudal vein. C. albicans exposed to sublethal photodynamic inactivation formed significantly less germ tube than untreated cells. In addition, mice infected with C. albicans submitted to sublethal PACT survived for a longer period of time than mice infected with untreated cells. The oxidative damage promoted by sublethal photodynamic inactivation inhibited virulence determinants and reduced in vivo pathogenicity of C. albicans.

  19. Neonatal malnutrition programs the oxidant function of macrophages in response to Candida albicans.

    Science.gov (United States)

    Costa, Thacianna Barreto Da; Morais, Natália Gomes De; Pedrosa, Amanda Lúcia F; De Albuquerque, Suênia Da Cunha G; De Castro, Maria Carolina A B; Pereira, Valéria Rêgo A; Cavalcanti, Milena De Paiva; De Castro, Célia Maria M B

    2016-06-01

    Experimental maternal nutrition restriction models are used to investigate short or long-term consequences of nutritional deficiency on puppies' growth. By assuming that the immune function is directly related to host's nutritional status, the current study aims to investigate the effects of neonatal malnutrition on oxidative stress and on the cell death of the alveolar macrophage after in vitro infection by Candida albicans. Wistar rats were suckled by mothers fed on diets containing 17% protein (Nourished group) or 8% protein (Malnourished group) in the current assay. Both groups received the standard diet used in the vivarium until adulthood, after weaning. The results showed that the offspring from mothers fed on low-protein diet presented lower body weight from 5 days of life on. Their low weight remained until adulthood when it was compared to that of rats in the nourished group. Superoxide and nitric oxide production was lower in malnourished animals and it was accompanied by low inducible nitric oxide synthase gene expression levels in systems in which the alveolar macrophages were challenged by immunogenic stimulus. No significant differences were observed in comparisons performed between the nourished and malnourished groups in any of the analyzed cell viability (apoptosis/necrosis) parameters. The fungal inoculum-stimulated system induced higher oxidative stress and cell death by necrosis. The current study demonstrated that dietary restriction during lactation alters the oxidant function of alveolar macrophages in puppies; It happens from the gene transcription step to the release of mediators, thus compromising the host's defenses against Candida albicans. It raises the possibility that Candida albicans may cease to be a commensal fungus to become a pathogen in offspring that have suffered nutritional deficiency during critical developmental periods, due to impaired immune responses. PMID:27001703

  20. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    Science.gov (United States)

    Ramirez, Lisa S; Wang, Jun

    2016-01-01

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

  1. SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae

    OpenAIRE

    Gale, Cheryl A.; Leonard, Michelle D.; Finley, Kenneth R.; Christensen, Leah; McClellan, Mark; Abbey, Darren; Kurischko, Cornelia; Bensen, Eric; Tzafrir, Iris; Kauffman, Sarah; Becker, Jeff; Berman, Judith

    2009-01-01

    The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other c...

  2. Application of microarray technology in pulmonary diseases

    OpenAIRE

    Patlakas George; Tzouvelekis Argyris; Bouros Demosthenes

    2004-01-01

    Abstract Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives.

  3. In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

    Directory of Open Access Journals (Sweden)

    Andreas Weinhäusel

    2012-06-01

    Full Text Available The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses. In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively. The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics.

  4. The EADGENE Microarray Data Analysis Workshop

    DEFF Research Database (Denmark)

    de Koning, Dirk-Jan; Jaffrézic, Florence; Lund, Mogens Sandø;

    2007-01-01

    Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from...... 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays...... statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful...

  5. Candida albicans keratitis in an immunocompromised patient

    Directory of Open Access Journals (Sweden)

    H Mohammed J Hassan

    2010-10-01

    Full Text Available H Mohammed J Hassan1, Theocharis Papanikolaou2, Georgios Mariatos1, Amany Hammad3, Hala Hassan41Ophthalmology Department, Barnsley Hospital NHS Foundation Trust, South Yorkshire, England, UK; 2Ophthalmology Department, Cambridge University Hospitals NHS Foundation Trust, England, UK; 3Ophthalmology Department, Rotherham Hospital NHS Foundation Trust, England, UK; 4Corneal and External Disease Service, Moorfields Eye Hospital NHS Foundation Trust, London, England, UKPurpose: When investigating a case of unexplained corneal ulceration, we need to think of fungal infection and any predisposing factors.Methods: A case study of a corneal ulceration in a patient who was HIV positive with a devastating visual outcome.Results: Therapeutic corneal graft was necessary due to corneal perforation. Immunocompromised state of patient was retrospectively diagnosed.Conclusions: Candida albicans keratitis is an opportunistic infection of a compromised cornea, and sometimes unknowingly compromised host, which can be initially misdiagnosed. Despite intensive antifungal therapy, occasionally patients require corneal grafting to improve vision, and before it is possible to establish an accurate diagnosis.Keywords: fungal keratitis, corneal perforation, keratoplasty, human immunodeficiency virus, HIV

  6. DNA Microarrays for Identifying Fishes

    Science.gov (United States)

    Nölte, M.; Weber, H.; Silkenbeumer, N.; Hjörleifsdottir, S.; Hreggvidsson, G. O.; Marteinsson, V.; Kappel, K.; Planes, S.; Tinti, F.; Magoulas, A.; Garcia Vazquez, E.; Turan, C.; Hervet, C.; Campo Falgueras, D.; Antoniou, A.; Landi, M.; Blohm, D.

    2008-01-01

    In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products. PMID:18270778

  7. Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

    Indian Academy of Sciences (India)

    Avmeet Kohli; Vinita Gupta; Shankarling Krishnamurthy; Seyed E Hasnain; Rajendra Prasad

    2001-09-01

    CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.

  8. Microarray analysis of gene expression profiles in ripening pineapple fruits

    Directory of Open Access Journals (Sweden)

    Koia Jonni H

    2012-12-01

    Full Text Available Abstract Background Pineapple (Ananas comosus is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study

  9. Activation of Rac1 by the Guanine Nucleotide Exchange Factor Dck1 Is Required for Invasive Filamentous Growth in the Pathogen Candida albicans

    OpenAIRE

    Hope, Hannah; Bogliolo, Stéphanie; Arkowitz, Robert A; Bassilana, Martine

    2008-01-01

    Rho G proteins and their regulators are critical for cytoskeleton organization and cell morphology in all eukaryotes. In the opportunistic pathogen Candida albicans, the Rho G proteins Cdc42 and Rac1 are required for the switch from budding to filamentous growth in response to different stimuli. We show that Dck1, a protein with homology to the Ced-5, Dock180, myoblast city family of guanine nucleotide exchange factors, is necessary for filamentous growth in solid media, similar to Rac1. Our ...

  10. Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells.

    Science.gov (United States)

    Mostefaoui, Yakout; Bart, Christian; Frenette, Michel; Rouabhia, Mahmoud

    2004-11-01

    We investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-alpha) and one chemokine (IL-8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-alpha mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius. IL-8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria. PMID:15469436

  11. Daya hambat xylitol dan nistation terhadap pertumbuhan Candida albicans (in vitro (Inhibition effect of xylitol and nistatin combination on Candida albicans growth (in vitro

    Directory of Open Access Journals (Sweden)

    Sarah Kartimah Djajusman

    2014-09-01

    Full Text Available Background: The growth of Candida albicans can be controlled by using antifungal such as nystatin. These days we found that using antifungal is not enough to control Candida albicans, we also have to control the intake of sugar by using xylitol. Purpose: Purpose of the study was to determine the optimal inhibitory concentration of xylitol-nystatin in the Candida albicans growth. Methods: This was an in-vitro study using an antimicrobial test of serial dilution with xylitol-nystatin and sucrose–nystatin consentration of 1%, 3%, 5%, 7%, 9%, and 10%.Growth inhibition of C. albicans was determined by the inhibition zone of xylitol + nystatin on C. albicans culture media (in vitro Results: The result of study was the inhibitory consentration of xylitol-nystatin to inhibit Candida albicans growth was 3%-10%. Conclusion: The study showed that combination of xylitol and nystation could inhibit the growth of Candida albicans.Latar belakang: Pertumbuhan Candida albicans dapat dikontrol dengan menggunakan antijamur seperti nistatin. Penggunakan antijamur saja tidak cukup untuk mengontrol Candida albicans, namun perlu pula mengontrol asupan gula dengan menggunakan xylitol. Tujuan: Tujuan dari penelitian ini adalah untuk menentukan konsentrasi hambat optimal xylitol-nistatin dalam pertumbuhan Candida albicans. Metode: Penelitian ini merupakan penelitian in vitro menggunakan uji antimikroba pengenceran serial dengan xylitol-nistatin dan nystatin-sukrosa konsentrasi 1%, 3 %, 5 %, 7%, 9%, dan 10%. Daya hambat pertumbuhan C. albicans diukur dari zona hambat xylitol + nistatin pada media kultur C. albicans (in vitro Hasil: Konsentrasi penghambatan xylitol-nistatin untuk menghambat pertumbuhan Candida albicans adalah 3-10%. Simpulan: Hasil penelitian menunjukkan bahwa kombinasi xylitol dan nystation bisa menghambat pertumbuhan Candida albicans.

  12. S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) Reduce In Vitro Inhibition by Fluconazole

    OpenAIRE

    Warrilow, Andrew G. S.; Mullins, Jonathan G. L.; Hull, Claire M.; Parker, Josie E.; Lamb, David C.; Kelly, Diane E.; Kelly, Steven L.

    2012-01-01

    The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type I...

  13. Oxidative Stress Responses in the Human Fungal Pathogen, Candida albicans

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    Alessandra da Silva Dantas

    2015-02-01

    Full Text Available Candida albicans is a major fungal pathogen of humans, causing approximately 400,000 life-threatening systemic infections world-wide each year in severely immunocompromised patients. An important fungicidal mechanism employed by innate immune cells involves the generation of toxic reactive oxygen species (ROS, such as superoxide and hydrogen peroxide. Consequently, there is much interest in the strategies employed by C. albicans to evade the oxidative killing by macrophages and neutrophils. Our understanding of how C. albicans senses and responds to ROS has significantly increased in recent years. Key findings include the observations that hydrogen peroxide triggers the filamentation of this polymorphic fungus and that a superoxide dismutase enzyme with a novel mode of action is expressed at the cell surface of C. albicans. Furthermore, recent studies have indicated that combinations of the chemical stresses generated by phagocytes can actively prevent C. albicans oxidative stress responses through a mechanism termed the stress pathway interference. In this review, we present an up-date of our current understanding of the role and regulation of oxidative stress responses in this important human fungal pathogen.

  14. MARS: Microarray analysis, retrieval, and storage system

    Directory of Open Access Journals (Sweden)

    Scheideler Marcel

    2005-04-01

    Full Text Available Abstract Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS, a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at http://genome.tugraz.at.

  15. Imaging combined autoimmune and infectious disease microarrays

    Science.gov (United States)

    Ewart, Tom; Raha, Sandeep; Kus, Dorothy; Tarnopolsky, Mark

    2006-09-01

    Bacterial and viral pathogens are implicated in many severe autoimmune diseases, acting through such mechanisms as molecular mimicry, and superantigen activation of T-cells. For example, Helicobacter pylori, well known cause of stomach ulcers and cancers, is also identified in ischaemic heart disease (mimicry of heat shock protein 65), autoimmune pancreatitis, systemic sclerosis, autoimmune thyroiditis (HLA DRB1*0301 allele susceptibility), and Crohn's disease. Successful antibiotic eradication of H.pylori often accompanies their remission. Yet current diagnostic devices, and test-limiting cost containment, impede recognition of the linkage, delaying both diagnosis and therapeutic intervention until the chronic debilitating stage. We designed a 15 minute low cost 39 antigen microarray assay, combining autoimmune, viral and bacterial antigens1. This enables point-of-care serodiagnosis and cost-effective narrowly targeted concurrent antibiotic and monoclonal anti-T-cell and anti-cytokine immunotherapy. Arrays of 26 pathogen and 13 autoimmune antigens with IgG and IgM dilution series were printed in triplicate on epoxysilane covalent binding slides with Teflon well masks. Sera diluted 1:20 were incubated 10 minutes, washed off, anti-IgG-Cy3 (green) and anti-IgM-Dy647 (red) were incubated for 5 minutes, washed off and the slide was read in an ArrayWoRx(e) scanning CCD imager (Applied Precision, Issaquah, WA). As a preliminary model for the combined infectious disease-autoimmune diagnostic microarray we surveyed 98 unidentified, outdated sera that were discarded after Hepatitis B antibody testing. In these, significant IgG or IgM autoantibody levels were found: dsDNA 5, ssDNA 11, Ro 2, RNP 7, SSB 4, gliadin 2, thyroglobulin 13 cases. Since control sera showed no autoantibodies, the high frequency of anti-DNA and anti-thyroglobulin antibodies found in infected sera lend increased support for linkage of infection to subsequent autoimmune disease. Expansion of the antigen

  16. DNA Microarrays in Herbal Drug Research

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    Preeti Chavan

    2006-01-01

    Full Text Available Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts.

  17. Subtype Identification of Avian Influenza Virus on DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    WANG Xiu-rong; YU Kang-zhen; DENG Guo-hua; SHI Rui; LIU Li-ling; QIAO Chuan-ling; BAO Hong-mei; KONG Xian-gang; CHEN Hua-lan

    2005-01-01

    We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus (AIV). The strains used in the experiment were A/Goose/Guangdong/1/96 (H5N1), A/African starling/983/79 (H7N1) and A/Turkey/Wiscosin/1/66 (H9N2). The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-1abeled fluorescent cDNAs,which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

  18. Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate

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    Apurva K Pathak

    2012-02-01

    Full Text Available OBJECTIVE: In polymicrobial biofilms bacteria extensively interact with Candida species, but the interaction among the different species of the Candida is yet to be completely evaluated. In the present study, the difference in biofilm formation ability of clinical isolates of four species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips was evaluated. MATERIAL AND METHODS: The species of Candida, isolated from multiple species oral candidiasis of the neutropenic patients, were used for the experiment. Organisms were cultured on Sabouraud dextrose broth with 8% glucose (SDB. Biofilm production on the acrylic resins strips was determined by crystal violet assay. Student's t-test and ANOVA were used to compare in vitro biofilm formation for the individual species of Candida and its different multi-species combinations. RESULTS: In the present study, differences between the mean values of the biofilm-forming ability of individual species (C. glabrata>C. krusei>C. tropicalis>C. albicans and in its multi-species' combinations (the highest for C. albicans with C. glabrata and the lowest for all the four species combination were reported. CONCLUSIONS: The findings of this study showed that biofilm-forming ability was found greater for non-Candida albicans Candida species (NCAC than for C. albicans species with intra-species variation. Presence of C. albicans in multi-species biofilms increased, whereas; C. tropicalis decreased the biofilm production with all other NCAC species.

  19. Macrophage phagocytosis of Candida albicans. An in vitro study

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    WEINFELD Ilan

    1999-01-01

    Full Text Available Considering the role of macrophages in relation to fungi and the various utilized methodologies, the authors established an in vitro model to evaluate macrophage phagocytosis of Candida albicans. Activated macrophages were obtained from the peritoneal cavity of isogenic mice (A/Sn. Two different strains of Candida albicans serotype A and serotype B with different levels of pathogenicity in vivo and other similar characteristics were utilized in the study. Several microscopic fields containing about 200 macrophages were counted. The percentage of macrophages phagocytizing at least one viable or nonviable yeast cell determined an average number of phagocytized yeasts. Neutral red and fluorescein diacetate plus ethidium bromide were used for staining. It is possible to conclude that this is an efficient model related to the used methodology. The average number of yeasts in both strains were similar when inside macrophages, and there was a higher percentage of C. albicans serotype A phagocytosis, which was not experimentally pathogenic in vivo.

  20. Interleukin 17-Mediated Host Defense against Candida albicans

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    Florian Sparber

    2015-08-01

    Full Text Available Candida albicans is part of the normal microbiota in most healthy individuals. However, it can cause opportunistic infections if host defenses are breached, with symptoms ranging from superficial lesions to severe systemic disease. The study of rare congenital defects in patients with chronic mucocutaneous candidiasis led to the identification of interleukin-17 (IL-17 as a key factor in host defense against mucosal fungal infection. Experimental infections in mice confirmed the critical role of IL-17 in mucocutaneous immunity against C. albicans. Research on mouse models has also contributed importantly to our current understanding of the regulation of IL-17 production by different cellular sources and its effector functions in distinct tissues. In this review, we highlight recent findings on IL-17-mediated immunity against C. albicans in mouse and man.

  1. Candida albicans mutant construction and characterization of selected virulence determinants.

    Science.gov (United States)

    Motaung, T E; Albertyn, J; Pohl, C H; Köhler, Gerwald

    2015-08-01

    Candida albicans is a diploid, polymorphic yeast, associated with humans, where it mostly causes no harm. However, under certain conditions it can cause infections ranging from superficial to life threatening. This ability to become pathogenic is often linked to the immune status of the host as well as the expression of certain virulence factors by the yeast. Due to the importance of C. albicans as a pathogen, determination of the molecular mechanisms that allow this yeast to cause disease is important. These studies rely on the ability of researchers to create deletion mutants of specific genes in order to study their function. This article provides a critical review of the important techniques used to create deletion mutants in C. albicans and highlights how these deletion mutants can be used to determine the role of genes in the expression of virulence factors in vitro.

  2. Oral candidiasis-adhesion of non-albicans Candida species

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    Bokor-Bratić Marija B.

    2008-01-01

    Full Text Available Oral candidiasis is an opportunistic infection caused primarily by Candida albicans. However, in recent years, species of non-albicans Candida have been implicated more frequently in mucosal infection. Candida species usually reside as commensal organisms and are part of normal oral microflora. Determining exactly how transformation from commensal to pathogen takes place and how it can be prevented is continuous challenge for clinical doctors. Candidal adherence to mucosal surfaces is considered as a critical initial step in the pathogenesis of oral candidiasis. Acrylic dentures, acting as reservoirs, play an important role in increasing the risk from Candida colonisation. Thus, this review discusses what is currently known about the adhesion of non-albicans Candida species of oral origin to buccal epithelial cells and denture acrylics.

  3. Relationship between salivary flow rates and Candida albicans counts.

    Science.gov (United States)

    Navazesh, M; Wood, G J; Brightman, V J

    1995-09-01

    Seventy-one persons (48 women, 23 men; mean age, 51.76 years) were evaluated for salivary flow rates and Candida albicans counts. Each person was seen on three different occasions. Samples of unstimulated whole, chewing-stimulated whole, acid-stimulated parotid, and candy-stimulated parotid saliva were collected under standardized conditions. An oral rinse was also obtained and evaluated for Candida albicans counts. Unstimulated and chewing-stimulated whole flow rates were negatively and significantly (p or = 500 count. Differences in stimulated parotid flow rates were not significant among different levels of Candida counts. The results of this study reveal that whole saliva is a better predictor than parotid saliva in identification of persons with high Candida albicans counts.

  4. Antimicrobial activity of calcium hydroxide and chlorhexidine on intratubular Candida albicans

    OpenAIRE

    Jacques Rezende Delgado, Ronan; Helena Gasparoto, Thaís; Renata Sipert, Carla; Ramos Pinheiro, Claudia; Gomes de Moraes, Ivaldo; Brandão Garcia, Roberto; Antônio Hungaro Duarte, Marco; Monteiro Bramante, Clóvis; Aparecido Torres, Sérgio; Pompermaier Garlet, Gustavo; Paula Campanelli, Ana; Bernardineli, Norberti

    2013-01-01

    This study investigated the efficacy of calcium hydroxide and chlorhexidine gel for the elimination of intratubular Candida albicans (C. albicans). Human single-rooted teeth contaminated with C. albicans were treated with calcium hydroxide, 2% chlorhexidine gel, calcium hydroxide plus 2% chlorhexidine gel, or saline (0.9% sodium chloride) as a positive control. The samples obtained at depths of 0–100 and 100–200 µm from the root canal system were analyzed for C. albicans load by counting the ...

  5. Effect of Xylitol on Candida albicans resistance in serum (in vitro study)

    OpenAIRE

    Ria Puspitawati; Theodorus Hedwin Kadrianto; Bachtiar, Boy M.; Lakshmi A. Leepel

    2013-01-01

    Xylitol is reported to inhibit the growth of C. albicans. Objectives: Investigating serum factor role in inhibiting the growth of C. albicans and the effect of 1%, 5%, 10% xylitol on C. albicans resistance in serum in vitro. Methods: Identification of C. albicans (oral swab of candidiasis patient) was conducted using CHROMAgar, confirmed by germ tube test. The cultures were serially diluted, inoculated in Saburoud Dextrose Broth (SDB) contained 0% (control), 1%, 5%, or 10% xylitol, and kept f...

  6. Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans.

    Science.gov (United States)

    Khan, Amber; Ahmad, Aijaz; Xess, Immaculata; Khan, Luqman A; Manzoor, Nikhat

    2014-03-15

    Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections. PMID:24252340

  7. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

    Science.gov (United States)

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M; Singh, Ashutosh; Coste, Alix T; Andes, David R; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-06-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  8. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    Science.gov (United States)

    Iborra, A; Sentandreu, R; Gozalbo, D

    1996-09-01

    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  9. Small molecule inhibitors of the Candida albicans budded-to-hyphal transition act through multiple signaling pathways.

    Directory of Open Access Journals (Sweden)

    John Midkiff

    Full Text Available The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT, is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.

  10. 3D Biomaterial Microarrays for Regenerative Medicine

    DEFF Research Database (Denmark)

    Gaharwar, Akhilesh K.; Arpanaei, Ayyoob; Andresen, Thomas Lars;

    2015-01-01

    Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem...

  11. SLIMarray: Lightweight software for microarray facility management

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    Marzolf Bruz

    2006-10-01

    Full Text Available Abstract Background Microarray core facilities are commonplace in biological research organizations, and need systems for accurately tracking various logistical aspects of their operation. Although these different needs could be handled separately, an integrated management system provides benefits in organization, automation and reduction in errors. Results We present SLIMarray (System for Lab Information Management of Microarrays, an open source, modular database web application capable of managing microarray inventories, sample processing and usage charges. The software allows modular configuration and is well suited for further development, providing users the flexibility to adapt it to their needs. SLIMarray Lite, a version of the software that is especially easy to install and run, is also available. Conclusion SLIMarray addresses the previously unmet need for free and open source software for managing the logistics of a microarray core facility.

  12. Polymer microarrays for cell based applications

    OpenAIRE

    Hansen, Anne Klara Brigitte

    2012-01-01

    The development and identification of new biomaterials that can replace specific tissues and organs is desirable. In the presented PhD thesis polymer microarrays were applied for the screening of polyacrylates and polyurethanes and evaluation for material discovery for applications in the life sciences. In the first part of the thesis, the largest polymer microarray ever made with more than 7000 features was fabricated and subsequently used for the screening of polyacrylates...

  13. Text Mining Perspectives in Microarray Data Mining

    OpenAIRE

    Natarajan, Jeyakumar

    2013-01-01

    Current microarray data mining methods such as clustering, classification, and association analysis heavily rely on statistical and machine learning algorithms for analysis of large sets of gene expression data. In recent years, there has been a growing interest in methods that attempt to discover patterns based on multiple but related data sources. Gene expression data and the corresponding literature data are one such example. This paper suggests a new approach to microarray data mining as ...

  14. Determination of strongly overlapping signaling activity from microarray data

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    Bidaut Ghislain

    2006-02-01

    Full Text Available Abstract Background As numerous diseases involve errors in signal transduction, modern therapeutics often target proteins involved in cellular signaling. Interpretation of the activity of signaling pathways during disease development or therapeutic intervention would assist in drug development, design of therapy, and target identification. Microarrays provide a global measure of cellular response, however linking these responses to signaling pathways requires an analytic approach tuned to the underlying biology. An ongoing issue in pattern recognition in microarrays has been how to determine the number of patterns (or clusters to use for data interpretation, and this is a critical issue as measures of statistical significance in gene ontology or pathways rely on proper separation of genes into groups. Results Here we introduce a method relying on gene annotation coupled to decompositional analysis of global gene expression data that allows us to estimate specific activity on strongly coupled signaling pathways and, in some cases, activity of specific signaling proteins. We demonstrate the technique using the Rosetta yeast deletion mutant data set, decompositional analysis by Bayesian Decomposition, and annotation analysis using ClutrFree. We determined from measurements of gene persistence in patterns across multiple potential dimensionalities that 15 basis vectors provides the correct dimensionality for interpreting the data. Using gene ontology and data on gene regulation in the Saccharomyces Genome Database, we identified the transcriptional signatures of several cellular processes in yeast, including cell wall creation, ribosomal disruption, chemical blocking of protein synthesis, and, criticially, individual signatures of the strongly coupled mating and filamentation pathways. Conclusion This works demonstrates that microarray data can provide downstream indicators of pathway activity either through use of gene ontology or transcription

  15. A New Endogenous Overexpression System of Multidrug Transporters of Candida albicans Suitable for Structural and Functional Studies

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    Atanu eBanerjee

    2016-03-01

    Full Text Available Fungal pathogens have a robust array of multidrug transporters which aid in active expulsion of drugs and xenobiotics to help them evade toxic effects of drugs. Thus, these transporters impose a major impediment to effective chemotherapy. Although the Saccharomyces cerevisiae strain AD1-8u- has catered well to the need of an over-expression system to study drug transport by multidrug transporters of Candida albicans, artefacts associated with a heterologous system could not be excluded. To avoid the issue, we exploited a azole-resistant clinical isolate of C. albicans to develop a new system devoid of three major multidrug transporters (Cdr1p, Cdr2p and Mdr1p for the over-expression of multidrug transporters under native hyperactive CDR1 promoter due to gain of function (GOF mutation in TAC1. The study deals with overexpression and functional characterization of representatives of two major classes of multidrug transporters, Cdr1p and Mdr1p, to prove the functionality of this newly developed endogenous expression system. Expression of native Cdr1 and Mdr1 protein in C. albicans cells was confirmed by confocal microscopy and immunodetection and resulted in increased resistance to the putative substrates as compared to control. The system was further validated by overexpressing a few key mutant variants of Cdr1p and Mdr1p. Together, our data confirms the utility of new endogenous overexpression system which is devoid of artifactual factors as most suited for functional characterization of multidrug transporter proteins of C. albicans.

  16. The Impact of Photobleaching on Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Marcel von der Haar

    2015-09-01

    Full Text Available DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results.

  17. DNA nanostructure-based universal microarray platform for high-efficiency multiplex bioanalysis in biofluids.

    Science.gov (United States)

    Li, Zhenhua; Zhao, Bin; Wang, Dongfang; Wen, Yanli; Liu, Gang; Dong, Haoqing; Song, Shiping; Fan, Chunhai

    2014-10-22

    Microarrays of biomolecules have greatly promoted the development of the fields of genomics, proteomics, and clinical assays because of their remarkably parallel and high-throughput assay capability. Immobilization strategies for biomolecules on a solid support surface play a crucial role in the fabrication of high-performance biological microarrays. In this study, rationally designed DNA tetrahedra carrying three amino groups and one single-stranded DNA extension were synthesized by the self-assembly of four oligonucleotides, followed by high-performance liquid chromatography purification. We fabricated DNA tetrahedron-based microarrays by covalently coupling the DNA tetrahedron onto glass substrates. After their biorecognition capability was evaluated, DNA tetrahedron microarrays were utilized for the analysis of different types of bioactive molecules. The gap hybridization strategy, the sandwich configuration, and the engineering aptamer strategy were employed for the assay of miRNA biomarkers, protein cancer biomarkers, and small molecules, respectively. The arrays showed good capability to anchor capture biomolecules for improving biorecognition. Addressable and high-throughput analysis with improved sensitivity and specificity had been achieved. The limit of detection for let-7a miRNA, prostate specific antigen, and cocaine were 10 fM, 40 pg/mL, and 100 nM, respectively. More importantly, we demonstrated that the microarray platform worked well with clinical serum samples and showed good relativity with conventional chemical luminescent immunoassay. We have developed a novel approach for the fabrication of DNA tetrahedron-based microarrays and a universal DNA tetrahedron-based microarray platform for the detection of different types of bioactive molecules. The microarray platform shows great potential for clinical diagnosis.

  18. Screening for C3 deficiency in newborns using microarrays.

    Directory of Open Access Journals (Sweden)

    Magdalena Janzi

    Full Text Available BACKGROUND: Dried blood spot samples (DBSS from newborns are widely used in neonatal screening for selected metabolic diseases and diagnostic possibilities for additional disorders are continuously being evaluated. Primary immunodeficiency disorders comprise a group of more than one hundred diseases, several of which are fatal early in life. Yet, a majority of the patients are not diagnosed due to lack of high-throughput screening methods. METHODOLOGY/PRINCIPAL FINDINGS: We have previously developed a system using reverse phase protein microarrays for analysis of IgA levels in serum samples. In this study, we extended the applicability of the method to include determination of complement component C3 levels in eluates from DBSS collected at birth. Normal levels of C3 were readily detected in 269 DBSS from healthy newborns, while no C3 was detected in sera and DBSS from C3 deficient patients. CONCLUSIONS/SIGNIFICANCE: The findings suggest that patients with deficiencies of specific serum proteins can be identified by analysis of DBSS using reverse phase protein microarrays.

  19. Daya hambat xylitol dan nistation terhadap pertumbuhan Candida albicans (in vitro) (Inhibition effect of xylitol and nistatin combination on Candida albicans growth (in vitro))

    OpenAIRE

    Sarah Kartimah Djajusman; Udijanto Tedjosasongko; Irmawati Irmawati

    2014-01-01

    Background: The growth of Candida albicans can be controlled by using antifungal such as nystatin. These days we found that using antifungal is not enough to control Candida albicans, we also have to control the intake of sugar by using xylitol. Purpose: Purpose of the study was to determine the optimal inhibitory concentration of xylitol-nystatin in the Candida albicans growth. Methods: This was an in-vitro study using an antimicrobial test of serial dilution with xylitol-nystatin and sucros...

  20. Expression, crystallization and preliminary X-ray data analysis of NT-Als9-2, a fungal adhesin from Candida albicans

    International Nuclear Information System (INIS)

    Details of the expression and crystallization of the N-terminal fragment of Als9-2, an adhesin from the human commensal/pathogenic fungus C. albicans, are reported. Preliminary analysis of the collected X-ray data is also discussed. Candida albicans is a common human fungal commensal that can also cause a range of infections from skin/mucosal ‘thrush’ to severe systemic candidiasis. Adherence to host cells is one of the key determinants of Candida pathogenesis. The Als family of surface proteins has been implicated in adhesion of C. albicans, yet limited information has been published on the structure and mechanism of these fungal adhesins. The N-terminal region of these proteins has been shown to possess adhesive properties, making it a possible target for new therapeutic strategies. Recombinant NT-Als9-2 from C. albicans (residues 18–329) was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.0 Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 34.73, b = 68.71, c = 120.03 Å, α = β = γ = 90° and one molecule in the asymmetric unit. Platinum-derivatized crystals belonged to the same space group, with similar unit-cell parameters, although they were not completely isomorphous

  1. Effects of ambroxol on Candida albicans growth and biofilm formation.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; José, Martínez-Sanmiguel Juan; Isela, Sánchez-Nájera Rosa; Claudio, Cabral-Romero

    2014-04-01

    Typically, the onset of candidiasis is characterised by the appearance of a biofilm of Candida albicans, which is associated with several diseases including oral candidiasis in young and elderly people. The objective of this work was to investigate the in vitro fungicidal activity as well as the antibiofilm activity of ambroxol (AMB) against C. albicans growth. In the present investigation, the fungicidal activity of AMB was established using the cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also the minimum inhibitory concentration (MIC) of AMB required to inhibit the fungal growth was determined. Simultaneously, the antibiofilm activity of AMB was evaluated using fluorescence microscopy. The study revealed that 2 mg ml(-1) of AMB exhibited higher fungicidal activity than 3.3 mg ml(-1) of terbinafine, one of most common commercial antifungals. A MIC of 1 mg ml(-1) was determined for AMB to interfere with C. albicans growth. Furthermore, AMB was found to be effective in inhibiting the biofilm formation of C. albicans and exerted its fungicidal activity against the fungal cells interspersed in the preformed biofilm. The study suggests a potential role of the mucolytic agent, AMB, as an interesting therapeutic alternative in the treatment of oral candidiasis.

  2. PERTUMBUHAN CANDIDA ALBICANS PADA PERMUKAAN POLIESTER EBP-2421

    Directory of Open Access Journals (Sweden)

    Widowati Siswomihardjo

    2015-08-01

    Full Text Available Acrylic resin has been the only polymeric material for denture base for many years. One of the requirements for an ideal polymeric denture base material. It should be resistant to bacterial growth. The growth of Candida albicans on the surface of dentures is a concern for many denture wearers. This organism often is associated with denture stomatitis. A preliminary study showed polyester EBP-2421, a polymeric material for statues can also be manipulated to denture base. This research examined the growth of Candida albicans on the surface of EBP-2421. Research was carried out on strips of polyester EBP-2421 and Selton acrylic resin. Strips were contaminated with Candida albicans for 24 hours. Examinations on polyester EBP-2421 and acrylic resin immersed in saliva significantly differ from the not immersed strips (p<0,05. The lowest frequency were Candida albicans adhered on stripes of polyester EBP-2421 immersed in saliva. This result related with the fact that polyester EBP-2421 has smoother surface topography than acrylic resin.

  3. Al-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation

    NARCIS (Netherlands)

    Bachtiar, Endang W.; Bachtiar, Boy M.; Jarosz, Lucja M.; Amir, Lisa R.; Sunarto, Hari; Ganin, Hadas; Meijler, Michael M.; Krom, Bastiaan P.

    2014-01-01

    Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (Al

  4. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Fikadu G Tafesse

    2015-10-01

    Full Text Available The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  5. Synergistic activity of rabbit granulocyte peptides against Candida albicans.

    OpenAIRE

    Lehrer, R I; Szklarek, D; Ganz, T; Selsted, M E

    1986-01-01

    Rabbit granulocytes contain six antimicrobial peptides that are structurally homologous to the human neutrophil "defensins." NP-5, a rabbit defensin, lacks significant activity against Candida albicans. Nevertheless, its addition to submicromolar concentrations of rabbit NP-1, NP-2, or NP-3a potentiates their candidacidal effect. Thus, granulocyte defensins can act synergistically against potential pathogens.

  6. Host defence against disseminated and invasive Candida albicans infections

    NARCIS (Netherlands)

    Vonk, Alouise Gabrielle

    2004-01-01

    The yeast Candida albicans is the primary etiologic agent of disseminated and invasive candidiasis. The incidence of disseminated and invasive candidiasis has paralleled the use of modern medical procedures that adversely affect the immune system, and highlights the difficulty of treating disseminat

  7. Chlorhexidine markedly potentiates the oxidants scavenging abilities of Candida albicans.

    Science.gov (United States)

    Ginsburg, I; Koren, E; Feuerstein, O; Zogakis, I P; Shalish, M; Gorelik, S

    2015-10-01

    The oxidant scavenging ability (OSA) of catalase-rich Candida albicans is markedly enhanced by chlorhexidine digluconate (CHX), polymyxin B, the bile salt ursodeoxycholate and by lysophosphatidylcholine, which all act as detergents facilitating the penetration of oxidants and their intracellular decomposition. Quantifications of the OSA of Candida albicans were measured by a highly sensitive luminol-dependent chemiluminescence assay and by the Thurman's assay, to quantify hydrogen peroxide (H2O2). The OSA enhancing activity by CHX depends to some extent on the media on which candida grew. The OSA of candida treated by CHX was modulated by whole human saliva, red blood cells, lysozyme, cationic peptides and by polyphenols. Concentrations of CHX, which killed over 95 % of Candida albicans cells, did not affect the cells' abilities to scavenge reactive oxygen species (ROS). The OSA of Candida cells treated by CHX is highly refractory to H2O2 (50 mM) but is strongly inhibited by hypochlorous acid, lecithin, trypan blue and by heparin. We speculate that similarly to catalase-rich red blood cells, Candida albicans and additional catalase-rich microbiota may also have the ability to scavenge oxidants and thus can protect catalase-negative anaerobes and facultative anaerobes cariogenic streptococci against peroxide and thus secure their survival in the oral cavity.

  8. Hyphal content determines the compression strength of Candida albicans biofilms

    NARCIS (Netherlands)

    Paramonova, Ekaterina; Krom, Bastiaan P.; van der Mei, Henny C.; Busscher, Henk J.; Sharma, Prashant K.

    2009-01-01

    Candida albicans is the most frequently isolated human fungal pathogen among species causing biofilm-related clinical infections. Mechanical properties of Candida biofilms have hitherto been given no attention, despite the fact that mechanical properties are important for selection of treatment or d

  9. Ocorrência de Candida albicans em intestinos de bovinos

    Directory of Open Access Journals (Sweden)

    Souza W.A.

    2003-01-01

    Full Text Available Foram realizadas a identificação e a sorotipagem de C. albicans isoladas de fezes de bovinos em amamentação natural. Para o isolamento, utilizou-se o meio seletivo e diferencial de Pagano Levin, adicionado de bifenilo na concentração final de 0,1%. De 210 bovinos inicialmente considerados, 70 adultos, 68 bezerros após o desmame e 72 bezerros em fase de amamentação natural, observou-se positividade para C. albicans somente em nove amostras de fezes de bezerros em fase de amamentação (12,5%. A determinação do sorotipo por meio de provas de aglutinação direta em lâmina, com soros monoespecíficos, revelou que a totalidade das amostras isoladas pertencia ao sorotipo A. O bifenilo na concentração de 0,1% mostrou-se inibitório para a maioria dos bolores sem, aparentemente, afetar a viabilidade de C. albicans. O isolamento de C. albicans somente a partir de fezes de bezerros em amamentação, provavelmente, está relacionado à dieta láctea.

  10. Resistance of Candida albicans biofilms to antifungal agents in vitro.

    OpenAIRE

    Hawser, S. P.; Douglas, L J

    1995-01-01

    Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by [3H]leucine incorporation and tetrazolium reduction assays. Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells. These findings were confirmed by scanning electron microscopy of the biofilms.

  11. Reduced virulence of Candida albicans mutants affected in multidrug resistance.

    OpenAIRE

    Becker, J. M.; Henry, L K; Jiang, W; Koltin, Y.

    1995-01-01

    Disruption of a multidrug resistance gene (CaMDR1) in Candida albicans resulted in mutant strains that colonized mouse kidneys to very high levels but were markedly reduced in their virulence. No obvious differences in several properties related to colonization and dissemination were noted among MDR+ or mdr- strains. These results suggest that specific fungal efflux pumps play a role in fungal pathogenicity.

  12. Genetic Relationship between Human and Animal Isolates of Candida albicans

    OpenAIRE

    Edelmann, Anke; Krüger, Monika; SCHMID, JAN

    2005-01-01

    Analyzing Candida albicans isolates from different human and animal individuals by Ca3 fingerprinting, we obtained no evidence for host-specific genotypes and for the existence of species-specific lineages, even though a certain degree of separation between human and animal isolates was found. Therefore, animals could potentially serve as reservoirs for human Candida infection.

  13. Effects of ambroxol on Candida albicans growth and biofilm formation.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; José, Martínez-Sanmiguel Juan; Isela, Sánchez-Nájera Rosa; Claudio, Cabral-Romero

    2014-04-01

    Typically, the onset of candidiasis is characterised by the appearance of a biofilm of Candida albicans, which is associated with several diseases including oral candidiasis in young and elderly people. The objective of this work was to investigate the in vitro fungicidal activity as well as the antibiofilm activity of ambroxol (AMB) against C. albicans growth. In the present investigation, the fungicidal activity of AMB was established using the cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also the minimum inhibitory concentration (MIC) of AMB required to inhibit the fungal growth was determined. Simultaneously, the antibiofilm activity of AMB was evaluated using fluorescence microscopy. The study revealed that 2 mg ml(-1) of AMB exhibited higher fungicidal activity than 3.3 mg ml(-1) of terbinafine, one of most common commercial antifungals. A MIC of 1 mg ml(-1) was determined for AMB to interfere with C. albicans growth. Furthermore, AMB was found to be effective in inhibiting the biofilm formation of C. albicans and exerted its fungicidal activity against the fungal cells interspersed in the preformed biofilm. The study suggests a potential role of the mucolytic agent, AMB, as an interesting therapeutic alternative in the treatment of oral candidiasis. PMID:24224742

  14. Zebrafish Egg Infection Model for Studying Candida albicans Adhesion Factors.

    Directory of Open Access Journals (Sweden)

    Yin-Zhi Chen

    Full Text Available Disseminated candidiasis is associated with 30-40% mortality in severely immunocompromised patients. Among the causal agents, Candida albicans is the dominant one. Various animal models have been developed for investigating gene functions in C. albicans. Zebrafish injection models have increasingly been applied in elucidating C. albicans pathogenesis because of the conserved immunity, prolific fecundity of the zebrafish and the low costs of care systems. In this study, we established a simple, noninvasive zebrafish egg bath infection model, defined its optimal conditions, and evaluated the model with various C. albicans mutant strains. The deletion of SAP6 did not have significant effect on the virulence. By contrast, the deletion of BCR1, CPH1, EFG1, or TEC1 significantly reduced the virulence under current conditions. Furthermore, all embryos survived when co-incubated with bcr1/bcr1, cph1/cph1 efg1/efg1, efg1/efg1, or tec1/tec1 mutant cells. The results indicated that our novel zebrafish model is time-saving and cost effective.

  15. Microarray analysis of E-box binding-related gene expression in young and replicatively senescent human fibroblasts.

    Science.gov (United States)

    Semov, Alexandre; Marcotte, Richard; Semova, Natalie; Ye, Xiangyun; Wang, Eugenia

    2002-03-01

    An E-box (CACGTG) designer microarray was developed to monitor a group of genes whose expressions share a particular regulatory mode. Sensitivity and specificity of microarray hybridization, as well as variability of microarray data, were evaluated. This designer microarray was used to generate expression profiles of E-box binding-related genes in WI-38 fibroblast cultures at three different growth states: low-passage replicating, low-passage contact-inhibited quiescent, and replicatively senescent. Microarray gene screening reveals that quiescent and senescent cells, in comparison with replicating ones, are characterized by downregulation of Pam, a protein associated with c-Myc, and upregulation of Mad family genes, Max dimerization proteins. Moreover, quiescence and senescence can be distinguished by increased expression of Irlb, c-Myc transcription factor, and Miz-1, c-Myc-interacting Zn finger protein 1, only in the former state. Senescence is characterized by downregulation of Id4, inhibitor of DNA binding 4, and Mitf, microphthalmia-associated transcription factor, in comparison with young replicating and quiescent states. Differential expression of genes detected by microarray hybridization was independently confirmed by reverse transcription polymerase chain reaction technique. Alterations in the expression of E-box-binding transcription factors and c-Myc-binding proteins demonstrate the importance of these genes in establishing the contact-inhibited quiescent or senescent phenotypes.

  16. HIV aspartyl protease inhibitors as promising compounds against Candida albicans

    Institute of Scientific and Technical Information of China (English)

    André; Luis; Souza; dos; Santos

    2010-01-01

    Cells of Candida albicans(C.albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated my coses of almost all inner organs,especially in immunocompromised patients.In this context,both the host immune status and the ability of C.albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance;in this last case,culminating in the establishment of successful infection knownas candidiasis.C.albicans possesses a potent arma-mentarium consisting of several virulence moleculesthat help the fungal cells to escape of the host immuneresponses.There is no doubt that the secretion of aspartyl-type proteases,designated as Saps,are one of the major virulence attributes produced by C.albicans cells,since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions.For these reasons,Saps clearly hold promise as new potential drug targets.Corroborating this hypothesis,the introduction of new anti-human immunodeficiency virus drugs of the as party l protease inhibitor-type(HIV PIs) have emerged as new agents for the inhibition of Saps.The introduction of HIV PIs has revolutionized the treatment of HIV disease,reducing opportunistic infections,especially candidiasis.The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status,but also as a result of direct inhibition of C.albicans Saps.In this article,we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C.albicans,focusing on the effects of these compounds on Sap activity,growth behavior,morphological architecture,cellular differentiation,fungal adhesion to animal cells and abiotic materials,modulation of virulence factors,experimental candidiasis infection,and their synergistic actions with classical antifungal agents.

  17. Photopatterning of Hydrogel Microarrays in Closed Microchips.

    Science.gov (United States)

    Gumuscu, Burcu; Bomer, Johan G; van den Berg, Albert; Eijkel, Jan C T

    2015-12-14

    To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications.

  18. A piglet model for studying Candida albicans colonization of the human oro-gastrointestinal tract.

    Science.gov (United States)

    Hoeflinger, Jennifer L; Coleman, David A; Oh, Soon-Hwan; Miller, Michael J; Hoyer, Lois L

    2014-08-01

    Pigs from a variety of sources were surveyed for oro-gastrointestinal (oro-GIT) carriage of Candida albicans. Candida albicans-positive animals were readily located, but we also identified C. albicans-free pigs. We hypothesized that pigs could be stably colonized with a C. albicans strain of choice, simply by feeding yeast cells. Piglets were farrowed routinely and remained with the sow for 4 days to acquire a normal microbiota. Piglets were then placed in an artificial rearing environment and fed sow milk replacer. Piglets were inoculated orally with one of three different C. albicans strains. Piglets were weighed daily, and culture swabs were collected to detect C. albicans orally, rectally and in the piglet's environment. Stable C. albicans colonization over the course of the study did not affect piglet growth. Necropsy revealed mucosally associated C. albicans throughout the oro-GIT with the highest abundance in the esophagus. Uninoculated control piglets remained C. albicans-negative. These data establish the piglet as a model to study C. albicans colonization of the human oro-GIT. Similarities between oro-GIT colonization in humans and pigs, as well as the ease of working with the piglet model, suggest its adaptability for use among investigators interested in understanding C. albicans-host commensal interactions.

  19. Intra-amniotic Candida albicans infection induces mucosal injury and inflammation in the ovine fetal intestine.

    Science.gov (United States)

    Nikiforou, Maria; Jacobs, Esmee M R; Kemp, Matthew W; Hornef, Mathias W; Payne, Matthew S; Saito, Masatoshi; Newnham, John P; Janssen, Leon E W; Jobe, Alan H; Kallapur, Suhas G; Kramer, Boris W; Wolfs, Tim G A M

    2016-01-01

    Chorioamnionitis is caused by intrauterine infection with microorganisms including Candida albicans (C.albicans). Chorioamnionitis is associated with postnatal intestinal pathologies including necrotizing enterocolitis. The underlying mechanisms by which intra-amniotic C.albicans infection adversely affects the fetal gut remain unknown. Therefore, we assessed whether intra-amniotic C.albicans infection would cause intestinal inflammation and mucosal injury in an ovine model. Additionally, we tested whether treatment with the fungistatic fluconazole ameliorated the adverse intestinal outcome of intra-amniotic C.albicans infection. Pregnant sheep received intra-amniotic injections with 10(7) colony-forming units C.albicans or saline at 3 or 5 days before preterm delivery at 122 days of gestation. Fetuses were given intra-amniotic and intra-peritoneal fluconazole treatments 2 days after intra-amniotic administration of C.albicans. Intra-amniotic C.albicans caused intestinal colonization and invasive growth within the fetal gut with mucosal injury and intestinal inflammation, characterized by increased CD3(+) lymphocytes, MPO(+) cells and elevated TNF-α and IL-17 mRNA levels. Fluconazole treatment in utero decreased intestinal C.albicans colonization, mucosal injury but failed to attenuate intestinal inflammation. Intra-amniotic C.albicans caused intestinal infection, injury and inflammation. Fluconazole treatment decreased mucosal injury but failed to ameliorate C.albicans-mediated mucosal inflammation emphasizing the need to optimize the applied antifungal therapeutic strategy. PMID:27411776

  20. Synergistic Effect of Fluconazole and Calcium Channel Blockers against Resistant Candida albicans.

    Directory of Open Access Journals (Sweden)

    Shuyuan Liu

    Full Text Available Candidiasis has increased significantly recently that threatens patients with low immunity. However, the number of antifungal drugs on the market is limited in comparison to the number of available antibacterial drugs. This fact, coupled with the increased frequency of fungal resistance, makes it necessary to develop new therapeutic strategies. Combination drug therapy is one of the most widely used and effective strategy to alleviate this problem. In this paper, we were aimed to evaluate the combined antifungal effects of four CCBs (calcium channel blockers, amlodipine (AML, nifedipine (NIF, benidipine (BEN and flunarizine (FNZ with fluconazole against C. albicans by checkerboard and time-killing method. In addition, we determined gene (CCH1, MID1, CNA1, CNB1, YVC1, CDR1, CDR2 and MDR1 expression by quantitative PCR and investigated the efflux pump activity of resistant candida albicans by rhodamine 6G assay to reveal the potential mechanisms. Finally, we concluded that there was a synergy when fluconazole combined with the four tested CCBs against resistant strains, with fractional inhibitory concentration index (FICI <0.5, but no interaction against sensitive strains (FICI = 0.56 ~ 2. The mechanism studies revealed that fluconazole plus amlodipine caused down-regulating of CNA1, CNB1 (encoding calcineurin and YVC1 (encoding calcium channel protein in vacuole membrane.

  1. Synergistic Effect of Fluconazole and Calcium Channel Blockers against Resistant Candida albicans.

    Science.gov (United States)

    Liu, Shuyuan; Yue, Longtao; Gu, Wenrui; Li, Xiuyun; Zhang, Liuping; Sun, Shujuan

    2016-01-01

    Candidiasis has increased significantly recently that threatens patients with low immunity. However, the number of antifungal drugs on the market is limited in comparison to the number of available antibacterial drugs. This fact, coupled with the increased frequency of fungal resistance, makes it necessary to develop new therapeutic strategies. Combination drug therapy is one of the most widely used and effective strategy to alleviate this problem. In this paper, we were aimed to evaluate the combined antifungal effects of four CCBs (calcium channel blockers), amlodipine (AML), nifedipine (NIF), benidipine (BEN) and flunarizine (FNZ) with fluconazole against C. albicans by checkerboard and time-killing method. In addition, we determined gene (CCH1, MID1, CNA1, CNB1, YVC1, CDR1, CDR2 and MDR1) expression by quantitative PCR and investigated the efflux pump activity of resistant candida albicans by rhodamine 6G assay to reveal the potential mechanisms. Finally, we concluded that there was a synergy when fluconazole combined with the four tested CCBs against resistant strains, with fractional inhibitory concentration index (FICI) fluconazole plus amlodipine caused down-regulating of CNA1, CNB1 (encoding calcineurin) and YVC1 (encoding calcium channel protein in vacuole membrane).

  2. A core filamentation response network in Candida albicans is restricted to eight genes.

    Directory of Open Access Journals (Sweden)

    Ronny Martin

    Full Text Available Although morphological plasticity is a central virulence trait of Candida albicans, the number of filament-associated genes and the interplay of mechanisms regulating their expression remain unknown. By correlation-based network modeling of the transcriptional response to different defined external stimuli for morphogenesis we identified a set of eight genes with highly correlated expression patterns, forming a core filamentation response. This group of genes included ALS3, ECE1, HGT2, HWP1, IHD1 and RBT1 which are known or supposed to encode for cell- wall associated proteins as well as the Rac1 guanine nucleotide exchange factor encoding gene DCK1 and the unknown function open reading frame orf19.2457. The validity of network modeling was confirmed using a dataset of advanced complexity that describes the transcriptional response of C. albicans during epithelial invasion as well as comparing our results with other previously published transcriptome studies. Although the set of core filamentation response genes was quite small, several transcriptional regulators are involved in the control of their expression, depending on the environmental condition.

  3. Single-cell force spectroscopy of the medically important Staphylococcus epidermidis-Candida albicans interaction

    Science.gov (United States)

    Beaussart, Audrey; Herman, Philippe; El-Kirat-Chatel, Sofiane; Lipke, Peter N.; Kucharíková, Soňa; van Dijck, Patrick; Dufrêne, Yves F.

    2013-10-01

    Despite the clinical importance of bacterial-fungal interactions, their molecular details are poorly understood. A hallmark of such medically important interspecies associations is the interaction between the two nosocomial pathogens Staphylococcus aureus and Candida albicans, which can lead to mixed biofilm-associated infections with enhanced antibiotic resistance. Here, we use single-cell force spectroscopy (SCFS) to quantify the forces engaged in bacterial-fungal co-adhesion, focusing on the poorly investigated S. epidermidis-C. albicans interaction. Force curves recorded between single bacterial and fungal germ tubes showed large adhesion forces (~5 nN) with extended rupture lengths (up to 500 nm). By contrast, bacteria poorly adhered to yeast cells, emphasizing the important role of the yeast-to-hyphae transition in mediating adhesion to bacterial cells. Analysis of mutant strains altered in cell wall composition allowed us to distinguish the main fungal components involved in adhesion, i.e. Als proteins and O-mannosylations. We suggest that the measured co-adhesion forces are involved in the formation of mixed biofilms, thus possibly as well in promoting polymicrobial infections. In the future, we anticipate that this SCFS platform will be used in nanomedicine to decipher the molecular mechanisms of a wide variety of pathogen-pathogen interactions and may help in designing novel anti-adhesion agents.

  4. Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.

    Science.gov (United States)

    Gazendam, Roel P; van de Geer, Annemarie; van Hamme, John L; Tool, Anton T J; van Rees, Dieke J; Aarts, Cathelijn E M; van den Biggelaar, Maartje; van Alphen, Floris; Verkuijlen, Paul; Meijer, Alexander B; Janssen, Hans; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-05-01

    Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content. PMID:26802050

  5. Study of Candida Albicans Vaginitis Model in Kunming Mice

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhuo; KONG Xiaofeng

    2007-01-01

    The model of vaginal candidiasis in Kunming mice was constructed in order to search for the optima construction conditions and provide an economic animal model of Candida albicans (C.albicans) vaginitis. Estrogen benzoate (E2) was given to mice at different concentrations ranging from 0.0 to 0.05 mg/mouse (4 levels) beginning 72 h prior to vaginal inoculation, then mice were inoculated intravaginally with various concentrations of stationary-phase C. albicans blastoconidia (ATCC90028) (5 levels) in 20 μL of phosphate-buffered saline (PBS) in each F2 level. General state,scores of genital pathology, the hyphae and vaginal fungal burden (CFU) in vaginal lavage fluid, the hydrops rate of uterus and vaginal tissues for pathological section in mice were observed and obtained at day 2, 4, 7, 14 and 21 after inoculation. The results showed the infection rate in mice was related to the dosage of E2 and concentration of C. albicans blastoconidia. Additionally there was better cross-effect between the two treated factors. The infection rate was about 80% on the day 4,and could reach 100% on the day 7 until the end of experiment after inoculated intravaginally in groups of E2I3, E2 0.025 mg/mouse injected hypodermically and inoculated intravaginally with 5×104 C. albicans blastoconidia, and large amount of hyphae and blastoconidia could be observe in superficial layer tissue and canal of vaginal by PAS. From the results in our experiment it was concluded that E2I3 was the optima construction condition in kunming mice.

  6. rapmad: Robust analysis of peptide microarray data

    Directory of Open Access Journals (Sweden)

    Rothermel Andrée

    2011-08-01

    Full Text Available Abstract Background Peptide microarrays offer an enormous potential as a screening tool for peptidomics experiments and have recently seen an increased field of application ranging from immunological studies to systems biology. By allowing the parallel analysis of thousands of peptides in a single run they are suitable for high-throughput settings. Since data characteristics of peptide microarrays differ from DNA oligonucleotide microarrays, computational methods need to be tailored to these specifications to allow a robust and automated data analysis. While follow-up experiments can ensure the specificity of results, sensitivity cannot be recovered in later steps. Providing sensitivity is thus a primary goal of data analysis procedures. To this end we created rapmad (Robust Alignment of Peptide MicroArray Data, a novel computational tool implemented in R. Results We evaluated rapmad in antibody reactivity experiments for several thousand peptide spots and compared it to two existing algorithms for the analysis of peptide microarrays. rapmad displays competitive and superior behavior to existing software solutions. Particularly, it shows substantially improved sensitivity for low intensity settings without sacrificing specificity. It thereby contributes to increasing the effectiveness of high throughput screening experiments. Conclusions rapmad allows the robust and sensitive, automated analysis of high-throughput peptide array data. The rapmad R-package as well as the data sets are available from http://www.tron-mz.de/compmed.

  7. "On-chip magnetic bead microarray using hydrodynamic focusing in a passive magnetic separator"

    DEFF Research Database (Denmark)

    Smistrup, Kristian; Kjeldsen, B.; Reimers, R.L.;

    2005-01-01

    Implementing DNA and protein microarrays into lab-on-a-chip systems can be problematic since these are sensitive to heat and strong chemicals. Here, we describe the functionalization of a microchannel with two types of magnetic beads using hydrodynamic focusing combined with a passive magnetic...

  8. Point-of-care vertical flow allergen microarray assay: proof of concept

    NARCIS (Netherlands)

    Chinnasamy, Thiruppathiraja; Segerink, Loes I.; Nystrand, Mats; Gantelius, Jesper; Andersson Svahn, Helene

    2014-01-01

    BACKGROUND: Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based

  9. Gene expression profiling in peanut using high density oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Burow Mark

    2009-06-01

    Full Text Available Abstract Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B, oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.

  10. DNA Microarrays in Comparative Genomics and Transcriptomics

    DEFF Research Database (Denmark)

    Willenbrock, Hanni

    2007-01-01

    data. For this, the DNA microarray technology has gained enormous popularity due to its ability to measure the presence or the activity of thousands of genes simultaneously. Microarrays for high throughput data analyses are not limited to a few organisms but may be applied to everything from bacteria...... at identifying the exact breakpoints where DNA has been gained or lost. In this thesis, three popular methods are compared and a realistic simulation model is presented for generating artificial data with known breakpoints and known DNA copy number. By using simulated data, we obtain a realistic evaluation...... of various strains of the bacteria, e.g. Escherichia coli, with regard to genes involved in pathogenesis. Finally, this thesis present results demonstrating that the gene expression level is sequence dependent, that is, it depends on both DNA structure and codon usage bias. Here, microarray data was used...

  11. Finding consistent disease subnetworks across microarray datasets

    Directory of Open Access Journals (Sweden)

    Soh Donny

    2011-11-01

    Full Text Available Abstract Background While contemporary methods of microarray analysis are excellent tools for studying individual microarray datasets, they have a tendency to produce different results from different datasets of the same disease. We aim to solve this reproducibility problem by introducing a technique (SNet. SNet provides both quantitative and descriptive analysis of microarray datasets by identifying specific connected portions of pathways that are significant. We term such portions within pathways as “subnetworks”. Results We tested SNet on independent datasets of several diseases, including childhood ALL, DMD and lung cancer. For each of these diseases, we obtained two independent microarray datasets produced by distinct labs on distinct platforms. In each case, our technique consistently produced almost the same list of significant nontrivial subnetworks from two independent sets of microarray data. The gene-level agreement of these significant subnetworks was between 51.18% to 93.01%. In contrast, when the same pairs of microarray datasets were analysed using GSEA, t-test and SAM, this percentage fell between 2.38% to 28.90% for GSEA, 49.60% tp 73.01% for t-test, and 49.96% to 81.25% for SAM. Furthermore, the genes selected using these existing methods did not form subnetworks of substantial size. Thus it is more probable that the subnetworks selected by our technique can provide the researcher with more descriptive information on the portions of the pathway actually affected by the disease. Conclusions These results clearly demonstrate that our technique generates significant subnetworks and genes that are more consistent and reproducible across datasets compared to the other popular methods available (GSEA, t-test and SAM. The large size of subnetworks which we generate indicates that they are generally more biologically significant (less likely to be spurious. In addition, we have chosen two sample subnetworks and validated them with

  12. Discovering biological progression underlying microarray samples.

    Directory of Open Access Journals (Sweden)

    Peng Qiu

    2011-04-01

    Full Text Available In biological systems that undergo processes such as differentiation, a clear concept of progression exists. We present a novel computational approach, called Sample Progression Discovery (SPD, to discover patterns of biological progression underlying microarray gene expression data. SPD assumes that individual samples of a microarray dataset are related by an unknown biological process (i.e., differentiation, development, cell cycle, disease progression, and that each sample represents one unknown point along the progression of that process. SPD aims to organize the samples in a manner that reveals the underlying progression and to simultaneously identify subsets of genes that are responsible for that progression. We demonstrate the performance of SPD on a variety of microarray datasets that were generated by sampling a biological process at different points along its progression, without providing SPD any information of the underlying process. When applied to a cell cycle time series microarray dataset, SPD was not provided any prior knowledge of samples' time order or of which genes are cell-cycle regulated, yet SPD recovered the correct time order and identified many genes that have been associated with the cell cycle. When applied to B-cell differentiation data, SPD recovered the correct order of stages of normal B-cell differentiation and the linkage between preB-ALL tumor cells with their cell origin preB. When applied to mouse embryonic stem cell differentiation data, SPD uncovered a landscape of ESC differentiation into various lineages and genes that represent both generic and lineage specific processes. When applied to a prostate cancer microarray dataset, SPD identified gene modules that reflect a progression consistent with disease stages. SPD may be best viewed as a novel tool for synthesizing biological hypotheses because it provides a likely biological progression underlying a microarray dataset and, perhaps more importantly, the

  13. The use of microarrays in microbial ecology

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

    2009-09-15

    Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer

  14. Prominent feature selection of microarray data

    Institute of Scientific and Technical Information of China (English)

    Yihui Liu

    2009-01-01

    For wavelet transform, a set of orthogonal wavelet basis aims to detect the localized changing features contained in microarray data. In this research, we investigate the performance of the selected wavelet features based on wavelet detail coefficients at the second level and the third level. The genetic algorithm is performed to optimize wavelet detail coefficients to select the best discriminant features. Exper-iments are carried out on four microarray datasets to evaluate the performance of classification. Experimental results prove that wavelet features optimized from detail coefficients efficiently characterize the differences between normal tissues and cancer tissues.

  15. Splicing-Sensitive DNA-Microarrays: Peculiarities and Applicationin Biomedical Research (Review

    Directory of Open Access Journals (Sweden)

    D.I. Knyazev

    2015-12-01

    Full Text Available Alternative splicing (АS provides a variety of protein and mature mRNA isoforms encoded by a single gene, and is the essential component of cell and tissue differentiation and functioning. DNA-microarrays are highly productive transcriptome research technique both at the level of total gene expression assessment and alternatively spliced mRNA isoforms exploration. The study of AS patterns requires thorough probe design to achieve appropriate accuracy of the analysis. There are two types of splicing-sensitive DNA-microarrays. The first type contain probes targeted to internal exonic sequences (exon bodies; the second type contain probes targeted to exon bodies and exon–exon and exon–intron junctions. So, the first section focused on probe sequence design, general features of splicing-sensitive DNA-microarrays and their main advantages and limitations. The results of AS research obtained using DNA-microarrays have been reviewed in special section. In particular, DNA-microarrays were used to reveal a number pre-mRNA processing and splicing mechanisms, to investigate AS patterns associated with cancer, cell and tissue differentiation. Splicing machinery regulation was demonstrated to be an essential step during carcinogenesis and differentiation. The examples of application of splicing-sensitive DNA-microarrays for diagnostic markers discovering and pathology mechanism elucidation were also reviewed. Investigations of AS role in pluripotency, stem cell commitment, immune and infected cells functioning during immune response are the promising future directions. Splicing-sensitive DNA-microarrays are relatively inexpensive but powerful research tool that give reason to suppose their introduction in clinical practice within the next few years.

  16. Comparative transcript profiling of Candida albicans and Candida dubliniensis identifies SFL2, a C. albicans gene required for virulence in a reconstituted epithelial infection model.

    LENUS (Irish Health Repository)

    Spiering, Martin J

    2010-02-01

    Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. DeltaDeltasfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the DeltaDeltasfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, DeltaDeltasfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

  17. Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results

    Directory of Open Access Journals (Sweden)

    Dai Yilin

    2012-06-01

    Full Text Available Abstract Background Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. Findings We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Conclusion Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

  18. The Potentials and Pitfalls of Microarrays in Neglected Tropical Diseases: A Focus on Human Filarial Infections.

    Science.gov (United States)

    Kwarteng, Alexander; Ahuno, Samuel Terkper

    2016-01-01

    Data obtained from expression microarrays enables deeper understanding of the molecular signatures of infectious diseases. It provides rapid and accurate information on how infections affect the clustering of gene expression profiles, pathways and networks that are transcriptionally active during various infection states compared to conventional diagnostic methods, which primarily focus on single genes or proteins. Thus, microarray technologies offer advantages in understanding host-parasite interactions associated with filarial infections. More importantly, the use of these technologies can aid diagnostics and helps translate current genomic research into effective treatment and interventions for filarial infections. Studying immune responses via microarray following infection can yield insight into genetic pathways and networks that can have a profound influence on the development of anti-parasitic vaccines. PMID:27600086

  19. Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Toepel Jörg

    2011-11-01

    Full Text Available Abstract Background Chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. The complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. Based on the completed annotation of Chlamydomonas reinhardtii a new microarray on an Agilent platform was designed using an extended JGI 3.1 genome data set which included 15000 transcript models. Results In total 44000 probes were determined (3 independent probes per transcript model covering 93% of the transcriptome. Alignment studies with the recently published AUGUSTUS 10.2 annotation confirmed 11000 transcript models resulting in a very good coverage of 70% of the transcriptome (17000. Following the estimation of 10000 predicted genes in Chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. Conclusions To demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and RNA-seq data. We could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport.

  20. Surface-activated microtiter-plate microarray for simultaneous CRP quantification and viral antibody detection.

    Science.gov (United States)

    Viitala, Sari M; Jääskeläinen, Anne J; Kelo, Eira; Sirola, Helena; Moilanen, Kirsi; Suni, Jukka; Vaheri, Antti; Vapalahti, Olli; Närvänen, Ale

    2013-02-01

    Microarrays are widely used in high-throughput DNA and RNA hybridization tests and recently adopted to protein and small molecule interaction studies in basic research and diagnostics. Parallel detection of serum antibodies and antigens has several potential applications in epidemiologic research, vaccine development, and in the diagnosis of allergies, autoimmunity, and infectious diseases. This study demonstrates an immobilization method for immunoassay-based microarray in conventional 96-well polystyrene plates for a serologic diagnostic method combined with quantitative C-reactive protein (CRP) assay. A synthetic peptide (HIV-1), a recombinant protein (Puumala hantavirus nucleocapsid), and purified virus preparations (Sindbis and adenoviruses) were used as antigens for virus-specific antibody detection and monoclonal anti-CRP antibody for antigen detection. The microarray was based on conventional enzyme immunoassays and densitometry from photographed results. Peptide and recombinant antigens functioned well, while whole virus antigens gave discrepant results in 1 out of 23 samples from the reference method, tested with human sera with various antibody responses. The CRP results were in concordance in the concentration range 0.5-150 mg/L with 2 commercially available CRP assays: ReaScan rapid test (R(2) = 0.9975) and Cobas 6000 analyzer (R(2) =0.9595). The results indicate that microtiter plates provide a promising platform for further development of microarrays for parallel antibody and antigen detection. PMID:23219230

  1. Recurrent Candida albicans Ventriculitis Treated with Intraventricular Liposomal Amphotericin B

    Directory of Open Access Journals (Sweden)

    Demet Toprak

    2015-01-01

    Full Text Available Central nervous system (CNS infection with Candida is rare but significant because of its high morbidity and mortality. When present, it is commonly seen among immunocompromised and hospitalized patients. Herein, we describe a case of a four-year-old boy with acute lymphoblastic leukemia (ALL who experienced recurrent Candida albicans meningitis. The patient was treated successfully with intravenous liposomal amphotericin B at first attack, but 25 days after discharge he was readmitted to hospital with symptoms of meningitis. Candida albicans was grown in CFS culture again and cranial magnetic resonance imaging (MRI showed ventriculitis. We administered liposomal amphotericin B both intravenously and intraventricularly and favorable result was achieved without any adverse effects. Intraventricular amphotericin B may be considered for the treatment of recurrent CNS Candida infections in addition to intravenous administration.

  2. Phenotypic consequences of LYS4 gene disruption in Candida albicans.

    Science.gov (United States)

    Gabriel, Iwona; Kur, Krzysztof; Laforce-Nesbitt, Sonia S; Pulickal, Anoop S; Bliss, Joseph M; Milewski, Sławomir

    2014-08-01

    A BLAST search of the Candida Genome Database with the Saccharomyces cerevisiae LYS4 sequence known to encode homoaconitase (HA) revealed ORFs 19.3846 and 19.11327. Both alleles of the LYS4 gene were sequentially disrupted in Candida albicans BWP17 cells using PCR-based methodology. The null lys4Δ mutant exhibited lysine auxotrophy in minimal medium but was able to grow in the presence of l-Lys and α-aminoadipate, an intermediate of the α-aminoadipate pathway, at millimolar concentrations. The presence of d-Lys and pipecolic acid did not trigger lys4Δ growth. The C. albicans lys4Δ mutant cells demonstrated diminished germination ability. However, their virulence in vivo in a murine model of disseminated neonatal candidiasis appeared identical to that of the wild-type strain. Moreover, there was no statistically significant difference in fungal burden of infected tissues between the strains.

  3. Increased Filamentous Growth of Candida albicans in Simulated Microgravity

    Institute of Scientific and Technical Information of China (English)

    Sara D. Altenburg; Sheila M. Nielsen-Preiss; Linda E. Hyman

    2008-01-01

    Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeasthyphal transition. The morphological response may have significant implications for astronauts' safety, as the fungal pathogen may become more virulent during spaceflight.

  4. Low virulent oral Candida albicans strains isolated from smokers.

    Science.gov (United States)

    de Azevedo Izidoro, Ana Claudia Santos; Semprebom, Andressa Marafon; Baboni, Fernanda Brasil; Rosa, Rosimeire Takaki; Machado, Maria Angela Naval; Samaranayake, Lakshman Perera; Rosa, Edvaldo Antonio Ribeiro

    2012-02-01

    It is widely accepted that tabagism is a predisposing factor to oral candidosis and cumulate data suggest that cigarette compounds may increase candidal virulence. To verify if enhanced virulence occurs in Candida albicans from chronic smokers, a cohort of 42 non-smokers and other of 58 smokers (all with excellent oral conditions and without signs of candidosis) were swabbed on tong dorsum and jugal mucosa. Results showed that oral candidal loads do not differ between smoker and non-smokers. Activities of secreted aspartyl-protease (Sap), phospholipase, chondroitinase, esterase-lipase, and haemolysin secretions were screened for thirty-two C. albicans isolates. There were detected significant increments in phospholipasic and chondroitinasic activities in isolates from non-smokers. For other virulence factors, no differences between both cohorts were achieved. PMID:21924704

  5. CaSfl1 plays a dual role in transcriptional regulation in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    ZHANG TingTing; LI Di; LI WanJie; WANG Yue; SANG JianLi

    2008-01-01

    As a newly identified transcription factor in Candida albcians, CaSfl1 has been shown to be involved in cell flocculation and filamentation and in the negative regulation of several genes involved in hyphal growth. In this study, we constructed Casfl1△/△ mutants and confirmed that deletion of this gene in-deed affected cell flocculation and filamentation. In addition, by RT-PCR we found that while Casfl1 repressed the expression of several hypha-specific genes including HWP1, ECE1, ALS1, ALS3, and FL08, it strongly activated the expression of the heat-shock protein genes HSP30 and HSP90 under certain stress conditions. Therefore, we propose that CaSfl1 can act as both positive and negative regulators, thereby playing a dual role in transcriptional controls in Candida albicans.

  6. Molecular Docking Evaluation of Imidazole Analogues as Potent Candida albicans 14α-Demethylase Inhibitors.

    Science.gov (United States)

    Rani, Nidhi; Kumar, Praveen; Singh, Randhir; Sharma, Ajay

    2015-01-01

    Candida albicans is one of the most important causes of life-threating fungal infections. Lanosterol 14α-demethylase (Cytochrome P450DM) is the target enzyme of azole antifungal agents. The study involved selection and modeling of the target enzyme followed by refinement of the model using molecular dynamic simulation. The modeled structure of enzyme was validated using Ramachandran plot and Sequence determination technique. A series of chlorosubstituted imidazole analogues were evaluated for Cytochrome P450 inhibitory activity using molecular docking studies. The imidazole analogues were prepared using Chem sketch and molecular docking was performed using Molergo Virtual Docker program. The docking study indicated that all the imidazole analogues (AN1-AN45) and standard drugs i.e., Ketoconazole, Clotrimazole and Miconazole have interaction with protein residue of 14α-demethylase, Heme cofactor and the water molecules present in the active site. PMID:26081558

  7. ANTAGONISTIC EFFECT OF EDIBLE MUSHROOM EXTRACT ON CANDIDA ALBICANS GROWTH

    Directory of Open Access Journals (Sweden)

    Paccola Edneia A. de Souza

    2001-01-01

    Full Text Available Five species of edible mushrooms, Lentinula edodes, Pleurotus ostreatus, Pholiota nameko, Macrolepiota bonaerensis and Agaricus blazei, were tested for their potential to inhibit the in vitro growth of the pathogenic yeast Candida albicans. Only L. edodes had a fungistatic effect on this human pathogen. The inhibitory compound was produced intra and extracellularly in submersed L. edodes culture, and was also present in fresh and dehydrated mushroom basidiocarps. The fungistatic compound was heat sensitive and lost activity after 72 hours.

  8. Microarray data mining with visual programming

    OpenAIRE

    Xu, Qikai; Curk, Tomaž; Shaulsky, Gad; Petrovič, Uroš; Bratko, Ivan; Zupan, Blaž; Demšar, Janez; Leban, Gregor

    2005-01-01

    Visual programming offers an intuitive means of combining known analysis and visualization methods into powerful applications. The system presented here enables users who are not programmers to manage microarray and genomic data flow and to customize their analyses by combining common data analysis tools to fit their needs.

  9. Shrinkage covariance matrix approach for microarray data

    Science.gov (United States)

    Karjanto, Suryaefiza; Aripin, Rasimah

    2013-04-01

    Microarray technology was developed for the purpose of monitoring the expression levels of thousands of genes. A microarray data set typically consists of tens of thousands of genes (variables) from just dozens of samples due to various constraints including the high cost of producing microarray chips. As a result, the widely used standard covariance estimator is not appropriate for this purpose. One such technique is the Hotelling's T2 statistic which is a multivariate test statistic for comparing means between two groups. It requires that the number of observations (n) exceeds the number of genes (p) in the set but in microarray studies it is common that n Hotelling's T2 statistic with the shrinkage approach is proposed to estimate the covariance matrix for testing differential gene expression. The performance of this approach is then compared with other commonly used multivariate tests using a widely analysed diabetes data set as illustrations. The results across the methods are consistent, implying that this approach provides an alternative to existing techniques.

  10. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    OpenAIRE

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S; Golova, Julia; Perov, Alexander; Protic, Miroslava; Robison, Richard; Schipma, Matthew; White, Amanda; Willse, Alan

    2006-01-01

    A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

  11. Single-species microarrays and comparative transcriptomics.

    Directory of Open Access Journals (Sweden)

    Frédéric J J Chain

    Full Text Available BACKGROUND: Prefabricated expression microarrays are currently available for only a few species but methods have been proposed to extend their application to comparisons between divergent genomes. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that the hybridization intensity of genomic DNA is a poor basis on which to select unbiased probes on Affymetrix expression arrays for studies of comparative transcriptomics, and that doing so produces spurious results. We used the Affymetrix Xenopus laevis microarray to evaluate expression divergence between X. laevis, X. borealis, and their F1 hybrids. When data are analyzed with probes that interrogate only sequences with confirmed identity in both species, we recover results that differ substantially analyses that use genomic DNA hybridizations to select probes. CONCLUSIONS/SIGNIFICANCE: Our findings have implications for the experimental design of comparative expression studies that use single-species microarrays, and for our understanding of divergent expression in hybrid clawed frogs. These findings also highlight important limitations of single-species microarrays for studies of comparative transcriptomics of polyploid species.

  12. Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans

    Science.gov (United States)

    Wang, Hong X.; Douglas, Lois M.; Veselá, Petra; Rachel, Reinhard; Malinsky, Jan; Konopka, James B.

    2016-01-01

    The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7. These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization. PMID:27009204

  13. Emergence of non-albicans candida species in neonatal candidemia

    Directory of Open Access Journals (Sweden)

    Deepak Juyal

    2013-01-01

    Full Text Available Background: Candida species are one of the most common causes of blood stream infections among neonates and account for 9-13% of such infections. Although Candida albicans remains the most common fungal isolate from neonatal candidemia, longitudinal studies have detected a shift towards non-albicans Candida (NAC species. Aim: To examine the prevalence and epidemiology of candidemia among infants admitted to our hospital. Materials and Methods: Blood samples were collected from 548 neonates and only those which yielded pure growth of Candida spp. were included in the study. The isolates were identified as per standard mycological techniques and antifungal susceptibility (AFS was done by disc diffusion method. Results: Of the total 132 neonates included in the study, NAC species were responsible for 80.30% cases with C. parapsilosis (25.0% and C. tropicalis (21.97% as the most predominant species; whereas 19.70% of cases were caused by C. albicans. AFS results revealed that 65.91, 73.49, and 96.21% isolates were sensitive to fluconazole (FLK, itraconazole (ITR, and amphotericin B (AMB, respectively. Conclusion: Candidemia in neonates is an ominous prognostic sign and is an important entity in our hospital. Strict infection control strategies, appropriate preventive and therapeutic measures such as prophylactic antifungal use and a restrictive policy of antibiotic use should be implemented.

  14. Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans.

    Science.gov (United States)

    Premachandra, Ilandari Dewage Udara Anulal; Scott, Kevin A; Shen, Chengtian; Wang, Fuqiang; Lane, Shelley; Liu, Haoping; Van Vranken, David L

    2015-10-01

    A spiroindolinone, (1S,3R,3aR,6aS)-1-benzyl-6'-chloro-5-(4-fluorophenyl)-7'-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3'-1H-indole]-2',4,6-trione, was previously reported to enhance the antifungal effect of fluconazole against Candida albicans. A diastereomer of this compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, was found to enhance the effect of fluconazole with an EC50 value of 300 pM against a susceptible strain of C. albicans and going as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole, with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for antifungal synergy.

  15. Distribution of Candida albicans genotypes among family members

    Science.gov (United States)

    Mehta, S. K.; Stevens, D. A.; Mishra, S. K.; Feroze, F.; Pierson, D. L.

    1999-01-01

    Thirty-three families (71 subjects) were screened for the presence of Candida albicans in mouthwash or stool specimens; 12 families (28 subjects) were culture-positive for this yeast. An enrichment procedure provided a twofold increase in the recovery of C. albicans from mouthwash specimens. Nine of the twelve culture-positive families had two positive members each, two families had three positive members each, and one family had four positive members. Genetic profiles were obtained by three methods: pulsed-field gel electrophoresis; restriction endonuclease analysis, and random amplification of polymorphic DNA analysis. DNA fingerprinting of C. albicans isolated from one body site three consecutive times revealed that each of the 12 families carried a distinct genotype. No two families shared the same strain, and two or more members of a family commonly shared the same strain. Intrafamily genotypic identity (i.e., each member within the family harbored the same strain) was demonstrated in six families. Genotypes of isolates from husband and wife differed from one another in five families. All three methods were satisfactory in determining genotypes; however, we concluded that restriction endonuclease analysis provided adequate resolving power.

  16. MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

    Directory of Open Access Journals (Sweden)

    Patricia de Souza Bonfim-Mendonca

    2013-12-01

    Full Text Available SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

  17. In vivo Models for Candida Albicans Biofilms Study

    Directory of Open Access Journals (Sweden)

    Wenrui Gu

    2016-03-01

    Full Text Available Biofilm is a common mode of fungal growth in clinical infection. In the mode of biofilm, Candida albicans tends to display high resistance to body immunity and antimicrobial agents, which has a significant impact on mortality. Biofilm models are essential tools to better understand the mechanisms of formation and resistance. Compared to in vitro models, in vivo models can better take into account the host immune system and are indispensable for the study of medical device related infection. The aim of this review is to summarize information related to the reported in vivo models of C. albicans biofilms, analyze the operating process and application of them, and compare their advantages and limitations. A literature search was performed from databases in Medline (PubMed, Web of Science, Science Direct, and Google scholar by applying some related search terms. The articles related to agriculture, ecology, and synthetic work and those using languages other than English have been excluded. The bibliographies of papers relating to the review subject were also searched for further relevant references. According to the common sites of C. albicans infection; three kinds of in vivo models are discussed in this review: oral mucosa model, vaginal mucosa model and implanted catheter model. The former two models can demonstrate the structure and composition of biofilms growing on the mucosa, and implanted catheter model represents different kinds of medical devices. To expedite the success of new treatments of infection, further refinement of in vivo models is an urgent need.

  18. Interactions of Candida albicans with host epithelial surfaces

    Directory of Open Access Journals (Sweden)

    David W. Williams

    2013-10-01

    Full Text Available Candida albicans is an opportunistic, fungal pathogen of humans that frequently causes superficial infections of oral and vaginal mucosal surfaces of debilitated and susceptible individuals. The organism is however, commonly encountered as a commensal in healthy individuals where it is a component of the normal microflora. The key determinant in the type of relationship that Candida has with its host is how it interacts with the epithelial surface it colonises. A delicate balance clearly exists between the potentially damaging effects of Candida virulence factors and the nature of the immune response elicited by the host. Frequently, it is changes in host factors that lead to Candida seemingly changing from a commensal to pathogenic existence. However, given the often reported heterogeneity in morphological and biochemical factors that exist between Candida species and indeed strains of C. albicans, it may also be the fact that colonising strains differ in the way they exploit resources to allow persistence at mucosal surfaces and as a consequence this too may affect the way Candida interacts with epithelial cells. The aim of this review is to provide an overview of some of the possible interactions that may occur between C. albicans and host epithelial surfaces that may in turn dictate whether Candida removal, its commensal persistence or infection follows.

  19. Role of Permutations in Significance Analysis of Microarray and Clustering of Significant Microarray Gene list

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    Tejashree Damle

    2012-03-01

    Full Text Available Microarray is the gene expression data that represent gene in different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. Significance analysis of microarrays (SAM is a statistical technique for determining whether changes in gene expression are statistically significant. During the SAM procedure permutation of microarray data is considered to observe the changes in the overall expression level of data. With increasing number of permutations false discovery rate for gene set varies. In our work we took microarray data of Normal Glucose Tolerance (NGT, and Diabetes Mellitus (DM Type II. In this paper we proposed the result of permutations during execution of SAM algorithm. The hierarchical clustering is applied for observing expression levels of significant data and visualize it with heat map.

  20. A comparative analysis of DNA barcode microarray feature size

    OpenAIRE

    Ammar, Ron; SMITH, ANDREW M.; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

    2009-01-01

    Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platfor...

  1. Facilitating functional annotation of chicken microarray data

    Directory of Open Access Journals (Sweden)

    Gresham Cathy R

    2009-10-01

    Full Text Available Abstract Background Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO. However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information. Results We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (AGOM tool to help researchers to quickly retrieve corresponding functional information for their dataset. Conclusion Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using AGOM tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and

  2. Identifying Fishes through DNA Barcodes and Microarrays

    Science.gov (United States)

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

    Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. PMID

  3. Identifying Fishes through DNA Barcodes and Microarrays.

    Directory of Open Access Journals (Sweden)

    Marc Kochzius

    Full Text Available BACKGROUND: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. METHODOLOGY/PRINCIPAL FINDINGS: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S, cytochrome b (cyt b, and cytochrome oxidase subunit I (COI for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90% renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. CONCLUSIONS/SIGNIFICANCE: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

  4. Background Adjustment for DNA Microarrays Using a Database of Microarray Experiments

    OpenAIRE

    Sui, Yunxia; Zhao, Xiaoyue; Speed, Terence P.; Wu, Zhijin

    2009-01-01

    DNA microarrays have become an indispensable technique in biomedical research. The raw measurements from microarrays undergo a number of preprocessing steps before the data are converted to the genomic level for further analysis. Background adjustment is an important step in preprocessing. Estimating background noise has been challenging because background levels vary a lot from probe to probe, yet there are limited observations on each probe. Most current methods have used the empirical Baye...

  5. Cell Wall Polysaccharides of Candida albicans Induce Mast Cell Degranulation in the Gut

    OpenAIRE

    Sakurai, Atsuko; Yamaguchi, Natsu; Sonoyama, Kei

    2012-01-01

    We investigated Candida albicans-induced mast cell degranulation in vitro and in vivo. Cell wall fraction but not culture supernatant and cell membrane fraction prepared from hyphally grown C. albicans induced β-hexosaminidase release in RBL-2H3 cells. Cell wall mannan and soluble β-glucan fractions also induced β-hexosaminidase release. Histological examination of mouse forestomach showed that C. albicans gut colonization induces mast cell degranulation. However, intragastric administration ...

  6. Funktionelle Analyse einer Familie von Oligopeptidtransportern des humanpathogenen Hefepilzes Candida albicans

    OpenAIRE

    Reuß, Oliver Rainer

    2006-01-01

    Der Hefepilz Candida albicans ist Teil der natürlichen Mikroflora auf den Schleimhäuten des Verdauungs- und Urogenitaltrakts der meisten gesunden Menschen. Allerdings kann C. albicans vor allem in immunsupprimierten Patienten auch schwerwiegende Infektionen verursachen. Diese reichen von oberflächlichen Mykosen bis hin zu lebensbedrohlichen systemischen Infektionen. C. albicans besitzt eine Reihe von Eigenschaften, die es diesem opportunistisch humanpathogenen Pilz ermöglichen unterschiedlich...

  7. Sputum Candida albicans presages FEV₁ decline and hospital-treated exacerbations in cystic fibrosis.

    LENUS (Irish Health Repository)

    Chotirmall, Sanjay H

    2010-11-01

    The role of Candida albicans in the cystic fibrosis (CF) airway is underexplored. Considered a colonizer, few question its pathogenic potential despite high isolation frequencies from sputum culture. We evaluated the frequency and identified the strongest predictors of C albicans colonization in CF. Independent associations of colonization with clinical outcomes were determined, and the longitudinal effects of C albicans acquisition on BMI and FEV₁ were evaluated.

  8. Control of Candida albicans Metabolism and Biofilm Formation by Pseudomonas aeruginosa Phenazines

    OpenAIRE

    Morales, Diana K.; Grahl, Nora; Okegbe, Chinweike; Dietrich, Lars E. P.; Jacobs, Nicholas J.; Hogan, Deborah A.

    2013-01-01

    ABSTRACT Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentra...

  9. Antifungal Activity of Bee Venom and Sweet Bee Venom against Clinically Isolated Candida albicans

    OpenAIRE

    Seung-Bae Lee

    2016-01-01

    Objectives: The purpose of this study was to investigate the antifungal effect of bee venom (BV) and sweet bee venom (SBV) against Candida albicans (C. albicans) clinical isolates. Methods: In this study, BV and SBV were examined for antifungal activities against the Korean Collection for Type Cultures (KCTC) strain and 10 clinical isolates of C. albicans. The disk diffusion method was used to measure the antifungal activity and minimum inhibitory concentration (MIC) assays were performed by ...

  10. Hydrophobic interaction in Candida albicans and Candida tropicalis adherence to various denture base resin materials.

    OpenAIRE

    Minagi, S; Miyake, Y; Inagaki, K; Tsuru, H; Suginaka, H

    1985-01-01

    The effects of hydrophobicities of substrate surfaces on microbial adherence were examined by using Candida albicans and Candida tropicalis and 21 denture base resin materials. With increasing surface free energy of resin plates, increasing adherence of C. albicans and decreasing adherence of C. tropicalis were observed. The surface free energy of C. albicans is higher than that of all resin material surfaces, and C. tropicalis has surface free energy lower than that of all materials used. In...

  11. Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure

    OpenAIRE

    Ophelders, Daan R. M. G.; Gussenhoven, Ruth; Lammens, Martin; Küsters, Benno; Kemp, Matthew W.; Newnham, John P; Payne, Matthew S.; Suhas G Kallapur; Jobe, Allan H.; Zimmermann, Luc J.; Boris W Kramer; Tim G A M Wolfs

    2016-01-01

    Background Intra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine mod...

  12. Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo

    OpenAIRE

    Lee, K K; MacCallum, D.M; Jacobsen, M.D.; Walker, L A; Odds, F C; Gow, N. A. R.; Munro, C.A.

    2012-01-01

    Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully tre...

  13. Effect of Xylitol on Candida albicans resistance in serum (in vitro study

    Directory of Open Access Journals (Sweden)

    Ria Puspitawati

    2013-07-01

    Full Text Available Xylitol is reported to inhibit the growth of C. albicans. Objectives: Investigating serum factor role in inhibiting the growth of C. albicans and the effect of 1%, 5%, 10% xylitol on C. albicans resistance in serum in vitro. Methods: Identification of C. albicans (oral swab of candidiasis patient was conducted using CHROMAgar, confirmed by germ tube test. The cultures were serially diluted, inoculated in Saburoud Dextrose Broth (SDB contained 0% (control, 1%, 5%, or 10% xylitol, and kept for 3 or 7 days. These inoculations were then exposed to either active or inactive serum (Fetal Bovine Serum heated in 65ºC for 30 minutes for 2 hours in 37ºC. The colony forming unit (CFU of C. albicans in Saburoud Dextrose Agar (SDA were counted after 2 days. C. albicans ATCC 10231 strain was used as a comparison. One-way ANOVA with 0.05 was used. Results: After 3 days cultured in media with or without xylitol, the CFU of C. albicans exposed to active serum were significantly lower than those exposed to inactive serum (p=0.032. Although not statistically significant (p=0.689, increased concentration of xylitol lead to increased resistance of C. albicans in active serum. Only 7 day exposure of 10% xylitol resulted in significantly higher growth of C. albicans (p=0.034. No significant difference of C. albicans CFU in active or inactive serum (p=0.404. Conclusion: Serum factor has role in inhibiting C. albicans growth in vitro. Exposure of 1%, 5%, or 10% xylitol for 3 or 7 days has no significant effect on C. albicans resistance in serum.DOI: 10.14693/jdi.v16i2.98

  14. Anti-Candida albicans activity of Pichia anomala as determined by a growth rate reduction assay.

    OpenAIRE

    Sawant, A D; Abdelal, A T; Ahearn, D G

    1988-01-01

    Killer toxin activity of Pichia anomala WC65 appeared fungicidal for P. bimundalis WC38 and fungistatic for Candida albicans RC1. Inhibitory activity against sensitive C. albicans showed a linear relationship between toxin concentrations and the inverse of the reduced growth rates. The plot of toxin concentrations against growth rates was hyperbolic, as is characteristic of saturation kinetics. Sensitivity of C. albicans to the toxin decreased with increased cell age. The measurement of growt...

  15. Self-Regulation of Candida albicans Population Size during GI Colonization

    OpenAIRE

    White, Sarah Jane; Rosenbach, Ari; Lephart, Paul; Nguyen, Diem; Benjamin, Alana; Tzipori, Saul; Whiteway, Malcolm; Mecsas, Joan; Kumamoto, Carol A.

    2007-01-01

    Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant ex...

  16. Effect of Nitric Oxide on the Antifungal Activity of Oxidative Stress and Azoles Against Candida albicans.

    Science.gov (United States)

    Li, De-Dong; Yang, Chang-Chun; Liu, Ping; Wang, Yan; Sun, Yan

    2016-06-01

    Nitric oxide (NO) is a small molecule with a wide range of biological activities in mammalian and bacteria. However, the role of NO in fungi, especially Candida albicans, is not clear. In this study, we confirmed the generation of endogenous NO in C. albicans, and found that the production of endogenous NO in C. albicans was associated with nitric oxide synthase pathway. Our results further indicated that the production of endogenous NO in C. albicans was reduced under oxidative stress such as menadione or H2O2 treatment. Meanwhile, exogenous NO donor, sodium nitroprusside (SNP), synergized with H2O2 against C. albicans. Interestingly, SNP could inhibit the antifungal effect of azoles against C. albicans in vitro, suggesting that NO might be involved in the resistance of C. albicans to antifungals. Collectively, this study demonstrated the production of endogenous NO in C. albicans, and indicated that NO may play an important role in the response of C. albicans to oxidative stress and azoles. PMID:27570314

  17. Antimicrobial blue light therapy for Candida albicans burn infection in mice

    Science.gov (United States)

    Zhang, Yunsong; Wang, Yucheng; Murray, Clinton K.; Hamblin, Michael R.; Gu, Ying; Dai, Tianhong

    2015-05-01

    In this preclinical study, we investigated the utility of antimicrobial blue light therapy for Candida albicans infection in acutely burned mice. A bioluminescent strain of C. albicans was used. The susceptibilities to blue light inactivation were compared between C. albicans and human keratinocyte. In vitro serial passaging of C. albicans on blue light exposure was performed to evaluate the potential development of resistance to blue light inactivation. A mouse model of acute thermal burn injury infected with the bioluminescent strain of C. albicans was developed. Blue light (415 nm) was delivered to mouse burns for decolonization of C. albicans. Bioluminescence imaging was used to monitor in real time the extent of fungal infection in mouse burns. Experimental results showed that C. albicans was approximately 42-fold more susceptible to blue light inactivation in vitro than human keratinocyte (P=0.0022). Serial passaging of C. albicans on blue light exposure implied a tendency for the fungal susceptibility to blue light inactivation to decrease with the numbers of passages. Blue light reduced fungal burden by over 4-log10 (99.99%) in acute mouse burns infected with C. albicans in comparison to infected mouse burns without blue light therapy (P=0.015).

  18. Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with Candida albicans.

    Science.gov (United States)

    Rast, Timothy J; Kullas, Amy L; Southern, Peter J; Davis, Dana A

    2016-01-01

    The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage.

  19. The role of pattern recognition receptors in the innate recognition of Candida albicans.

    Science.gov (United States)

    Zheng, Nan-Xin; Wang, Yan; Hu, Dan-Dan; Yan, Lan; Jiang, Yuan-Ying

    2015-01-01

    Candida albicans is both a commensal microorganism in healthy individuals and a major fungal pathogen causing high mortality in immunocompromised patients. Yeast-hypha morphological transition is a well known virulence trait of C. albicans. Host innate immunity to C. albicans critically requires pattern recognition receptors (PRRs). In this review, we summarize the PRRs involved in the recognition of C. albicans in epithelial cells, endothelial cells, and phagocytic cells separately. We figure out the differential recognition of yeasts and hyphae, the findings on PRR-deficient mice, and the discoveries on human PRR-related single nucleotide polymorphisms (SNPs).

  20. Genome-Wide Chromatin Immunoprecipitation in Candida albicans and Other Yeasts

    Science.gov (United States)

    Lohse, Matthew B.; Kongsomboonvech, Pisiwat; Madrigal, Maria; Hernday, Aaron D.; Nobile, Clarissa J.

    2016-01-01

    Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments. PMID:26483022

  1. Application of DNA microarray in the identification of Candida spp. and mutations of ERG11 gene resulting in fluconazole resistance%DNA芯片鉴定念珠菌种和氟康唑耐药基因ERG11突变

    Institute of Scientific and Technical Information of China (English)

    徐永豪; 王克玉; 李颖; 陈腊梅; 苏英; 孙青; 李春阳

    2009-01-01

    目的 建立DNA芯片技术鉴定念珠菌种和氟康唑耐药基因ERG11突变.方法 根据6种常见念珠菌内转录间隔(ITS2)区种特异性序列和白念珠菌ERG11基因中已证实可导致对氟康唑耐药的6种突变序列设计探针,制备DNA芯片,鉴定12条50 bp的念珠菌种特异性序列和ERG11突变序列及34株念珠菌(其中白念珠菌29株,热带念珠菌、光滑念珠菌、都柏林念珠菌、近平滑念珠菌和克柔念珠菌各1株).结果 ①芯片玤确鉴定12条人工合成序列;②正确鉴定34株试验菌株的菌种;③正确鉴定29株白念珠菌ERG11基因中可致耐药的已知突变.敏感性和特异性均为100%.结论 用DNA芯片进行念珠菌菌种鉴定和自念珠菌ERG11突变筛查,结果可靠.%Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains

  2. Viral diagnosis in Indian livestock using customized microarray chips.

    Science.gov (United States)

    Yadav, Brijesh S; Pokhriyal, Mayank; Ratta, Barkha; Kumar, Ajay; Saxena, Meeta; Sharma, Bhaskar

    2015-01-01

    Viral diagnosis in Indian livestock using customized microarray chips is gaining momentum in recent years. Hence, it is possible to design customized microarray chip for viruses infecting livestock in India. Customized microarray chips identified Bovine herpes virus-1 (BHV-1), Canine Adeno Virus-1 (CAV-1), and Canine Parvo Virus-2 (CPV-2) in clinical samples. Microarray identified specific probes were further confirmed using RT-PCR in all clinical and known samples. Therefore, the application of microarray chips during viral disease outbreaks in Indian livestock is possible where conventional methods are unsuitable. It should be noted that customized application requires a detailed cost efficiency calculation.

  3. Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

    Science.gov (United States)

    Grubaugh, Nathan D; Petz, Lawrence N; Melanson, Vanessa R; McMenamy, Scott S; Turell, Michael J; Long, Lewis S; Pisarcik, Sarah E; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L; Lee, John S

    2013-02-01

    Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

  4. SDS-Page and numerical analysis of Candida albicans from human oral cavity and other anatomical sites Eletroforese de proteínas totais e análise numérica de Candida albicans isolada da cavidade oral e outros sítios anatômicos de humanos

    Directory of Open Access Journals (Sweden)

    Cristina Crespo Rodrigues

    2004-06-01

    Full Text Available The aim of the present research was to evaluate the protein polymorphism degrees among forty-eight C. albicans isolates from fourteen anatomical sites of clinical patients by polyacrylamide gel electrophoresis (SDS-PAGE and numerical analyzes, in order to identify subspecies and their similarities in some infectious niches. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed in cold saline solution. The whole-cell proteins were extracted by cell disruption using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrixes and dendrograms were generated by application of similarity coefficient of simple matching and UPGMA algorithm, respectively. The results obtained showed several C. albicans subtypes and their similarity degrees (80% to 100%. Such data showed that same patients may be infected by two or more C. albicans subtypes in certain anatomical sites (i.e. only in oral cavity of immunocompromised patients, blood, or tracheal secretion, or yet, two or more patients can be infected in identical anatomical sites (i.e. bronchial washing, urine, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva with a same C. albicans subtype. However, two or more patients also can show infections in corresponding sites (i.e. oral cavity of immunocompromised patients, blood, oropharyngeal secretion, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva by different C. albicans subtypes. Besides, two or more patients also can be infected with identical or different C. albicans subtypes in different anatomical sites (i.e.1. identical subtypes in vaginal secretion, tracheal secretion, and urine; abdominal secretion and spittle; drainage and oral cavity; catheter and healthy saliva - i.e.2. subtypes different in bronchial washing, oropharyngeal

  5. Microarray tools to unveil viral-microbe interactions in nature

    Directory of Open Access Journals (Sweden)

    Fernando eSantos

    2014-07-01

    Full Text Available The interactions between viruses and their microbial hosts play a central role in the control of microbial communities in nature. However, the study of such interactions within the uncultured majority is technically very challenging. Here, we review how microarray tools can be used to analyze the interactions between viruses and their microbial hosts in nature, away from laboratory pure culture-based models. We show examples of how DNA arrays have been used to study the expression of viral assemblages in natural samples, and to assign viruses to hosts within uncultured communities. Finally, we briefly discuss the possibilities of protein and glycan arrays to gain insight into the ways microbes interact with their viruses.

  6. Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity

    Science.gov (United States)

    Sabino, Caetano Padial; Fuchs, Beth Burgwyn; Tegos, George P.; Mylonakis, Eleftherios; Hamblin, Michael R.; Ribeiro, Martha Simões

    2013-01-01

    The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm2, 9 to 27 J/cm2). In vitro, we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo, we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells (P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast (P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections. PMID:23129051

  7. Antimicrobial blue light inactivation of Candida albicans: In vitro and in vivo studies.

    Science.gov (United States)

    Zhang, Yunsong; Zhu, Yingbo; Chen, Jia; Wang, Yucheng; Sherwood, Margaret E; Murray, Clinton K; Vrahas, Mark S; Hooper, David C; Hamblin, Michael R; Dai, Tianhong

    2016-07-01

    Fungal infections are a common cause of morbidity, mortality and cost in critical care populations. The increasing emergence of antimicrobial resistance necessitates the development of new therapeutic approaches for fungal infections. In the present study, we investigated the effectiveness of an innovative approach, antimicrobial blue light (aBL), for inactivation of Candida albicans in vitro and in infected mouse burns. A bioluminescent strain of C. albicans was used. The susceptibilities to aBL (415 nm) were compared between C. albicans and human keratinocytes. The potential development of aBL resistance by C. albicans was investigated via 10 serial passages of C. albicans on aBL exposure. For the animal study, a mouse model of thermal burn infected with the bioluminescent C. albicans strain was used. aBL was delivered to mouse burns approximately 12 h after fungal inoculation. Bioluminescence imaging was performed to monitor in real time the extent of infection in mice. The results obtained from the studies demonstrated that C. albicans was approximately 42-fold more susceptible to aBL than human keratinocytes. Serial passaging of C. albicans on aBL exposure implied a tendency of reduced aBL susceptibility of C. albicans with increasing numbers of passages; however, no statistically significant difference was observed in the post-aBL survival rate of C. albicans between the first and the last passage (P>0.05). A single exposure of 432 J/cm(2) aBL reduced the fungal burden in infected mouse burns by 1.75-log10 (P=0.015). Taken together, our findings suggest aBL is a potential therapeutic for C. albicans infections. PMID:26909654

  8. The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Rebecca A Hall

    Full Text Available The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26. Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.

  9. Undetected sex chromosome aneuploidy by chromosomal microarray.

    Science.gov (United States)

    Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

    2012-11-01

    We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.

  10. A Gene Expression Barcode for Microarray Data

    OpenAIRE

    Zilliox, Michael J.; Irizarry, Rafael A.

    2007-01-01

    The ability to measure genome-wide expression holds great promise for characterizing cells and distinguishing diseased from normal tissues. Thus far, microarray technology has only been useful for measuring relative expression between two or more samples, which has handicapped its ability to classify tissue types. This paper presents the first method that can successfully predict tissue type based on data from a single hybridization. A preliminary web-tool is available at http://rafalab.jhsph...

  11. Pineal Function: Impact of Microarray Analysis

    OpenAIRE

    Klein, David C.; Bailey, Michael J; Carter, David A.; Kim, Jong-So; Shi, Qiong; Ho, Anthony; Chik, Constance; Gaildrat, Pascaline; Morin, Fabrice; Ganguly, Surajit; Rath, Martin F.; Møller, Morten; Sugden, David; Rangel, Zoila G.; Peter J Munson

    2009-01-01

    Microarray analysis has provided a new understanding of pineal function by identifying genes that are highly expressed in this tissue relative to other tissues and also by identifying over 600 genes that are expressed on a 24-hour schedule. This effort has highlighted surprising similarity to the retina and has provided reason to explore new avenues of study including intracellular signaling, signal transduction, transcriptional cascades, thyroid/retinoic acid hormone signaling, metal biology...

  12. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

    Directory of Open Access Journals (Sweden)

    Shantelle L LaFayette

    Full Text Available Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC, which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which

  13. Metadata Management and Semantics in Microarray Repositories

    Science.gov (United States)

    Kocabaş, F; Can, T; Baykal, N

    2011-01-01

    The number of microarray and other high-throughput experiments on primary repositories keeps increasing as do the size and complexity of the results in response to biomedical investigations. Initiatives have been started on standardization of content, object model, exchange format and ontology. However, there are backlogs and inability to exchange data between microarray repositories, which indicate that there is a great need for a standard format and data management. We have introduced a metadata framework that includes a metadata card and semantic nets that make experimental results visible, understandable and usable. These are encoded in syntax encoding schemes and represented in RDF (Resource Description Frame-word), can be integrated with other metadata cards and semantic nets, and can be exchanged, shared and queried. We demonstrated the performance and potential benefits through a case study on a selected microarray repository. We concluded that the backlogs can be reduced and that exchange of information and asking of knowledge discovery questions can become possible with the use of this metadata framework. PMID:24052712

  14. Microarrays for rapid identification of plant viruses.

    Science.gov (United States)

    Boonham, Neil; Tomlinson, Jenny; Mumford, Rick

    2007-01-01

    Many factors affect the development and application of diagnostic techniques. Plant viruses are an inherently diverse group that, unlike cellular pathogens, possess no nucleotide sequence type (e.g., ribosomal RNA sequences) in common. Detection of plant viruses is becoming more challenging as globalization of trade, particularly in ornamentals, and the potential effects of climate change enhance the movement of viruses and their vectors, transforming the diagnostic landscape. Techniques for assessing seed, other propagation materials and field samples for the presence of specific viruses include biological indexing, electron microscopy, antibody-based detection, including enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and microarray detection. Of these, microarray detection provides the greatest capability for parallel yet specific testing, and can be used to detect individual, or combinations of viruses and, using current approaches, to do so with a sensitivity comparable to ELISA. Methods based on PCR provide the greatest sensitivity among the listed techniques but are limited in parallel detection capability even in "multiplexed" applications. Various aspects of microarray technology, including probe development, array fabrication, assay target preparation, hybridization, washing, scanning, and interpretation are presented and discussed, for both current and developing technology.

  15. Chicken sperm transcriptome profiling by microarray analysis.

    Science.gov (United States)

    Singh, R P; Shafeeque, C M; Sharma, S K; Singh, R; Mohan, J; Sastry, K V H; Saxena, V K; Azeez, P A

    2016-03-01

    It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from 10 fertile males and used for RNA preparation. Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 × 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 21,639 predominantly nuclear-encoded transcripts in chicken sperm. The majority (66.55%) of the sperm transcripts were shared with the testes, while surprisingly, 33.45% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility.

  16. Integrating data from heterogeneous DNA microarray platforms.

    Science.gov (United States)

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

  17. Candida albicans biofilm development in vitro for photodynamic therapy study

    International Nuclear Information System (INIS)

    Photodynamic therapy (PDT) is a phototherapy based on the use of a photo sensitizer (PS) in the presence of low intensity light with resonant wavelength of absorption of the PS and biological systems that can raise awareness, generating reactive oxygen species. Studies show that PDT has a lethal effect on Candida albicans. The biofilm formed by C. albicans is the cause of infections associated with medical devices such as catheters, with a proven resistance to antifungal agents, and the removal of the catheter colonized almost always is necessary. However, few studies in literature report the behavior and response of biofilm organized by C. albicans against PDT. The aims of this study were to develop a methodology for in vitro biofilm formation of C. albicans, evaluate the sensitivity of the biofilm of C. albicans to antimicrobial photodynamic therapy using PS as the methylene blue (MB) and hypocrellin B: La+3 (HBLa+3) and analyze the biofilm by Optical Coherence Tomography (OCT). For biofilm formation, discs were made from elastomeric silicone catheters. The PS were dissolved in solution of PBS, and the MB had two different concentrations tested in the biofilm: 100μM and 1mM; HBLa+3 only one of 10μM. The irradiation of both dyes with the microorganism was done by two different LEDs, one with red emission at λ = 630nm ± 20nm and the other one blue emission at λ = 460nm ± 30nm. We performed a curve of survival fraction versus time of irradiation of each sample with biofilm and suspension of the microorganism in the yeast form to verify the susceptibility of the front PDT. The yeast showed 100% reduction using both PS, but at different times of irradiation (30s to HBLa+3 and 6 min for the MB at 100μM). When the therapy was applied in biofilm, the MB 100μM did not show any significant reduction, while at concentration of 1mM was reduced by 100% after 6 min of irradiation. The HBLa+3 biofilm group showed a lower reduction in the concentration of 10μM in

  18. Candida albicans biofilm on titanium: effect of peroxidase precoating

    Directory of Open Access Journals (Sweden)

    Mohamed Ahariz

    2010-08-01

    Full Text Available Mohamed Ahariz1, Philippe Courtois1,21Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, 2UER de Biologie Médicale, Haute Ecole Francisco Ferrer, Brussels, BelgiumAbstract: The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2–21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 106 blastoconidia per gram of titanium powder (n = 30 and 0.50 ± 0.04 × 106 blastoconidia per cm² of titanium foil (n = 12. The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate, Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated and liquid environment (containing peroxidase substrates to limit C. albicans biofilm formation.Keywords: adhesion, material, oral, yeast

  19. Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion.

    Science.gov (United States)

    Gil-Bona, Ana; Monteoliva, Lucía; Gil, Concha

    2015-10-01

    Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.

  20. Identification of superficial Candida albicans germ tube antigens in a rabbit model of disseminated candidiasis. A proteomic approach.

    Science.gov (United States)

    Sáez-Rosón, Aranzazu; Sevilla, María-Jesús; Moragues, María-Dolores

    2014-03-01

    The diagnosis of invasive candidiasis remains a clinical challenge. The detection by indirect immunofluorescence of Candida albicans germ-tube-specific antibodies (CAGTA), directed against germ-tube surface antigens, is a useful diagnostic tool that discriminates between colonization and invasion. However, the standardization of this technique is complicated by its reliance on subjective interpretation. In this study, the antigenic recognition pattern of CAGTA throughout experimental invasive candidiasis in a rabbit animal model was determined by means of 2D-PAGE, Western blotting, and tandem mass spectrometry (MS/MS). Seven proteins detected by CAGTA were identified as methionine synthase, inositol-3-phosphate synthase, enolase 1, alcohol dehydrogenase 1,3-phosphoglycerate kinase, 14-3-3 (Bmhl), and Egd2. To our knowledge, this is the first report of antibodies reacting with Bmhl and Egd2 proteins in an animal model of invasive candidiasis. Although all of the antigens were recognized by CAGTA in cell-wall dithiothreitol extracts of both germ tubes and blastospores of C. albicans, immunoelectron microscopy study revealed their differential location, as the antigens were exposed on the germ-tube cell-wall surface but hidden in the inner layers of the blastospore cell wall. These findings will contribute to developing more sensitive diagnostic methods that enable the earlier detection of invasive candidiasis.

  1. Development of a membrane based detection of Candida albicans

    OpenAIRE

    Almeida, Catarina Guerreiro Silva de

    2015-01-01

    Tese de mestrado integrado em Engenharia Biomédica e Biofísica , apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015 Candida é uma família de fungos, normalmente, presente na flora gastrointestinal, nos orgãos genitais, no sistema respiratório e na pele de pessoas saudáveis e, até determinada quantidade, não trazem nenhum risco. Apenas 17 espécies de Candida podem ser consideradas como patogénicas para o ser humano e, dentro deste grupo, Candida albicans é a esp...

  2. Activation and binding of C3 by Candida albicans.

    OpenAIRE

    Kozel, T R; Brown, R R; Pfrommer, G S

    1987-01-01

    Interaction with components of the complement system is an important aspect of the pathogenesis of infection by Candida albicans. The key role of C3 as an opsonic ligand and as an element in amplification of complement activation led us to examine several factors that influence the activation and binding of C3 cleavage fragments to the yeast. Activation and binding of C3 were determined by use of normal human serum containing 125I-labeled C3. Incubation of yeast-phase cells in 20% serum led t...

  3. [Value of the microarray for the study of Laboratory Animal Allergy (LAA)].

    Science.gov (United States)

    D'Ovidio, Maria Concetta; Martini, Agnese; Melis, Paola; Signorini, Stefano

    2011-01-01

    Since 1989, the National Institute for Occupational Safety and Health (NIOSH) considers the Laboratory Animal Allergy - LAA a risk for workers and in 1998 the LAA has been recognized as occupational risk in the USA. Rat and mouse are the most source of allergens, not so much for the higher power of allergy respect to the other animals, but because represent the more utilized species in the research. Most of the allergens are members of the lipocalin superfamily, small extracellular proteins represented by at least 50 proteins that mainly bind or carry small hydrophobic molecules. The recent and innovative molecular techniques, as the microarray, have allow the characterization of numerous allergens. The protein microarray gives the possibility to study of IgE profile for each individual, simultaneos analysis of a wide number of parameters concerning the allergy, giving new diagnostic and therapeutic opportunities for the allergies. In the study of occupational allergy--as LAA--the protein microarray could improve: the identification and characterization of new allergens; the individuation of susceptible workers; the study of immunological responses in exposed workers; the strategies of prevention and protection; the environmental and housing conditions. The participation, formation and information of the workers could improve the behavioural and occupational practices, the use of personal and collective protective devices in order to reduce the exposure to LAA in occupational context.

  4. 建立白色念珠菌蛋白指纹库的研究%A study on establishment of peptide mapping database of Candida albicans

    Institute of Scientific and Technical Information of China (English)

    杨永长; 喻华; 刘华; 肖代雯; 黄文芳

    2014-01-01

    目的:探讨白色念珠菌蛋白指纹库的建立,为白色念珠菌感染快速诊断奠定基础。方法收集96株临床分离的白色念珠菌,提取DNA ,采用聚合酶链反应(PCR)扩增其ITS1-5.8S-ITS2基因片段,利用限制性内切酶对其进行鉴定。应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)仪检测念珠菌蛋白指纹,Ciphergen ProteinChip软件自动采集数据,筛选稳定表达蛋白峰建立白色念珠菌蛋白指纹库,运用经准确鉴定的念珠菌对建立的蛋白指纹库进行验证。结果限制性片段长度多态性分析证实临床分离的所有菌株均为白色念珠菌。SELDI-TOF-MS芯片能捕获15个蛋白峰,其中5个蛋白峰在所有白色念珠菌中均稳定表达,利用相似性分析软件建立白色念珠菌蛋白指纹库,白色念珠菌蛋白指纹与建立数据库的相似性大于95%,而其他种类念珠菌蛋白指纹与数据库的相似性均小于50%。结论白色念珠菌蛋白指纹库的建立为快速诊断白色念珠菌感染提供了理论依据。%Objective To explore the establishment of peptide mapping database of Candida albicans ,laying the foundation for rapid diagnosis of Candida albicans infection .Methods 96 Candida albicans were collected clinically ,and its DNA was extracted . Polymerase chain reaction(PCR) was used to amplify the ITS1-5 .8S-ITS2 gene fragments and restriction endonucleases were a-dopted to identify them .Surface enhanced laser desorption ionization-time of flight-mass spectrometry(SELDI-TOF-MS) instrument was applied to detect the Candida albicans peptide mapping ,and Ciphergen ProteinChip software was used to collect data automati-cally .The established peptide mapping database was verified by confirmed Candida .Results According to restriction fragment length polymorphism analysis ,96 strains were confirmed as Candida albicans .15 peptide peaks were captured by SELDI-TOF-MS chips .Five

  5. Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms of Candida albicans.

    Science.gov (United States)

    Marriott, M S

    1975-01-01

    It has been possible to induce the yeast-mycelium transformation in Candida albicans by growth of the organism under completely defined conditions in batch culture. Protoplasts have been obtained from the two forms by using a lytic enzyme preparation from Streptomyces violaceus. A plasma membrane fraction was prepared by osmotic lysis of these protoplasts and fractionated by using a combination of differential and discontinuous sucrose density-gradient flotation centrifugation. The purity of this fraction was determined by radioactive dansylation and iodination of plasma membranes of intact protoplasts followed by localization of the radioactivity upon fractionation. This procedure demonstrated less than 4% contamination of the plasma membrane fraction with other cell membranes. Chemical analysis of this fraction revealed that the major components were protein and lipid. Membranes from the yeast form contained (w/w): 50% protein, 45% lipid, 9% carbohydrate and 0.3% nucleic acid. Plasma membranes from the mycelial form contained significantly more carbohydrate and were found to be composed of (w/w): 43% protein, 31% lipid, 25% carbohydrate and 0.5% nucleic acid. Marked differences were also observed between the phospholipid, free and esterified sterols, and total fatty acids of membranes from the two forms of the organism. PMID:1089750

  6. Effect of Xylitol with Various Concentration and Duration on the Growth of Candida albicans (In Vitro study)

    OpenAIRE

    Lakshmi A. Leepel; Shandy Sastra; Ria Puspitawati; Bachtiar, Boy M.

    2012-01-01

    The growth of C. albicans is influenced by glucose intake. Xylitol is commonly used as sugar substitute. Reported effective concentrations of xylitol in reducing C. albicans growth in vitro were varied, 1%, 5%, and 10%. Objectives: Investigate the effect of different concentration and duration of xylitol exposure in inhibiting C. albicans growth in vitro. Method: Identification of C. albicans from oral swab of a male candidiasis patient was conducted using CHROMagar, confirmed by germ tube te...

  7. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

    Directory of Open Access Journals (Sweden)

    Humidah Alanazi

    2014-01-01

    Full Text Available The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P<0.01 sensitive to oxidation but significantly (P<0.01 resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P<0.01 slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

  8. Autophagy is redundant for the host defense against systemic Candida albicans infections

    NARCIS (Netherlands)

    Smeekens, S.P.; Malireddi, R.K.; Plantinga, T.S.; Buffen, K.; Oosting, M.; Joosten, L.A.B.; Kullberg, B.J.; Perfect, J.R.; Scott, W.K.; Veerdonk, F.L. van de; Xavier, R.J.; Vosse, E. van de; Kanneganti, T.D.; Johnson, M.D.; Netea, M.G.

    2014-01-01

    Autophagy has been demonstrated to play an important role in the immunity against intracellular pathogens, but very little is known about its role in the host defense against fungal pathogens such as Candida albicans. Therefore, the role of autophagy for the host defense against C. albicans was asse

  9. Candida albicans septicemia in a premature infant successfully treated with oral fluconazole

    DEFF Research Database (Denmark)

    Bodé, S; Pedersen-Bjergaard, Lars; Hjelt, K

    1992-01-01

    A premature male infant, birth-weight 1460 g, was treated successfully for a Candida albicans septicemia with orally administered fluconazole for 20 days. Dosage was 5 mg/kg/day. No side effects were seen. Fluconazole may present a major progress in treatment of invasive C. albicans infections...

  10. Synthesis of Melanin Pigment by Candida albicans In Vitro and during Infection

    OpenAIRE

    Morris-Jones, Rachael; Gomez, Beatriz L.; Diez, Soraya; Uran, Martha; Morris-Jones, Stephen D.; Casadevall, Arturo; Nosanchuk, Joshua D.; Hamilton, Andrew J.

    2005-01-01

    Melanins are implicated in the pathogenesis of several important human diseases. This study confirmed the presence of melanin particles in Candida albicans in vitro and during infection. Dark particles were isolated from the digestion of C. albicans cultures and from infected tissue, as established by electron microscopy and immunofluorescence techniques.

  11. Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue

    NARCIS (Netherlands)

    L.M. Schlecht; B.M. Peters; B.P. Krom; J.A. Freiberg; G.M. Hänsch; S.G. Filler; M.A. Jabra-Rizk; M.E. Shirtliff

    2015-01-01

    Candida albicans and Staphylococcus aureus are often co-isolated in cases of biofilm-associated infections. C. albicans can cause systemic disease through morphological switch from the rounded yeast to the invasive hyphal form. Alternatively, systemic S. aureus infections arise from seeding through

  12. Manipulation of host diet to reduce gastrointestinal colonization by the opportunistic pathogen Candida albicans

    Science.gov (United States)

    Candida albicans, the most common human fungal pathogen, can cause systemic infections with a mortality rate of ~40%. Infections arise from colonization of the gastrointestinal (GI) tract, where C. albicans is part of the normal microflora. Reducing colonization in at-risk patients using antifungal ...

  13. Efek Penambahan Glukosa pada Saburoud Dextrose Broth terhadap Pertumbuhan Candida albicans (Uji In Vitro

    Directory of Open Access Journals (Sweden)

    Lakshmi A. Leepel

    2012-10-01

    Full Text Available High carbohydrate intake is one of predisposing factors of oral candidiasis. Objective: Investigating the effect of 1%,5%,10% glucose addition on the growth of C.albicans in vitro. Method: C.albicans sample was taken from oral swab of a male oral candidiasis patient. Identification of C.albicans was conducted using CHROMagar and confirmed by germ tube formation in serum. As a comparison, C.albicans ATCC10231 was used. After 2 days the cultures were serially diluted and inoculated in SDB without glucose, and with 1%,5%,10% addditional glucose, kept for 3 and 7 days in room temperature, then inoculated in SDA. The CFU/ml were counted after 2 days. ANOVA with α0.05 was used. Result: Statisticaly, additional 1% glucose for 3 days lead to significant decreased of growth of both clinical strain and ATCC 10231 C. albicans. However, only additional 5% and 10% glucose in clinical isolate for 7 days increased the growth of C.albicans significantly. Conclusion: The effect of additional glucose on the increased growth of C.albicans in vitro is influenced by the concentration, exposure duration of glucose, and by the strain of C.albicans.DOI: 10.14693/jdi.v16i1.14

  14. Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm.

    Science.gov (United States)

    Martins, Carlos Henrique Gomes; Pires, Regina Helena; Cunha, Aline Oliveira; Pereira, Cristiane Aparecida Martins; Singulani, Junya de Lacorte; Abrão, Fariza; Moraes, Thais de; Mendes-Giannini, Maria José Soares

    2016-04-01

    Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm.

  15. Pengaruh Perendaman Basis Gigitiruan Resin Akrilik Polimerisasi Panas Dalam Ekstrak Kayu Manis Terhadap Jumlah Candida albicans

    OpenAIRE

    Siahaan, Grace Asima

    2015-01-01

    Resin akrilik polimerisasi panas merupakan bahan yang sering digunakan dalam proses pembuatan basis gigitiruan. Bahan ini memilik sifat kemis dan biologis yang berhubungan dengan pembentukan koloni Candida albicans .Terbentuknya koloni Candida albicans pada basis gigitiruan akan mengakibatkan terjadinya denture stomatitis pada pasien pemakai gigitiruan. Oleh karena itu, dokter gigi memiliki tanggung jawab memberikan instruksi kepada pasien agar menjaga kebersihan gigitiruan dan kebersihan ron...

  16. Cigarette smoke-exposed Candida albicans increased chitin production and modulated human fibroblast cell responses.

    Science.gov (United States)

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew; Rouabhia, Mahmoud

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers. PMID:25302312

  17. Candida albicans Carriage in Children with Severe Early Childhood Caries (S-ECC) and Maternal Relatedness

    Science.gov (United States)

    Xiao, Jin; Moon, Yonghwi; Li, Lihua; Rustchenko, Elena; Wakabayashi, Hironao; Zhao, Xiaoyi; Feng, Changyong; Gill, Steven R.; McLaren, Sean; Malmstrom, Hans; Ren, Yanfang; Quivey, Robert

    2016-01-01

    Introduction Candida albicans has been detected together with Streptococcus mutans in high numbers in plaque-biofilm from children with early childhood caries (ECC). The goal of this study was to examine the C. albicans carriage in children with severe early childhood caries (S-ECC) and the maternal relatedness. Methods Subjects in this pilot cross-sectional study were recruited based on a convenient sample. DMFT(S)/dmft(s) caries and plaque scores were assessed during a comprehensive oral exam. Social-demographic and related background information was collected through a questionnaire. Saliva and plaque sample from all children and mother subjects were collected. C. albicans were isolated by BBL™ CHROMagar™ and also identified using germ tube test. S. mutans was isolated using Mitis Salivarius with Bacitracin selective medium and identified by colony morphology. Genetic relatedness was examined using restriction endonuclease analysis of the C. albicans genome using BssHII (REAG-B). Multilocus sequence typing was used to examine the clustering information of isolated C. albicans. Spot assay was performed to examine the C. albicans Caspofungin susceptibility between S-ECC children and their mothers. All statistical analyses (power analysis for sample size, Spearman’s correlation coefficient and multiple regression analyses) were implemented with SAS 9.4 Results A total of 18 S-ECC child-mother pairs and 17 caries free child-mother pairs were enrolled in the study. Results indicated high C. albicans carriage rate in the oral cavity (saliva and plaque) of both S-ECC children and their mothers (>80%). Spearman’s correlation coefficient also indicated a significant correlation between salivary and plaque C. albicans and S. mutans carriage (pcaries severity (pcaries-free children). Among 18 child-mother pairs, >60% of them demonstrated identical C. albicans REAG-B pattern. C. albicans isolated from >65% of child-mother pairs demonstrated similar susceptibility to

  18. PENGHAMBATAN CAJUPUTS CANDY TERHADAP VIABILITAS KHAMIR Candida albicans SECARA IN VITRO [Inhibition of Cajuputs Candy Toward the Viability of Candida albicans by using In Vitro Assay

    OpenAIRE

    C. Hanny Wijaya 2); A. Fieki Rachmatillah1); Bachtiar, Boy M.

    2014-01-01

    The utilization of cajuput essential oil as a flavor in candy may produce a physiological active added value. Some compounds of cajuput plant (Melaleuca cajuputi L) have been reported for their anti-microbial activities. Candida albicans is a normal commensal organism in human mouth. However, it may become virulent and responsible for oral diseases known as oral candidiasis. This study aimed to determine the effect of cajuput and peppermint oil in cajuputs candy in inhibiting the C. albicans ...

  19. Anti-fungal activity of Morinda citrifolia (noni extracts against Candida albicans: An in vitro study

    Directory of Open Access Journals (Sweden)

    K Barani

    2014-01-01

    Full Text Available Aim: The aim of this study was to investigate the anti-fungal activity of Morinda citrifolia fruit extract on Candida albicans. Materials and Methods: Juice extract from M. citrifolia fruit was lyophilized and used in anti-fungal testing. Anti-fungal activity of M. citrifolia fruit extract against C. albicans was tested in vitro at various concentrations. The inhibitory effect of M. citrifolia extract on C. albicans was determined by agar culture and applied broth dilution test. Results: M. citrifolia extract at 1000 μg/ml concentration effectively inhibited the growth of C. albicans (16.6 ± 0.3 compared with the positive control - amphotericin B (20.6 ± 0.6. It was found to be a dose-dependent reaction. Conclusion: M. citrifolia fruit extract had an anti-fungal effect on C. albicans and the inhibitory effect varied with concentration.

  20. Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

    Directory of Open Access Journals (Sweden)

    Nobumasa Hitoshi

    2007-04-01

    Full Text Available Abstract Background Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. Results Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. Conclusion Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross

  1. Activation of the Cph1-dependent MAP kinase signaling pathway induces white-opaque switching in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Bernardo Ramírez-Zavala

    Full Text Available Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white to an elongated cell type (opaque, which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11(ΔN467 efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.

  2. AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

    Science.gov (United States)

    te Riet, Joost; Reinieren-Beeren, Inge; Figdor, Carl G; Cambi, Alessandra

    2015-11-01

    The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs.

  3. Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

    DEFF Research Database (Denmark)

    Novak, Jaroslav P; Kim, Seon-Young; Xu, Jun;

    2006-01-01

    BACKGROUND: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have...... technology give "true" representation of physical processes, involved in measurement of RNA abundance. REVIEWERS: This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser)....

  4. Effect of Candida albicans on Intestinal Ischemia-reperfusion Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Lei Yan; Chun-Rong Wu; Chen Wang; Chun-Hui Yang; Guang-Zhi Tong; Jian-Guo Tang

    2016-01-01

    Background:Inflammation is supposed to play a key role in the pathophysiological processes of intestinal ischemia-reperfusion injury (IIRI),and Candida albicans in human gut commonly elevates inflammatory cytokines in intestinal mucosa.This study aimed to explore the effect of C albicans on IIRI.Methods:Fifty female Wistar rats were divided into five groups according to the status of C albicans infection and IIRI operation:group blank and sham;group blank and IIRI;group cefoperazone plus IIRI;group C.albicans plus cefoperazone and IIRI (CCI);and group C.albicans plus cefoperazone and sham.The levels of inflammatory factors tumor necrosis factor (TNF)-α,interleukin (IL)-6,IL-1β,and diamine oxidase (DAO) measured by enzyme-linked immunosorbent assay were used to evaluate the inflammation reactivity as well as the integrity of small intestine.Histological scores were used to assess the mucosal damage,and the C albicans blood translocation was detected to judge the permeability of intestinal mucosal barrier.Results:The levels of inflammatory factors TNF-α,IL-6,and IL-1β in serum and intestine were higher in rats undergone both C.albicans infection and IIRI operation compared with rats in other groups.The levels of DAO (serum:44.13 ± 4.30 pg/ml,intestine:346.21 ± 37.03 pg/g) and Chiu scores (3.41 ± 1.09) which reflected intestinal mucosal disruption were highest in group CCI after the operation.The number of C.albicans translocated into blood was most in group CCI ([33.80 ± 6.60] × 102 colony forming unit (CFU)/ml).Conclusion:Intestinal C.albicans infection worsened the IIRI-induced disruption of intestinal mucosal barrier and facilitated the subsequent C.albicans translocation and dissemination.

  5. Analysis of genital Candida albicans infection by rapid microsatellite markers genotyping

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-min; MEI Xing-yu; GAO Fei; HUO Ke-ke; SHEN Liang-liang; QIN Hai-hong; WU Zhou-wei; ZHENG Jie

    2007-01-01

    Background Candida albicans (C. albicans) infection, often occurring in genital candidiasis, has increased dramatically recently. Developing an efficient C. albicans typing method may contribute to understanding its epidemiological characteristics and guiding efficient treatment. We used rapid microsatellite genotyping assay for interstrain differentiation of C. albicans isolates and explored some characteristics of its spread.Methods DNA was extracted from C. albicans isolates from gentalia, recta and mouths of 39 female cases and 27 male cases of genital candidiasis. Three fluorescent primers for the microsatellite markers in conserved genes (CDC3, EF3and HIS3) of C. albicans were used to amplify the isolates DNA by PCR. Fluorescent signals were read with an automatic sequencer and analyzed with GeneScan software.Results Analysis of the three microsatellites markers showed 18 gene allelic associations in genital C. albicans infected patients: 10 allelic associations in female and 11 allelic associations in male, of which 3 allelic associations shared by both genders covered 71% of infections. The most dominant allele association of pathogenic strains for both genders was 116:124, 122:131,160:200 that covered about 50% of infection. Gentalia and recta shared the same strains in 80%of female patients, but in only 3.8% of male patients. There were 2.7% female patients, but no males, with same strain in both gentalia and mouths. Five of seven genital C. albicans infected couples had the same allelic associations of which 4were the dominant pathogenic C. albicans susceptible for both genders.Conclusions The predominant allelic association of the pathogenic strain in genital C. albicans infection is 116:124,122:131, 160:200. Vaginal pathogenic strains are probably maintained from the rectal reservoir. Pathogenic strains of male patients are probably from frequent sexual intercourse. The aggressiveness of some strains varies with gender.

  6. Candida albicans exposures, sex specificity and cognitive deficits in schizophrenia and bipolar disorder

    Science.gov (United States)

    Severance, Emily G; Gressitt, Kristin L; Stallings, Catherine R; Katsafanas, Emily; Schweinfurth, Lucy A; Savage, Christina L; Adamos, Maria B; Sweeney, Kevin M; Origoni, Andrea E; Khushalani, Sunil; Leweke, F Markus; Dickerson, Faith B; Yolken, Robert H

    2016-01-01

    Immune aberrations in schizophrenia and bipolar disorder have led to the hypotheses that infectious agents or corresponding immune responses might contribute to psychiatric etiopathogeneses. We investigated case–control differences in exposure to the opportunistic fungal pathogen, Candida albicans, and examined associations with cognition, medication, lifestyle, and somatic conditions. We quantified C. albicans IgG antibodies in two cohorts totaling 947 individuals and evaluated odds ratios (OR) of exposure with psychiatric disorder using multivariate regressions. The case–control cohort included 261 with schizophrenia, 270 with bipolar disorder, and 277 non-psychiatric controls; the second included 139 with first-episode schizophrenia, 78 of whom were antipsychotic naive. No differences in C. albicans exposures were found until diagnostic groups were stratified by sex. In males, C. albicans seropositivity conferred increased odds for a schizophrenia diagnosis (OR 2.04–9.53, P⩽0.0001). In females, C. albicans seropositivity conferred increased odds for lower cognitive scores on Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) in schizophrenia (OR 1.12, P⩽0.004), with significant decreases on memory modules for both disorders (P⩽0.0007–0.03). C. albicans IgG levels were not impacted by antipsychotic medications. Gastrointestinal (GI) disturbances were associated with elevated C. albicans in males with schizophrenia and females with bipolar disorder (P⩽0.009–0.02). C. albicans exposure was associated with homelessness in bipolar males (P⩽0.0015). In conclusion, sex-specific C. albicans immune responses were evident in psychiatric disorder subsets. Inquiry regarding C. albicans infection or symptoms may expedite amelioration of this treatable comorbid condition. Yeast exposure as a risk factor for schizophrenia and its associated cognitive and GI effects require further investigation including the possible contribution of

  7. A novel immune evasion strategy of candida albicans: proteolytic cleavage of a salivary antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Timothy F Meiller

    Full Text Available Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps, involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the

  8. Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans.

    Science.gov (United States)

    Barbosa, Júnia Oliveira; Rossoni, Rodnei Dennis; Vilela, Simone Furgeri Godinho; de Alvarenga, Janaína Araújo; Velloso, Marisol dos Santos; Prata, Márcia Cristina de Azevedo; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.

  9. Candida albicans exposures, sex specificity and cognitive deficits in schizophrenia and bipolar disorder.

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    Severance, Emily G; Gressitt, Kristin L; Stallings, Catherine R; Katsafanas, Emily; Schweinfurth, Lucy A; Savage, Christina L; Adamos, Maria B; Sweeney, Kevin M; Origoni, Andrea E; Khushalani, Sunil; Leweke, F Markus; Dickerson, Faith B; Yolken, Robert H

    2016-01-01

    Immune aberrations in schizophrenia and bipolar disorder have led to the hypotheses that infectious agents or corresponding immune responses might contribute to psychiatric etiopathogeneses. We investigated case-control differences in exposure to the opportunistic fungal pathogen, Candida albicans, and examined associations with cognition, medication, lifestyle, and somatic conditions. We quantified C. albicans IgG antibodies in two cohorts totaling 947 individuals and evaluated odds ratios (OR) of exposure with psychiatric disorder using multivariate regressions. The case-control cohort included 261 with schizophrenia, 270 with bipolar disorder, and 277 non-psychiatric controls; the second included 139 with first-episode schizophrenia, 78 of whom were antipsychotic naive. No differences in C. albicans exposures were found until diagnostic groups were stratified by sex. In males, C. albicans seropositivity conferred increased odds for a schizophrenia diagnosis (OR 2.04-9.53, P⩽0.0001). In females, C. albicans seropositivity conferred increased odds for lower cognitive scores on Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) in schizophrenia (OR 1.12, P⩽0.004), with significant decreases on memory modules for both disorders (P⩽0.0007-0.03). C. albicans IgG levels were not impacted by antipsychotic medications. Gastrointestinal (GI) disturbances were associated with elevated C. albicans in males with schizophrenia and females with bipolar disorder (P⩽0.009-0.02). C. albicans exposure was associated with homelessness in bipolar males (P⩽0.0015). In conclusion, sex-specific C. albicans immune responses were evident in psychiatric disorder subsets. Inquiry regarding C. albicans infection or symptoms may expedite amelioration of this treatable comorbid condition. Yeast exposure as a risk factor for schizophrenia and its associated cognitive and GI effects require further investigation including the possible contribution of gut

  10. Antibody microarray profiling of osteosarcoma cell serum for identifying potential biomarkers.

    Science.gov (United States)

    Zhu, Zi-Qiang; Tang, Jin-Shan; Gang, Duan; Wang, Ming-Xing; Wang, Jian-Qiang; Lei, Zhou; Feng, Zhou; Fang, Ming-Liang; Yan, Lin

    2015-07-01

    The aim of the present study was to identify biomarkers in osteosarcoma (OS) cell serum by antibody microarray profiling, which may be used for OS diagnosis and therapy. An antibody microarray was used to detect the expression levels of cytokines in serum samples from 20 patients with OS and 20 healthy individuals. Significantly expressed cytokines in OS serum were selected when P2. An enzyme-linked immunosorbent assay (ELISA) was used to validate the antibody microarray results. Finally, classification accuracy was calculated by cluster analysis. Twenty one cytokines were significantly upregulated in OS cell serum samples compared with control samples. Expression of interleukin-6, monocyte chemoattractant protein-1, tumor growth factor-β, growth-related oncogene, hepatocyte growth factor, chemokine ligand 16, Endoglin, matrix metalloproteinase-9 and platelet-derived growth factor-AA was validated by ELISAs. OS serum samples and control samples were distinguished by significantly expressed cytokines with an accuracy of 95%. The results demonstrated that expressed cytokines identified by antibody microarray may be used as biomarkers for OS diagnosis and therapy.

  11. Measuring information flow in cellular networks by the systems biology method through microarray data.

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    Chen, Bor-Sen; Li, Cheng-Wei

    2015-01-01

    In general, it is very difficult to measure the information flow in a cellular network directly. In this study, based on an information flow model and microarray data, we measured the information flow in cellular networks indirectly by using a systems biology method. First, we used a recursive least square parameter estimation algorithm to identify the system parameters of coupling signal transduction pathways and the cellular gene regulatory network (GRN). Then, based on the identified parameters and systems theory, we estimated the signal transductivities of the coupling signal transduction pathways from the extracellular signals to each downstream protein and the information transductivities of the GRN between transcription factors in response to environmental events. According to the proposed method, the information flow, which is characterized by signal transductivity in coupling signaling pathways and information transductivity in the GRN, can be estimated by microarray temporal data or microarray sample data. It can also be estimated by other high-throughput data such as next-generation sequencing or proteomic data. Finally, the information flows of the signal transduction pathways and the GRN in leukemia cancer cells and non-leukemia normal cells were also measured to analyze the systematic dysfunction in this cancer from microarray sample data. The results show that the signal transductivities of signal transduction pathways change substantially from normal cells to leukemia cancer cells.

  12. An expanded regulatory network temporally controls Candida albicans biofilm formation.

    Science.gov (United States)

    Fox, Emily P; Bui, Catherine K; Nett, Jeniel E; Hartooni, Nairi; Mui, Michael C; Andes, David R; Nobile, Clarissa J; Johnson, Alexander D

    2015-06-01

    Candida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free-floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C. albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time-dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points.

  13. Candida albicans menengitis in a newborn with classical galactosemia

    Directory of Open Access Journals (Sweden)

    Hüseyin Altunhan

    2012-12-01

    Full Text Available Classical galactosemia is a rarely seen carbohydrate metabolismdisorder. The frequency of sepsis significantlyincreases in patients with galactosemia. The most commonagent causing sepsis is E. coli. Sepsis due to fungusin patients with galactosemia is rarely reported. Candidais an important cause of sepsis in newborn intensive careunits especially in newborns with underlying risk factorssuch as prematurity and low birth weight. Although themost common etiologic agent of sepsis is E. coli in caseswith galactosemia, it should be kept in mind that candidamay also be causative agent of sepsis and meningitis inthese patients even though there is no underlying risk factor.Also the clinical and laboratory findings of candidiasismay be obscure. For this reason, especially in newborncandida meningitis, the index of suspicion should be kepthigh for early diagnosis and treatment. In such patientscerebrospinal fluid analysis, culture and brain imagingshould be done necessarily, because early diagnosis andtreatment will be life saving. In this article we reported agalactosemia case with the diagnosis of meningitis andCandida albicans grown in his blood culture derived onthe fourth day of admission to clinic.Key words: Candida albicans, galactosemia, meningitis,newborn, sepsis

  14. Rat indwelling urinary catheter model of Candida albicans biofilm infection.

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    Nett, Jeniel E; Brooks, Erin G; Cabezas-Olcoz, Jonathan; Sanchez, Hiram; Zarnowski, Robert; Marchillo, Karen; Andes, David R

    2014-12-01

    Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida, exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract.

  15. Candida albicans susceptibility to lactoperoxidase-generated hypoiodite

    Directory of Open Access Journals (Sweden)

    Mohamed Ahariz

    2010-08-01

    Full Text Available Mohamed Ahariz1, Philippe Courtois21Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, Belgium; 2Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, Belgium and UER de Biologie Médicale, Haute Ecole Francisco Ferrer, Brussels, BelgiumAbstract: In vivo, lactoperoxidase produces hypothiocyanite (OSCN- from thiocyanate (SCN- in the presence of hydrogen peroxide (H2O2; in vitro, iodide (I- can be oxidized into hypoiodite (OI- by this enzyme. The aim of this study was to compare in vitro the anti-Candida effect of iodide versus thiocyanate used as lactoperoxidase substrate to prevent Candida biofilms development. Candida albicans ATCC 10231 susceptibility upon both peroxidase systems was tested in three different experimental designs: (i in a liquid culture medium, (ii in an interface model between solid culture medium and gel containing the enzymic systems, (iii in a biofilm model onto titanium and acrylic resin. Yeast growth in liquid medium was monitored by turbidimetry at 600 nm. Material-adherent yeast biomass was evaluated by the tetrazolium salt MTT method. The iodide-peroxidase system has been shown to inhibit Candida biofilm formation at lower substrate concentrations (~200 fold less H2O2 donor and for longer incubation periods than the thiocyanate-peroxidase system. In conclusion, efficiency of lactoperoxidase-generated OI- to prevent C. albicans biofilm development allows refining iodine antifungal use in ex vivo conditions.Keywords: denture, iodide, oral, peroxidase, saliva, titanium

  16. Feasibility study on blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies. Final report

    International Nuclear Information System (INIS)

    The final report on the feasibility of blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies covers the following topics: blood samples; methodologies: 2D gel electrophoresis; protein identification using MALDI-MS; accomplishment and evaluation of the proteomics and cDNA microarray analysis.

  17. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Bader Oliver

    2008-07-01

    Full Text Available Abstract Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Results In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

  18. Formation and characterization of DNA microarrays at silicon nitride substrates.

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    Manning, Mary; Redmond, Gareth

    2005-01-01

    A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.

  19. Extended -Regular Sequence for Automated Analysis of Microarray Images

    Directory of Open Access Journals (Sweden)

    Jin Hee-Jeong

    2006-01-01

    Full Text Available Microarray study enables us to obtain hundreds of thousands of expressions of genes or genotypes at once, and it is an indispensable technology for genome research. The first step is the analysis of scanned microarray images. This is the most important procedure for obtaining biologically reliable data. Currently most microarray image processing systems require burdensome manual block/spot indexing work. Since the amount of experimental data is increasing very quickly, automated microarray image analysis software becomes important. In this paper, we propose two automated methods for analyzing microarray images. First, we propose the extended -regular sequence to index blocks and spots, which enables a novel automatic gridding procedure. Second, we provide a methodology, hierarchical metagrid alignment, to allow reliable and efficient batch processing for a set of microarray images. Experimental results show that the proposed methods are more reliable and convenient than the commercial tools.

  20. Competitive Interactions between C. albicans, C. glabrata and C. krusei during Biofilm Formation and Development of Experimental Candidiasis.

    Science.gov (United States)

    Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; dos Santos, Jéssica Diane; de Barros, Patrícia Pimentel; Prata, Márcia Cristina de Azevedo; Anbinder, Ana Lia; Fuchs, Beth Burgwyn; Jorge, Antonio Olavo Cardoso; Mylonakis, Eleftherios; Junqueira, Juliana Campos

    2015-01-01

    In this study, we evaluated the interactions between Candida albicans, Candida krusei and Candida glabrata in mixed infections. Initially, these interactions were studied in biofilms formed in vitro. CFU/mL values of C. albicans were lower in mixed biofilms when compared to the single biofilms, verifying 77% and 89% of C. albicans reduction when this species was associated with C. glabrata and C. krusei, respectively. After that, we expanded this study for in vivo host models of experimental candidiasis. G. mellonella larvae were inoculated with monotypic and heterotypic Candida suspensions for analysis of survival rate and quantification of fungal cells in the haemolymph. In the groups with single infections, 100% of the larvae died within 18 h after infection with C. albicans. However, interaction groups achieved 100% mortality after 72 h of infection by C. albicans-C. glabrata and 96 h of infection by C. albicans-C. krusei. C. albicans CFU/mL values from larvae hemolymph were lower in the interacting groups compared with the monoespecies group after 12 h of infection. In addition, immunosuppressed mice were also inoculated with monotypic and heterotypic microbial suspensions to induce oral candidiasis. C. albicans CFU/mL values recovered from oral cavity of mice were higher in the group with single infection by C. albicans than the groups with mixed infections by C. albicans-C. glabrata and C. albicans-C. krusei. Moreover, the group with single infection by C. albicans had a higher degree of hyphae and epithelial changes in the tongue dorsum than the groups with mixed infections. We concluded that single infections by C. albicans were more harmful for animal models than mixed infections with non-albicans species, suggesting that C. albicans establish competitive interactions with C. krusei and C. glabrata during biofilm formation and development of experimental candidiasis.