Supernatants from culture of type I collagen-stimulated PBMC from patients with cutaneous systemic sclerosis versus localized scleroderma demonstrate suppression of MMP-1 by fibroblasts.

Science.gov (United States)

Brown, Monica; Postlethwaite, Arnold E; Myers, Linda K; Hasty, Karen A

2012-06-01

Systemic sclerosis (SSc) is a chronic fibrosing disease characterized by vasculopathy, autoimmunity, and an accumulation of collagen in tissues. Numerous studies have shown that compared to healthy or diseased controls, the peripheral blood mononuclear cells (PBMC) from patients with SSc produce a variety of cytokines or proliferate when cultured with solubilized type I collagen (CI) or constituent α1(II) and α2(I) polypeptide chains. The purpose of this study was to determine whether PBMC isolated from patients with SSc and cultured in vitro with soluble CI elaborated soluble mediators that inhibit the production of collagenase (i.e., matrix metalloproteinase, MMP-1) by fibroblasts. Supernatants of CI-stimulated PBMC from juvenile and adult diffuse cutaneous (dc)SSc patients significantly reduced MMP-1 production by SSc dermal fibroblasts, while supernatants of CI-stimulated PBMC from patients with localized scleroderma (LS) did not. CI-stimulated PBMC culture supernatants from patients with dcSSc in contrast to patients with LS exhibited increased levels of platelet-derived growth factor (PDGF)-AA, PDGF-BB, TNF-α, IL-13, and EGF. Prolonged culture of SSc dermal fibroblasts with recombinant PDGF-BB or IL-13 inhibited the induction of MMP-1 in response to subsequent TNF-α stimulation. These data suggest that therapies aimed at reducing these cytokines may decrease collagen accumulation in SSc, preventing the development of chronic fibrosis.

Transformation of carbon tetrachloride and chloroform by trichloroethene respiring anaerobic mixed cultures and supernatant.

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Vickstrom, Kyle E; Azizian, Mohammad F; Semprini, Lewis

2017-09-01

Carbon tetrachloride (CT) and chloroform (CF) were transformed in batch reactor experiments conducted with anaerobic dechlorinating cultures and supernatant (ADC + S) harvested from continuous flow reactors. The Evanite (EV) and Victoria/Stanford (VS) cultures, capable of respiring trichloroethene (TCE), 1,2-cis-dichloroethene (cDCE), and vinyl chloride (VC) to ethene (ETH), were grown in continuous flow reactors receiving an influent feed of saturated TCE (10 mM; 60 mEq) and formate (45 mM; 90 mEq) but no CT or CF. Cells and supernatant were harvested from the chemostats and inoculated into batch reactors at the onset of each experiment. CT transformation was complete following first order kinetics with CF, DCM and CS 2 as the measurable transformation products, representing 20-40% of the original mass of CT, with CO 2 likely the unknown transformation product. CF was transformed to DCM and likely CO 2 at an order of magnitude rate lower than CT, while DCM was not further transformed. An analytical first order model including multiple key reactions effectively simulated CT transformation, product formation and transformation, and provided reasonable estimates of transformation rate coefficients. Biotic and abiotic treatments indicated that CT was mainly transformed via abiotic processes. However, the presence of live cells was associated with the transformation of CF to DCM. In biotic tests both TCE and CT were simultaneously transformed, with TCE transformed to ETH and approximately 15-53% less CF formed via CT transformation. A 14-day exposure to CF (CF max  = 1.4 μM) reduced all rates of chlorinated ethene respiration by a factor of 10 or greater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antifungal performance of extracellular chitinases and culture supernatants of Streptomyces galilaeus CFFSUR-B12 against Mycosphaerella fijiensis Morelet.

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Castillo, Benjamín Moreno; Dunn, Michael F; Navarro, Karina Guillén; Meléndez, Francisco Holguín; Ortiz, Magdalena Hernández; Guevara, Sergio Encarnación; Palacios, Graciela Huerta

2016-03-01

The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62% of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

Science.gov (United States)

Shankar, Jayendra; Walker, Rachel G; Wilkinson, Mark C; Ward, Deborah; Horsburgh, Malcolm J

2012-07-01

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

[Contribution of blue-green pigments to hemolytic activity of Pseudomonas aeruginosa cultural fluid].

Science.gov (United States)

Pyzh, A É; Nikandrov, V N

2011-01-01

To assess the contribution of blue-green pigments of Pseudomonas aeruginosa to hemolytic activity of its cultural fluid. MATERIALS AND METHODS. Eight hospital strains and reference strain ATCC 15442 were used. Growth dynamics of strains as well as features of accumulation of hemolytic and phospholipase activity were studied. Purified samples of pyoverdin and pyocyanin were extracted by gel-chromatography and chloroform extraction methods. Hemolytic and lecitinase activities of the samples as well as effect of active oxygen scavengers and chelating agents on these activities were studied. Dynamics of accumulation of hemolytic activity significantly differed from that of phospholipase activity when strains were grown in liquid medium. Chromatographic separation of the pigments from cultural fluid supernatants sharply reduced its hemolytic activity. Purified samples of pyoverdin and pyocyanin were capable to lyse erythrocytes and chicken egg lecitin. These characteristics of the pigments were inhibited by nitroblue tetrazolium and sensitive to chelating agents. Conclusion. Pyoverdin and pyocyanin of pathogenic strains of P. aeruginosa are capable to lyse erythrocytes and suspension of purified chicken egg lecitin, they contribute to total hemolytic activity of pathogenic strains of Pseudomonas, which is not determined only by phospholipase C produced by microorganism. Lytic activity of the pigments is blocked by nitroblue tetrazolium and susceptible to some chelating agents. Apparently, this activity is mediated by superoxide radical and determined by presence of metals with transient valence in pigments' molecules.

Effect of bacterial components of mixed culture supernatants of planktonic and biofilm Pseudomonas aeruginosa with commensal Escherichia coli on the neutrophil response in vitro.

Science.gov (United States)

Maslennikova, Irina L; Kuznetsova, Marina V; Nekrasova, Irina V; Shirshev, Sergei V

2017-11-30

Pseudomonas aeruginosa (PA) responsible for acute and chronic infections often forms a well-organized bacterial population with different microbial species including commensal strains of Escherichia coli. Bacterial extracellular components of mixed culture can modulate the influence of bacteria on the neutrophil functions. The objective of this study was to compare the effect of pyocyanin, pyoverdine, LPS, exopolysaccharide of single species and mixed culture supernatants of PA strains and E. coli K12 on microbicidal, secretory activity of human neutrophils in vitro. Bacterial components of E. coli K12 in mixed supernatants with 'biofilm' PA strains (PA ATCC, PA BALG) enhanced short-term microbicidal mechanisms and inhibited neutrophil secretion delayed in time. The influence of 'planktonic' PA (PA 9-3) exometabolites in mixed culture is almost mimicked by E. coli K12 effect on functional neutrophil changes. This investigation may help to understand some of the mechanisms of neutrophil response to mixed infections of different PA with other bacteria species. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

Science.gov (United States)

Meza-Lucas, Antonio; Pérez-Villagómez, María-Fernanda; Martínez-López, José-Patricio; García-Rodea, Ricardo; Martínez-Castelán, María-Guadalupe; Escobar-Gutiérrez, Alejandro; de-la-Rosa-Arana, Jorge-Luis; Villanueva-Zamudio, Altagracia

2016-09-01

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

Use of QuantiFERON®-TB Gold in-tube culture supernatants for measurement of antibody responses.

Directory of Open Access Journals (Sweden)

Simon G Kimuda

Full Text Available QuantiFERON®-TB Gold in-tube (QFT-GIT supernatants may be important samples for use in assessment of anti-tuberculosis (TB antibodies when only limited volumes of blood can be collected and when a combination of antibody and cytokine measurements are required. These analytes, when used together, may also have the potential to differentiate active pulmonary TB (APTB from latent TB infection (LTBI. However, few studies have explored the use of QFT-GIT supernatants for investigations of antibody responses. This study determined the correlation and agreement between anti-CFP-10 and anti-ESAT-6 antibody concentrations in QFT-GIT nil supernatant and serum pairs from 68 TB household contacts. We also explored the ability of Mycobacterium tuberculosis (M.tb specific antibodies, or ratios of antibody to interferon gamma (IFN-γ in QFT-GIT supernatants, to differentiate 97 APTB cases from 58 individuals with LTBI. Sputum smear microscopy was used to define APTB, whereas the QFT-GIT and tuberculin skin test were used to define LTBI. There were strong and statistically significant correlations between anti-CFP-10 and anti-ESAT-6 antibodies in unstimulated QFT-GIT supernatants and sera (r = 0.89; p<0.0001 for both, and no significant differences in antibody concentration between them. Anti-CFP-10 & anti-ESAT-6 antibodies differentiated APTB from LTBI with sensitivities of 88.7% & 71.1% and specificities of 41.4% & 51.7% respectively. Anti-CFP-10 antibody/M.tb specific IFN-γ and anti-ESAT-6 antibody/M.tb specific IFN-γ ratios had sensitivities of 48.5% & 54.6% and specificities of 89.7% and 75.9% respectively. We conclude that QFT-GIT nil supernatants may be used in the place of sera when measuring antibody responses, reducing blood volumes needed for such investigations. Antibodies in QFT-GIT nil supernatants on their own discriminate APTB from LTBI with high sensitivity but have poor specificity, whereas the reverse is true when antibodies are used in

Growth Inhibition of Cronobacter sakazakii in Experimentally Contaminated Powdered Infant Formula by Kefir Supernatant.

Science.gov (United States)

Kim, Dong-Hyeon; Chon, Jung-Whan; Kang, Il-Byeong; Kim, Hyunsook; Kim, Hong-Seok; Song, Kwang-Young; Seo, Kun-Ho

2015-09-01

Kefir is a type of fermented milk containing lactic and acetic acid bacteria and yeast. In this study, we evaluated the antimicrobial activity of kefir supernatant against Cronobacter sakazakii in powdered infant formula (PIF). In a spot-on-lawn test, the growth of 20 C. sakazakii strains, including 10 clinical and 10 food isolates, was completely inhibited in the presence of kefir supernatant. Significant differences in the diameters of inhibition zones were observed upon treatment with kefir compared with the results for Lactobacillus kefiri and Candida kefyr culture supernatants or solutions of lactic and acetic acid and ethyl alcohol in the agar well diffusion test (P < 0.05). The addition of 100 μl of kefir supernatant to 1 ml of nutrient broth completely inhibited the growth of C. sakazakii, as evaluated by spectrophotometry. The antimicrobial activity of kefir supernatant in experimentally contaminated PIF was also tested; we found no viable C. sakazakii cells remaining in PIF rehydrated with 30% kefir supernatant solution for 1 h, demonstrating that the antimicrobial activity of kefir supernatant against C. sakazakii could be applied in real food samples.

Injection of embryo culture supernatant to the endometrial cavity does not affect outcomes in IVF/ICSI or oocyte donation cycles: a randomized clinical trial.

Science.gov (United States)

Prapas, Yannis; Petousis, Stamatios; Panagiotidis, Yannis; Gullo, Giuseppe; Kasapi, Lia; Papadeothodorou, Achilleas; Prapas, Nikos

2012-06-01

To evaluate whether intrauterine injection of embryo culture supernatant before embryo transfer has any impact on pregnancy and implantation rates. A total of 400 cycles, of which 200 IVF/ICSI and 200 oocyte donor (OD), were randomly assigned to have their uterine cavity injected (group I) or not (group II). Primary endpoints to be studied were pregnancy and implantation rates. Clinical pregnancy rate per transfer (47.87%, 90/188 versus 48.45%, 94/194) based on transvaginal scan findings at 7 weeks of gestation and implantation rate (25.6% versus 26.5%) were similar in the two groups. The day of embryo transfer, day 3 or day 5, did not affect the final outcome. Injection of embryo culture supernatant into the uterine cavity, 30 min before the embryo transfer on either day 3 or 5, neither improves nor adversely affects the pregnancy rate in IVF/ICSI or oocyte donation cycles. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

In vitro and in vivo antitumor effects of 50 to 100-KDa components from B16 melanoma culture supernatant.

Science.gov (United States)

Qin, Ying-Song; Zhang, X U; Zhang, Xiang-Yu

2015-07-01

The development of immunological therapies for melanoma has been of considerable concern in recent years. Whole tumor cell lysates have been used to develop antitumor vaccines, but the effective components of the lysates have not been identified. In the present study, protein elements were purified from the B16 supernatant to analyze the in vitro chemotaxis towards mouse spleen lymphocytes using a Boyden chamber. Prior to establishing a B16 melanoma model, C57BL/6 mice were vaccinated with these proteins, and melanoma growth, tumor appearance time and behavioral changes were observed. Next, the cytotoxicity and subsets of the tumor infiltrating lymphocytes, and the histological characteristics of the melanoma were analyzed. The isolated purified fragments of B16 melanoma culture supernatant had strong antitumor effects. The possible antitumor mechanism was delineated, and was identified to possibly be through the activation of cluster of differentiation 8-positive T cells and the promotion of B16 cell differentiation. These methods will provide a novel insight into understanding antitumor immunological mechanisms and provide a potential avenue for immunotherapy.

Bacterial Population in Intestines of Litopenaeus vannamei Fed Different Probiotics or Probiotic Supernatant.

Science.gov (United States)

Sha, Yujie; Liu, Mei; Wang, Baojie; Jiang, Keyong; Qi, Cancan; Wang, Lei

2016-10-28

The interactions of microbiota in the gut play an important role in promoting or maintaining the health of hosts. In this study, in order to investigate and compare the effects of dietary supplementation with Lactobacillus pentosus HC-2 (HC-2), Enterococcus faecium NRW-2, or the bacteria-free supernatant of a HC-2 culture on the bacterial composition of Litopenaeus vannamei , Illumina sequencing of the V1-V2 region of the 16S rRNA gene was used. The results showed that unique species exclusively existed in specific dietary groups, and the abundance of Actinobacteria was significantly increased in the intestinal bacterial community of shrimp fed with the bacteria-free supernatant of an HC-2 culture compared with the control. In addition, the histology of intestines of the shrimp from the four dietary groups was also described, but no obvious improvements in the intestinal histology were observed. The findings in this work will help to promote the understanding of the roles of intestinal bacteria in shrimps when fed with probiotics or probiotic supernatant.

Ascitic Fluid Culture In Cirrhotic Patients With Spontaneous Bacterial Peritonitis

International Nuclear Information System (INIS)

Sajjad, M.; Khan, Z.A.; Khan, M.S.

2016-01-01

Objective: To determine the frequency and compare the culture yield of bacterial isolation by conventional and blood culture BACTEC bottle techniques in cirrhotic patients with spontaneous bacterial peritonitis (SBP). Study Design: Cross-sectional comparative study. Place and Duration of Study: Pathology Department, Bannu Medical College, Bannu, KPK, from January 2012 to December 2013. Methodology: Paracentesis of 20 ml of ascitic fluid tapped from cirrhotic patients with SBP was carried out by a single technologist. The analysis included differential leukocyte count (DLC), while 5 ml each of the fluid was inoculated into conventional culture media and BACTEC blood culture bottle. All the data were analysed on (SPSS) version 16 to determine frequencies with percentages and mean values with standard deviation. Chi-square test was used for comparing the yield of conventional and blood culture bottle methods. P-value was considered significant if < 0.05. Results: In 105 cases of ascitic fluid analyses, 27 (25.72 percent) had positive ascitic fluid culture whereas 78 (74.28 percent) had negative ascitic fluid culture. Ascitic fluid culture was positive in 6 cases by conventional culture media and in 27 cases by BACTEC culture bottle media (p < 0.001). Bacterial isolation was obtained by both culture methods in 6 cases (p < 0.001). Conclusion: Direct bedside inoculation of ascitic fluid by BACTEC culture bottle method has better yield as compared to conventional culture method. (author)

Determination of drugs in biological fluids by high-performance liquid chromatography with on-line sample processing.

Science.gov (United States)

Oertel, R; Richter, K; Gramatté, T; Kirch, W

1998-02-27

An automated two column HPLC system with the new packing material LiChrospher RP-18 ADS (alkyl-diol-silica) was tested for the determination of several drugs and metabolites (talinolol, celiprolol, metoprolol, oxprenolol, triamterene, trimethoprim, tiracizine, articaine, detajmium, ajmaline, lamotrigine) in various biological fluids (serum, urine, intestinal aspirates, supernatants of cell cultures and supernatants after protein denaturation). The method allows the direct injection of biological fluids into a reversed-phase HPLC system and on-line clean-up and sample enrichment by a column-switching technique. Precision, accuracy and sensitivity were similar to conventional assays as described in the literature. With this new method it was possible to measure drug concentrations in various biological fluids without changing the sample preparation procedure. In some cases an additional sample preparation like protein denaturation or solid-phase extraction was advantageous to enhance the sensitivity of the method and the life-time of the ADS column.

Analysis of glutathione in supernatants and lysates of a human proximal tubular cell line from perfusion culture upon intoxication with cadmium chloride by HPLC and LC-ESI-MS

NARCIS (Netherlands)

Hahn, Hans; Huck, Christian W; Rainer, Matthias; Najam-ul-Haq, Muhammad; Bakry, Rania; Abberger, Thomas; Jennings, Paul; Pfaller, Walter; Bonn, Günther K

A simple and highly effective reversed-phase (RP) high-performance liquid chromatography (HPLC) method is described for analysing glutathione (GSH) and glutathione disulfide (GSSG) in out-flowing supernatants and lysates of perfusion cell cultures of human kidney cells (HK-2 cells) continuously

Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

Science.gov (United States)

Abe, P M; Kendall, C J; Stauffer, L R; Holland, J W

1979-01-01

Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.

Inhibition of the early stage of Salmonella enterica serovar Enteritidis biofilm development on stainless steel by cell-free supernatant of a Hafnia alvei culture.

Science.gov (United States)

Chorianopoulos, Nikos G; Giaouris, Efstathios D; Kourkoutas, Yiannis; Nychas, George-John E

2010-03-01

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

Science.gov (United States)

Lee, Jin Ju; Lim, Jeong Ju; Kim, Dae Geun; Simborio, Hannah Leah; Kim, Dong Hyeok; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

2014-09-01

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

International Nuclear Information System (INIS)

Minagawa, T.; Nakaya, C.; Iida, H.

1974-01-01

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

Supernatant treatment system design through testing

International Nuclear Information System (INIS)

Ploetz, D.K.; Leonard, I.M.

1988-12-01

The main purpose of the Supernatant Treatment System (STS) is to remove more than 99.9 percent of the radioactive cesium (Cs-137) from the high-level waste stored in tank 8D-2. Cesium removal is accomplished in the STS by processing the supernatant (liquid) portion of the high-level waste through three or four ion exchange columns filled with zeolite. After treatment in the STS, the decontaminated supernatant is processed as low-level waste and finally encapsulated in cement for eventual disposal. The Cs-137 removed from the waste and absorbed onto zeolite ion exchange material is temporarily stored in tank 8D-1 until it can be encapsulated in glass and disposed of as high-level waste. This report discusses construction and testing of the STS. Design of the STS was started in 1982 in parallel with the selection of the ion exchange material. The construction of this system was accomplished in five phase in parallel with completion of design to allow for faster completion of the project. The existing high-level waste storage tanks -- 8D-1, 8D-2, and 8D-3 -- required major renovations to permit transfer of the high-level waste from tank 8D-2 to tank 8D-1, to house the components that comprise the STS in tank 8D-1, and to store decontaminated wastes in tank 8D-3. Testing in the STS started before construction was complete and was accomplished by first testing components individually. Then the system was retested using simulated supernatant. Integrated testing of the whole Integrated Radwaste Treatment System (IRTS), which includes the STS, Liquid Waste Treatment System (LWTS), Cement Solidification System (CSS), and the Drum Cell, was also performed using simulated supernatant. Finally, slightly radioactive condensate water from tank 8D-1 was processed. After successfully completing this testing, the STS started operations with radioactive supernatant on May 23, 1988. 21 refs., 33 figs., 21 tabs

Maxisorp RAST. A sensitive method for detection of antigen-specific human IgE in culture fluids

DEFF Research Database (Denmark)

Poulsen, L K; Pedersen, M F; Malling, H J

1989-01-01

For determination of allergen-specific IgE in cell culture supernatants and other highly diluted IgE preparations a radioallergosorbent test (RAST) based on high adsorption polystyrene test tubes has been developed ("Maxisorp RAST"). Cladosporium herbarum extract was used as a model allergen...

Evaluation of the Antibody in Lymphocyte Supernatant Assay to Detect Active Tuberculosis.

Directory of Open Access Journals (Sweden)

Margaretha Sariko

Full Text Available We aimed to evaluate the antibody in lymphocyte supernatant (ALS assay as a biomarker to diagnose tuberculosis among adults from Tanzania with and without HIV.Adults admitted with suspicion for tuberculosis had sputa obtained for GeneXpert MTB/RIF, acid-fast bacilli smear and mycobacterial culture; blood was obtained prior to treatment initiation and after 4 weeks. Adults hospitalized with non-infectious conditions served as controls. Peripheral blood mononuclear cells were cultured unstimulated for 72 hours. Anti-mycobacterial antibodies were measured from culture supernatants by ELISA, using BCG vaccine as the coating antigen. Median ALS responses were compared between cases and controls at baseline and between cases over time.Of 97 TB cases, 85 were microbiologically confirmed and 12 were clinically diagnosed. Median ALS responses from TB cases (0.366 OD from confirmed cases and 0.285 from clinical cases were higher compared to controls (0.085, p<0.001. ALS responses did not differ based on HIV status, CD4 count or sputum smear status. Over time, the median ALS values declined significantly (0.357 at baseline; 0.198 after 4-weeks, p<0.001.Robust ALS responses were mounted by patients with TB regardless of HIV status, CD4 count, or low sputum bacillary burden, potentially conferring a unique niche for this immunologic biomarker for TB.

In vitro culture of mouse embryos amniotic fluid ID human

African Journals Online (AJOL)

1989-07-15

Jul 15, 1989 ... Because human amniotic fluid is a physiological, balanced ultrafiltrate, it has been considered as an inexpensive alternative culture medium in. IVF. A study of the development of mouse embryos in human amniotic fluid was undertaken to assess the suitability of this as an optional culture medium in human ...

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

Science.gov (United States)

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis Tratamento de soro com sobrenadante de cultura de Candida albicans reduz a fagocitose soro-dependente

Directory of Open Access Journals (Sweden)

Aderbal Antonio dos Santos

2002-01-01

Full Text Available Candida albicans is a potent activator of the complement system, and heat labile opsonins produced by activation of C3 (C3b and iC3b enhance phagocytosis of C. albicans mediated by complement receptors. In this study we treated mouse serum with supernatants from cultures of a protease producer strain of C. albicans and evaluated the ability of this serum to enhance phagocytosis of C. albicans. Cell-free supernatants from cultures of C. albicans were concentrated 5 fold and added to mouse serum for 30 min at 37ºC, before using this serum for opsonization of glutaraldehyde-fixed yeast cells. We observed that normal mouse serum increased about 3 fold the phagocytosis of C. albicans by mice peritoneal macrophages, whereas supernatant-treated serum did not increase phagocytosis. This effect of supernatants on serum was prevented by addition of pepstatin (5 µg/ ml; an inhibitor of C. albicans acid proteases to the medium. Serum treated with supernatants from cultures of a protease-deficient mutant of C. albicans also increased about 3 fold phagocytosis of the yeast. These results suggest that a protease produced by C. albicans causes proteolysis of serum opsonins, thereby reducing the phagocytosis of the yeast.Candida albicans é um potente ativador do sistema complemento, e opsoninas lábeis ao calor produzidas por ativação de C3 (C3b e iC3b aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de células obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37ºC, antes deste soro ser usado para opsonização de C. albicans na forma de levedura e fixadas em glutaraldeido. Nós observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por

Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

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Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

2018-06-01

Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp.

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Oleas, Gabriela; Callegari, Eduardo; Sepulveda, Romina; Eyzaguirre, Jaime

2016-01-01

The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

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Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J; Romero, Fernando; Gil, Angel

2013-01-01

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro

Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

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Miriam Bermudez-Brito

Full Text Available Dendritic cells (DCs constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS have immunomodulatory effects in human intestinal-like dendritic cells (DCs and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down

Sphingolipid metabolism correlates with cerebrospinal fluid Beta amyloid levels in Alzheimer's disease.

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Alfred N Fonteh

Full Text Available Sphingolipids are important in many brain functions but their role in Alzheimer's disease (AD is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but

The yield of different pleural fluid volumes for Mycobacterium tuberculosis culture.

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von Groote-Bidlingmaier, Florian; Koegelenberg, Coenraad Frederik; Bolliger, Chris T; Chung, Pui Khi; Rautenbach, Cornelia; Wasserman, Elizabeth; Bernasconi, Maurizio; Friedrich, Sven Olaf; Diacon, Andreas Henri

2013-03-01

We prospectively compared the culture yields of two pleural fluid volumes (5 and 100 ml) inoculated in liquid culture medium in 77 patients of whom 58 (75.3%) were diagnosed with pleural tuberculosis. The overall fluid culture yield was high (60.3% of cases with pleural tuberculosis). The larger volume had a faster time to positivity (329 vs 376 h, p=0.055) but its yield was not significantly higher (53.5% vs 50%; p=0.75). HIV-positive patients were more likely to have positive cultures (78.9% vs 51.5%; p=0.002).

Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

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Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

2015-01-01

Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

Treatment of supernatant from sewage sludge by elctron beam irradiation

International Nuclear Information System (INIS)

Arai, Hidehiko; Sugiyama, Masashi; Shimizu, Ken.

1988-01-01

Part of the results was presented on the investigation of treatment of supernatant from sewage sludge by combination of electron beam irradiation and microbiological treatment. Supernatant is electron-beam irradiated after microbiologically treated, and then treated microbiologically again. Based this method, by irradiation of 10 kGy, chemical oxygen demand (COD) in supernatant can be decreased lower than 30 ppm. Moreover, electron-beam irradiation induces remarkable decolorization and deodorization. (author)

Enrichment methodology to increase the positivity of cultures from body fluids

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Alessandra Valle Daur

Full Text Available Isolation and identification of etiological agents found in body fluids can be of critical importance for the recovery of patients suffering from potentially-severe infections, which are often followed by serious sequels. Eighty-two samples of different body fluids were analyzed using two different methods: (1 the conventional culture method (agar plating and (2 the enrichment culture technique, using the Bact/Alert® blood culture bottle. The number of positive cultures increased on average from 9.7% to 23.1% with the enrichment culture technique. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were the most frequently isolated bacteria. The enrichment method could provide a more accurate means the identifying etiological agents.

Analysis of culture-dependent versus culture-independent techniques for identification of bacteria in clinically obtained bronchoalveolar lavage fluid.

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Dickson, Robert P; Erb-Downward, John R; Prescott, Hallie C; Martinez, Fernando J; Curtis, Jeffrey L; Lama, Vibha N; Huffnagle, Gary B

2014-10-01

The diagnosis and management of pneumonia are limited by the use of culture-based techniques of microbial identification, which may fail to identify unculturable, fastidious, and metabolically active viable but unculturable bacteria. Novel high-throughput culture-independent techniques hold promise but have not been systematically compared to conventional culture. We analyzed 46 clinically obtained bronchoalveolar lavage (BAL) fluid specimens from symptomatic and asymptomatic lung transplant recipients both by culture (using a clinical microbiology laboratory protocol) and by bacterial 16S rRNA gene pyrosequencing. Bacteria were identified in 44 of 46 (95.7%) BAL fluid specimens by culture-independent sequencing, significantly more than the number of specimens in which bacteria were detected (37 of 46, 80.4%, P ≤ 0.05) or "pathogen" species reported (18 of 46, 39.1%, P ≤ 0.0001) via culture. Identification of bacteria by culture was positively associated with culture-independent indices of infection (total bacterial DNA burden and low bacterial community diversity) (P ≤ 0.01). In BAL fluid specimens with no culture growth, the amount of bacterial DNA was greater than that in reagent and rinse controls, and communities were markedly dominated by select Gammaproteobacteria, notably Escherichia species and Pseudomonas fluorescens. Culture growth above the threshold of 10(4) CFU/ml was correlated with increased bacterial DNA burden (P Microbiology. All Rights Reserved.

Half-liter supernatant sampler system engineering work plan

International Nuclear Information System (INIS)

Ritter, G.A.

1995-01-01

The Tank Waste Remediation System (TWRS) pretreatment facility project W-236B, known as the Initial Pretreatment Module (IPM), requires samples of supernatants and sludges from 200 Area tank farms for planned hot testing work in support of IPM design. The IPM project has proposed the development of several new sampler systems. These systems include a 0.5-l supernatant sampler, 3-l and 25-l supernatant and sludge samplers, and a 4,000-l sampler system. The 0.5-l sampler will support IPM sampling needs in the 1 to 3 l range starting in late fiscal year 1995. This sampler is intended to be used in conjunction with the existing 100 ml bottle-on-a-string. The 3-l and 25-l systems will be based on the Savannah River Site's sampler system and will support IPM sampling needs in the 3 to 100 liter range. Most of the hot testing required for design of the IPM must be accomplished in the next 3 years. This work plan defines the tasks associated with the development of a 0.5-l sampler system. This system will be referred to as the Half-Liter Supernatant Sampler System (HLSSS). Specifically, this work plan will define the scope of work, identify organizational responsibilities, identify major technical requirements, describe configuration control and verification requirements, and provide estimated costs and schedule. The sampler system will be fully operational, including trained staff and operating procedures, upon completion of this task

A study of chromosomal aberrations in amniotic fluid cell cultures.

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Wolstenholme, J; Crocker, M; Jonasson, J

1988-06-01

This paper represents the analysis of 1916 routine amniotic fluid specimens harvested by an in situ fixation technique in a prospective study with regard to cultural chromosome anomalies. Excluding constitutional abnormalities, 2.9 per cent of 19,432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 per cent) and chromatid/chromosome breaks and gaps (18 per cent) being the most frequent, followed by interchange aberrations (13 per cent) and trisomy (5 per cent). No case was found of more than one colony from the same culture showing the same anomaly without it being present in other cultures from the same fluid. The wholly abnormal colonies had a surplus of trisomies and from the mathematical considerations presented one may infer that these are likely to reflect the presence of abnormal cells in the amniotic fluid. Partly abnormal colonies appeared at a frequency that would correspond to virtual absence of selection against chromosomally abnormal cells when cultured in vitro. The aberrations found were similar to those seen as single cell anomalies, except for chromatid breaks and exchanges. The data suggest a basic preferential induction of trisomy for chromosomes 2, 18, 21, and the Y-chromosome. Structural aberrations showed a marked clustering of breakpoints around the centromeres. The frequency of mutant cells was low (1.4 X 10(-3)) before culture was initiated. At harvest, the frequency of abnormal cells was much higher (3 X 10(-2)) corresponding to 3 X 10(-3) mutations per cell per generation accumulating over approximately ten generations in vitro.

Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

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DA Ribeiro

2011-01-01

Full Text Available Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40% among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60°C and 100°C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2% were positive for ST II (estB and two (5% positive for αHly (hlyA. Gene amplification of SLT (stx 1, stx 2, ST I (estA, LT (eltI, eltII, EAST1 (astA, EHly (enhly and plasmid-encoded enterotoxin (pet were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

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Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10 10 /ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

Revisiting tuberculous pleurisy: pleural fluid characteristics and diagnostic yield of mycobacterial culture in an endemic area.

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Ruan, Sheng-Yuan; Chuang, Yu-Chung; Wang, Jann-Yuan; Lin, Jou-Wei; Chien, Jung-Yien; Huang, Chun-Ta; Kuo, Yao-Wen; Lee, Li-Na; Yu, Chong-Jen J

2012-09-01

Tuberculous pleurisy is traditionally indicated by extreme lymphocytosis in pleural fluid and low yield of effusion culture. However, there is considerable inconsistency among previous study results. In addition, these data should be updated due to early effusion studies and advances in culture methods. From January 2004 to June 2009, patients with tuberculous pleurisy were retrospectively identified from the mycobacteriology laboratories and the pathology and tuberculosis registration databases of two hospitals in Taiwan where tuberculosis is endemic. Pleural fluid characteristics and yields of mycobacterial cultures using liquid media were evaluated. A total of 382 patients with tuberculous pleurisy were identified. The median lymphocyte percentage of total cells in pleural fluids was 84% (IQR 64-95%) and 17% of cases had a lymphocyte percentage of pleural biopsy specimens. The degree of lymphocyte predominance in tuberculous pleurisy was lower than was previously thought. The lymphocyte percentage in pleural fluid was negatively associated with the probability of a positive effusion culture. With the implementation of a liquid culture method, the sensitivity of effusion culture was much higher than has been previously reported, and the combination of effusion and sputum cultures provided a good diagnostic yield.

Inoculation of peritoneal dialysate fluid into blood culture bottles ...

African Journals Online (AJOL)

The aim of the study was to determine if direct inoculation of peritoneal fluid into Bactec blood culture bottles would improve the positive bacteriological yield compared with conventional techniques in continuous ambulatory peritoneal dialysis (CAPD) patients with peritonitis. All patients presenting with suspected peritonitis ...

Effect of lidocaine 2% on bacterial culture of bronchial fluid

International Nuclear Information System (INIS)

Samet, M.; Meybodi, F.A.A.; Mokarianpour, T.; Fallah, T.; Mongabadi, F.D.; Ayatollahi, J.; Shahcherghi, S.H.; Yazdi, M.H.A

2017-01-01

Objective:To evaluate the action of 2% lidocaine on the culture results of bronchial fluid in patients suspected of having lower respiratory tract infections. Study Design:Cross-sectional analytical study. Place and Duration of Study:Shahid Sadoughi Hospital, Yazd, Iran, from November 2014 to November 2015. Methodology:Patients suspected of lower respiratory tract infections referred to bronchoscopy unit of the Hospital were included. Those with incomplete questionnaire and bronchoscopy contraindication were excluded. Bronchial fluid was aspirated before and after local application of 2% lidocaine and cultured, according to the suspected clinical diagnosis. Finally, statistical analysis was performed using SPSS software, version 17.0. For statistical comparisons, McNemar's test was used. Level of significance was kept at p <0.05. Results:The mean age of the study population was 51.83 +-15.93 with a range of 25 - 80 years. Out of 130 patients, 60 patients had positive culture results. Nineteen (31.7%) cases had positive culture for tuberculosis and 41 (63.3%) cases had positive results for other bacteria before intervention that did not change after using 2% lidocaine (p=1). In 70 (53.84%) cases, results were negative before and after use of 2% lidocaine. Conclusion:No significant difference was found between culture results before and after the use of lidocaine. Therefore, lidocaine can be used during bronchoscopy to increase patient tolerance. (author)

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

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Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

2018-02-01

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel approach to enhance biological nutrient removal using a culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (Rpf) in SBR process.

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Liu, Yindong; Su, Xiaomei; Lu, Lian; Ding, Linxian; Shen, Chaofeng

2016-03-01

A culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (SRpf) was used to enhance the biological nutrient removal of potentially functional bacteria. The obtained results suggest that SRpf accelerated the start-up process and significantly enhanced the biological nutrient removal in sequencing batch reactor (SBR). PO4 (3-)-P removal efficiency increased by over 12 % and total nitrogen removal efficiency increased by over 8 % in treatment reactor acclimated by SRpf compared with those without SRpf addition. The Illumina high-throughput sequencing analysis showed that SRpf played an essential role in shifts in the composition and diversity of bacterial community. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrient removal, were greatly abundant after SRpf addition. This study demonstrates that SRpf acclimation or addition might hold great potential as an efficient and cost-effective alternative for wastewater treatment plants (WWTPs) to meet more stringent operation conditions and legislations.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

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Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

Chromosomal studies on 2 mL of celomic fluid obtained during the fifth week of development in the timed-pregnant baboon model.

Science.gov (United States)

Santolaya-Forgas, Joaquin; De Leon-Luis, Juan; Shen, Zhion; McCorquodale, Maureen

2005-09-01

To determine if chromosomal studies could be performed using 2 mL of celomicfluid obtained during the fifth postfertilization week in pregnant baboons. Nine ultrasound-guided celocenteses were performed. The initial 0.5 mL of celomic fluid was discarded to decrease maternal cell contamination. Approximately 2 mL of celomic fluid was then collected. The fluid was centrifuged and the supernatant removed to a final volume of 0.5 mL. The celomic fluid sample was placed in either a small plastic flaskette chamber slide with a mix of 0.5 mL celomic fluid, 1 mL of Amniomax, and 1 mL of usedfibroblast culture medium to spread on the entire surface (n=4), or a 3.5 x 1-cm plastic Petri dish with a 24 x 30-mm glass coverslip to keep the 0.5 mL celomic fluid mixed with 1 mL of Amniomax (Invitrogen, Carlsbad, California) within a 1 cm2 area (n=5). The medium was changed on day 5 and thereafter every second to third day. The cells were harvested when the number of cells appeared sufficient for chromosomal analysis. Standard chromosomal studies were possible in 5 of the 9 celomicfluid samples. Mean (+/-SD) celomic fluid volume used for culture was 1.85 +/- 0.3 mL. Mean (+/-SD) time to karyotype result was 18.8 +/- 1.8 days. The findings of this study suggest that there are living cells at 36-42 days of embryonic development in the extraembryonic celomic fluid of primates and that they can be cultured for chromosomal studies. However, significant improvements in understanding the biology of cells present at 5 weeks after fertilization in celomic fluid are needed to improve culture conditions.

Optimizing the multimodal approach to pancreatic cyst fluid diagnosis: developing a volume-based triage protocol.

Science.gov (United States)

Chai, Siaw Ming; Herba, Karl; Kumarasinghe, M Priyanthi; de Boer, W Bastiaan; Amanuel, Benhur; Grieu-Iacopetta, Fabienne; Lim, Ee Mun; Segarajasingam, Dev; Yusoff, Ian; Choo, Chris; Frost, Felicity

2013-02-01

The objective of this study was to develop a triage algorithm to optimize diagnostic yield from cytology, carcinoembryonic antigen (CEA), and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) testing on different components of a single pancreatic cyst fluid specimen. The authors also sought to determine whether cell block supernatant was suitable for CEA and KRAS testing. Fifty-four pancreatic cysts were triaged according to a volume-dependent protocol to generate fluid (neat and supernatant) and cell block specimens for cytology, comparative CEA, and KRAS testing. Follow-up histology, diagnostic cytology, or a combined clinicopathologic interpretation was recorded as the final diagnosis. There were 26 mucinous cystic lesions and 28 nonmucinous cystic lesions with volumes ranging from 0.3 mL to 55 mL. Testing different components of the specimens (cell block, neat, and/or supernatant) enabled all laboratory investigations to be performed on 50 of 54 cyst fluids (92.6%). Interpretive concordance was observed in 17 of 17 cases (100%) and in 35 of 40 cases (87.5%) that had multiple components tested for CEA and KRAS mutations, respectively. An elevated CEA level (>192 ng/mL) was the most sensitive test for the detection of a mucinous cystic lesion (62.5%) versus KRAS mutation (56%) and "positive" cytology (61.5%). KRAS mutations were identified in 2 of 25 mucinous cystic lesions (8%) in which cytology and CEA levels were not contributory. A volume-based protocol using different components of the specimen was able to optimize diagnostic yield in pancreatic cyst fluids. KRAS mutation testing increased diagnostic yield when combined with cytology and CEA analysis. The current results demonstrated that supernatant is comparable to neat fluid and cell block material for CEA and KRAS testing. Copyright © 2012 American Cancer Society.

Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

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Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

2009-01-01

To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

Proteomics of apheresis platelet supernatants during routine storage: Gender-related differences.

Science.gov (United States)

Dzieciatkowska, Monika; D'Alessandro, Angelo; Burke, Timothy A; Kelher, Marguerite R; Moore, Ernest E; Banerjee, Anirban; Silliman, Christopher C; West, Bernadette F; Hansen, Kirk C

2015-01-01

Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender. The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet

Test procedures and instructions for Hanford tank waste supernatant cesium removal

Energy Technology Data Exchange (ETDEWEB)

Hendrickson, D.W., Westinghouse Hanford

1996-05-31

This document provides specific test procedures and instructions to implement the test plan for the preparation and conduct of a cesium removal test using Hanford Double-Shell Slurry Feed supernatant liquor from tank 251-AW-101 in a bench-scale column.Cesium sorbents to be tested include resorcinol-formaldehyde resin and crystalline silicotitanate. The test plan for which this provides instructions is WHC-SD-RE-TP-022, Hanford Tank Waste Supernatant Cesium Removal Test Plan.

Differential diagnosis of amyotrophic lateral sclerosis from Guillain-Barré syndrome by quantitative determination of TDP-43 in cerebrospinal fluid.

Science.gov (United States)

Hosokawa, Masato; Arai, Tetsuaki; Yamashita, Makiko; Tsuji, Hiroshi; Nonaka, Takashi; Masuda-Suzukake, Masami; Tamaoka, Akira; Hasegawa, Masato; Akiyama, Haruhiko

2014-05-01

The aim of this study was to investigate whether an increased level of TAR DNA-binding protein 43 (TDP-43) in the cerebrospinal fluid (CSF) could be a biomarker for amyotrophic lateral sclerosis (ALS) and facilitate differential diagnosis of ALS from peripheral motor neuropathy. TDP-43 is the major constituent of neuronal and glial inclusions that neuropathologically characterize both ALS and tau-negative frontotemporal lobar degeneration. Recent discoveries of various missense mutations in the TDP-43 gene in familial ALS indicate a pivotal role of the aberrant accumulation of TDP-43 in neurodegeneration. Increased TDP-43 in the CSF could be a hallmark of ALS and other TDP-43 proteinopathy. Sandwich enzyme-linked immunosorbent assay (ELISA) was established to measure the concentration of TDP-43 in biological fluids. Culture supernatants of cells transfected with various TDP-43 constructs were used to confirm that the ELISA detected TDP-43. TDP-43 in the culture supernatant of TDP-43 transfected cells was detected by immunoprecipitation with subsequent immunoblotting and concentrations were successfully measured by sandwich ELISA. We then measured TDP-43 concentrations in the CSF of patients with ALS and Guillain-Barré syndrome (GBS). TDP-43 concentrations in CSF were significantly higher in ALS than in GBS (p = 0.016). The sensitivity of the diagnostic test was 71.4% and the specificity was 84.6%. Quantitative determination of TDP-43 concentrations in the CSF by sandwich ELISA is a potential laboratory test for differentiating ALS from peripheral motor neuropathies such as GBS.

Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157:H7 enhances the role of organic acids production as a factor for pathogen control

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Larissa B. Poppi

2015-04-01

Full Text Available Many attempts have been made to establish the control of foodborne pathogens through Lactobacillus isolates and their metabolism products with success being obtained in several situations. The aim of this study was to investigate the antagonistic effect of eight Lactobacillus isolates, including L. casei subsp. pseudoplantarum, L. plantarum, L. reuteri and L. delbrueckii subsp. delbrueckii, on the pathogenic Escherichia colistrain O157:H7. The inhibitory effect of pure cultures and two pooled cultures supernatants of Lactobacillus on the growth of pathogenic bacteria was evaluated by the spot agar method and by monitoring turbidity. Antimicrobial activity was confirmed for L. reuteri and L. delbrueckii subsp. delbrueckii and for a pool of lactic acid bacteria. The neutralized supernatant of the pool exerted a higher antimicrobial activity than that of the individual strains. Furthermore, D-lactic acid and acetic acid were produced during growth of the Lactobacillus isolates studied.

Fate of cyanobacteria in drinking water treatment plant lagoon supernatant and sludge

International Nuclear Information System (INIS)

Pestana, Carlos J.; Reeve, Petra J.; Sawade, Emma; Voldoire, Camille F.; Newton, Kelly; Praptiwi, Radisti; Collingnon, Lea; Dreyfus, Jennifer; Hobson, Peter; Gaget, Virginie; Newcombe, Gayle

2016-01-01

In conventional water treatment processes, where the coagulation and flocculation steps are designed to remove particles from drinking water, cyanobacteria are also concentrated into the resultant sludge. As a consequence, cyanobacteria-laden sludge can act as a reservoir for metabolites such as taste and odour compounds and cyanotoxins. This can pose a significant risk to water quality where supernatant from the sludge treatment facility is returned to the inlet to the plant. In this study the complex processes that can take place in a sludge treatment lagoon were investigated. It was shown that cyanobacteria can proliferate in the conditions manifest in a sludge treatment lagoon, and that cyanobacteria can survive and produce metabolites for at least 10 days in sludge. The major processes of metabolite release and degradation are very dependent on the physical, chemical and biological environment in the sludge treatment facility and it was not possible to accurately model the net effect. For the first time evidence is provided to suggest that there is a greater risk associated with recycling sludge supernatant than can be estimated from the raw water quality, as metabolite concentrations increased by up to 500% over several days after coagulation, attributed to increased metabolite production and/or cell proliferation in the sludge. - Highlights: • Cyanobacteria in water treatment sludge significantly impact supernatant quality • Cyanobacteria can survive, and thrive, in sludge lagoon supernatant and in treatment sludge • Metabolite concentrations in cyanobacteria in sludge can increase up to 500% • The risk associated with supernatant recycling was assessed relative to available treatment barriers

Fate of cyanobacteria in drinking water treatment plant lagoon supernatant and sludge

Energy Technology Data Exchange (ETDEWEB)

Pestana, Carlos J.; Reeve, Petra J.; Sawade, Emma [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia); Voldoire, Camille F. [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia); École Européenne de Chimie, Polymères et Matériaux (ECPM), Strasbourg 67087 (France); Newton, Kelly; Praptiwi, Radisti [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia); Collingnon, Lea [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia); École Européenne de Chimie, Polymères et Matériaux (ECPM), Strasbourg 67087 (France); Dreyfus, Jennifer [Allwater, Adelaide Services Alliance, Wakefield St, Adelaide, SA 5001 (Australia); Hobson, Peter [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia); Gaget, Virginie [University of Adelaide, Ecology and Environmental Sciences, School of Biological Sciences, Adelaide, SA 5005 (Australia); Newcombe, Gayle, E-mail: gayle.newcombe@sawater.com.au [Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000 (Australia)

2016-09-15

In conventional water treatment processes, where the coagulation and flocculation steps are designed to remove particles from drinking water, cyanobacteria are also concentrated into the resultant sludge. As a consequence, cyanobacteria-laden sludge can act as a reservoir for metabolites such as taste and odour compounds and cyanotoxins. This can pose a significant risk to water quality where supernatant from the sludge treatment facility is returned to the inlet to the plant. In this study the complex processes that can take place in a sludge treatment lagoon were investigated. It was shown that cyanobacteria can proliferate in the conditions manifest in a sludge treatment lagoon, and that cyanobacteria can survive and produce metabolites for at least 10 days in sludge. The major processes of metabolite release and degradation are very dependent on the physical, chemical and biological environment in the sludge treatment facility and it was not possible to accurately model the net effect. For the first time evidence is provided to suggest that there is a greater risk associated with recycling sludge supernatant than can be estimated from the raw water quality, as metabolite concentrations increased by up to 500% over several days after coagulation, attributed to increased metabolite production and/or cell proliferation in the sludge. - Highlights: • Cyanobacteria in water treatment sludge significantly impact supernatant quality • Cyanobacteria can survive, and thrive, in sludge lagoon supernatant and in treatment sludge • Metabolite concentrations in cyanobacteria in sludge can increase up to 500% • The risk associated with supernatant recycling was assessed relative to available treatment barriers.

Fluorescence Spectrum and Decay Measurement for Hsil VS Normal Cytology Differentiation in Liquid Pap Smear Supernatant

Science.gov (United States)

Vaitkuviene, A.; Gegzna, V.; Juodkazis, S.; Jursenas, S.; Miasojedovas, S.; Kurtinaitiene, R.; Rimiene, J.; Vaitkus, J.

2009-06-01

Cervical smear material contains endo and exocervical cells, mucus and inflammative, immune cells in cases of pathology. Just not destroyed keratinocytes lay on the glass for microscopy. Liquid cytology supernatant apart other diagnostics could be used for photodiagnostic. The spectroscopic parameters suitable for Normal and HSIL cytology groups supernatant differentiation are demonstrated. The dried liquid PAP supernatant fractions—sediment and liquid were investigated. Excitation and emission matrices (EEM), supernatant fluorescence decay measured under 280 nm diode short pulse excitation and fluorescence spectroscopy by excitation with 355 nm laser light were analyzed. The differences between Normal and HSIL groups were statistically proven in the certain spectral regions. Fluorescence decay peculiarities show spectral regions consisting of few fluorophores. Obtained results on fluorescence differences in Normal and HSIL groups' supernatant shows the potency of photodiagnosis application in cervical screening.

Cultural factors influencing dietary and fluid restriction behaviour: perceptions of older Chinese patients with heart failure.

Science.gov (United States)

Rong, Xiaoshan; Peng, Youqing; Yu, Hai-Ping; Li, Dan

2017-03-01

To explore the cultural factors related to dietary and fluid restriction behaviours among older Chinese patients. Excess dietary sodium and fluid intake are risk factors contributing to the worsening and rehospitalisation for heart failure in older patients. Managing the complex fluid and diet requirements of heart failure patients is challenging and is made more complicated by cultural variations in self-management behaviours in response to a health threat. Qualitative study using semi-structured in interviews and framework analysis. The design of this study is qualitative descriptive. Semi-structured in-depth interviews were conducted with 15 heart failure patients. Data were analysed through content analysis. Seven cultural themes emerged from the qualitative data: the values placed on health and illness, customary way of life, preference for folk care and the Chinese healthcare system, and factors related to kinship and social ties, religion, economics and education. Dietary change and management in response to illness, including heart failure, is closely related to individuals' cultural background. Healthcare providers should have a good understanding of cultural aspects that can influence patients' conformity to medical recommendations. Heart failure patients need support that considers their cultural needs. Healthcare providers must have a good understanding of the experiences of people from diverse cultural backgrounds. © 2016 John Wiley & Sons Ltd.

Assessment of chemical composition and microbiological properties of titanium nickelide supernatant

Directory of Open Access Journals (Sweden)

Uruzbaev R.M.

2018-03-01

Full Text Available According to the World Health Organization, in the structure of internal injuries, burns occur in 2.1 per 1000 adults. In practice, all victims after stabilization of the condition are treated surgically with the autodermoplasty, which in the late period is accompanied by a rough scarring, sometimes with the deformation of the damaged area. In addition, infectious complications worsen and aggravate the wound healing process, and also significantly prolong the epithelialization time. A wound is usually infected by opportunistic pathogenic microflora resistant to various groups of antibiotics and antiseptics, by which the affected areas are treated. Modern researches are aimed at finding ways to accelerate regenerative processes of the affected area, to avoid surgical treatment and prevent infection contamination. The object of this study was titanium nickelide supernatant. Currently, there are no published data on the chemical composition of the supernatant, as well as its microbiological safety. This alloy is actively and successfully used in traumatology, dentistry and other fields of medicine. The supernatant was prepared under sterile conditions at a rate of 10 g of titanium nickelide powder per 1 liter of distilled water. In the course of the studies it was proved that nickel and titanium ions are present in the solution, in addition, its bactericidal and bacteriostatic properties are reflected. Thus, the supernatant of nitinol can be used in biological environment and is safe for them.

Differential effects of catecholamines on in vitro growth of pathogenic bacteria

Science.gov (United States)

Belay, Tesfaye; Sonnenfeld, Gerald

2002-01-01

Supplementation of minimal medium inoculated with bacterial cultures with norepinephrine, epinephrine, dopamine, or isoproterenol resulted in marked increases in growth compared to controls. Norepinephrine and dopamine had the greatest enhancing effects on growth of cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae, while epinephrine and isoproterenol also enhanced growth to a lesser extent. The growth of Escherichia coli in the presence of norepinephrine was greater than growth in the presence of the three other neurochemicals used in the study. Growth of Staphylococcus aureus was also enhanced in the presence of norepinephrine, but not to the same degree as was the growth of gram negative bacteria. Addition of culture supernatants from E. coli cultures that had been grown in the presence of norepinephrine was able to enhance the growth of K. pneumoniae. Addition of the culture supernatant fluid culture from E. coli cultures that had been grown in the presence of norepinephrine did not enhance growth of P. aeruginosa or S. aureus. Culture supernatant fluids from bacteria other than E. coli grown in the presence of norepinephrine were not able to enhance the growth of any bacteria tested. The results suggest that catecholamines can enhance growth of pathogenic bacteria, which may contribute to development of pathogenesis; however, there is no uniform effect of catecholamines on bacterial growth.

Low Fluid Shear Culture of Staphylococcus Aureus Represses hfq Expression and Induces an Attachment-Independent Biofilm Phenotype

Science.gov (United States)

Ott, C. Mark; Castro, S. L.; Nickerson, C. A.; Nelman-Gonzalez, M.

2011-01-01

Background: The opportunistic pathogen, Staphylococcus aureus, experiences fluctuations in fluid shear during infection and colonization of a human host. Colonization frequently occurs at mucus membrane sites such as in the gastrointestinal tract where the bacterium may experience low levels of fluid shear. The response of S. aureus to low fluid shear remains unclear. Methods: S. aureus was cultured to stationary phase using Rotating-Wall Vessel (RWV) bioreactors which produce a physiologically relevant low fluid shear environment. The bacterial aggregates that developed in the RWV were evaluated by electron microscopy as well as for antibiotic resistance and other virulence-associated stressors. Genetic expression profiles for the low-shear cultured S. aureus were determined by microarray analysis and quantitative real-time PCR. Results: Planktonic S. aureus cultures in the low-shear environment formed aggregates completely encased in high amounts of extracellular polymeric substances. In addition, these aggregates demonstrated increased antibiotic resistance indicating attachment-independent biofilm formation. Carotenoid production in the low-shear cultured S. aureus was significantly decreased, and these cultures displayed an increased susceptibility to oxidative stress and killing by whole blood. The hfq gene, associated with low-shear growth in Gram negative organisms, was also found to be down-regulated in S. aureus. Conclusions: Collectively, this data suggests that S. aureus decreases virulence characteristics in favor of a biofilm-dwelling colonization phenotype in response to a low fluid shear environment. Furthermore, the identification of an Hfq response to low-shear culture in S. aureus, in addition to the previously reported responses in Gram negative organisms, strongly suggests an evolutionarily conserved response to mechanical stimuli among structurally diverse prokaryotes.

Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

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Perno Carlo

2002-09-01

Full Text Available Abstract Background Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M. Results Flow cytometric analysis (FACS of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite and by abnormal formation of malondialdehyde (MDA. Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining. Conclusions Taken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.

Lactobacillus rhamnosus GG supernatant enhance neonatal resistance to systemic Escherichia coli K1 infection by accelerating development of intestinal defense

OpenAIRE

Xiaolong He; Qing Zeng; Santhosh Puthiyakunnon; Zhijie Zeng; Weijun Yang; Jiawen Qiu; Lei Du; Swapna Boddu; Tongwei Wu; Danxian Cai; Sheng-He Huang; Hong Cao

2017-01-01

The objective of this study was to determine whether Lactobacillus rhamnosus GG culture supernatant (LCS) has a preventive effect against gut-derived systemic neonatal Escherichia coli (E. coli) K1 infection. The preventive effects were evaluated in human colonic carcinoma cell line Caco-2 and neonatal rat models. Our in vitro results showed that LCS could block adhesion, invasion and translocation of E. coli K1 to Caco-2 monolayer via up-regulating mucin production and maintaining intestinal...

Compounds from Lactobacillus plantarum culture supernatants with potential pro-healing and anti-pathogenic properties in skin chronic wounds.

Science.gov (United States)

Ramos, Alberto N; Sesto Cabral, Maria E; Arena, Mario E; Arrighi, Carlos F; Arroyo Aguilar, Abel A; Valdéz, Juan C

2015-03-01

It is necessary to advance the field of alternative treatments for chronic wounds that are financially accessible to the least economically developed countries. Previously we demonstrated that topical applications of Lactobacillus plantarum culture supernatants (LAPS) on human-infected chronic wounds reduce the pathogenic bioburden, the amount of necrotic tissue, and the wound area, as well as promote debridement, granulation tissue, and wound healing. To study LAPS chemically and biologically and to find potential molecules responsible for its pro-healing and anti-pathogenic properties in chronic wounds. (1) Chemical analysis: extracts were subjected to a column chromatography and the fractions obtained were studied by GCMS. (2) Quantification: dl-lactic acid (commercial kit), phenolic compounds (Folin-Ciocalteu), H2O2 (micro-titration), and cations (flame photometry). (3) Biological analysis: autoinducers type 2 (AI-2) (Vibrio harveyi BB170 bioassay), DNAase activity (Agar DNAase), and Pseudomonas aeruginosa biofilm inhibition (crystal violet technique). According to its biological activity, the most significant molecules found by GCMS were the following: antimicrobials (mevalonolactone, 5-methyl-hydantoine, benzoic acid, etc.); surfactants (di-palmitin, distearin, and 1,5-monolinolein); anesthetics (barbituric acid derivatives), and AI-2 precursors (4,5-dihydroxy-2,3-pentanedione and 2-methyl-2,3,3,4-tetrahydroxytetrahydrofurane). Concentrations measured (µg/mL): DL-lactic acid (11.71 ± 1.53) and H2O2 (36 ± 2.0); phenolic compounds (485.2 ± 15.20); sodium (370 ± 17); potassium 920 ± 24); calcium (20 ± 4); and magnesium (15 ± 3). DNAase from LAPS had activity on genomic DNA from PMNs and P. aeruginosa. The molecules and biological activities found in LAPS could explain the observed effects in human chronic wounds.

Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

International Nuclear Information System (INIS)

Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B.

1990-01-01

B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients

Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

Energy Technology Data Exchange (ETDEWEB)

Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

1990-10-01

B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

Test procedures and instructions for Hanford complexant concentrate supernatant cesium removal using CST

Energy Technology Data Exchange (ETDEWEB)

Hendrickson, D.W.

1997-01-08

This document provides specific test procedures and instructions to implement the test plan for the preparation and conduct of a cesium removal test, using Hanford Complexant Concentrate supernatant liquor from tank 241-AN-107, in a bench-scale column. The cesium sorbent to be tested is crystalline silicotitanate. The test plan for which this provides instructions is WHC-SD-RE-TP-023, Hanford Complexant Concentrate Supernatant Cesium Removal Test Plan.

Procalcitonin levels in patients with positive blood culture, positive body fluid culture, sepsis, and severe sepsis: a cross-sectional study.

Science.gov (United States)

Yu, Ying; Li, Xia-Xi; Jiang, Ling-Xiao; Du, Meng; Liu, Zhan-Guo; Cen, Zhong-Ran; Wang, Hua; Guo, Zhen-Hui; Chang, Ping

2016-01-01

Numerous investigations on procalcitonin (PCT) have been carried out, although few with large sample size. To deal with the complexity of sepsis, an understanding of PCT in heterogeneous clinical conditions is required. Hospitalized patients aged 10-79 years were included in this retrospective and cross-sectional study. PCT tests were assayed within 2 days of blood culture. A total of 2952 cases (from 2538 patients) were enrolled in this study, including 440 cases in the 'positive BC' group, 123 cases in the 'positive body fluid culture' group, and 2389 cases in the 'negative all culture' group. Median PCT values were 4.53 ng/ml, 2.95 ng/ml, and 0.49 ng/ml, respectively. Median PCT values in the gram-negative BC group and gram-positive BC group, respectively, were 6.99 ng/ml and 2.96 ng/ml. Median PCT values in the 'positive hydrothorax culture' group, 'positive ascites culture' group, 'positive bile culture' group, and 'positive cerebrospinal fluid culture' group, respectively, were 1.39 ng/ml, 8.32 ng/ml, 5.98 ng/ml, and 0.46 ng/ml. In all, 357 cases were classified into the 'sepsis' group, 150 of them were classified into the 'severe sepsis' group. Median PCT values were 5.63 ng/ml and 11.06 ng/ml, respectively. PCT could be used in clinical algorithms to diagnose positive infections and sepsis. Different PCT levels could be related to different kinds of microbemia, different infection sites, and differing severity of sepsis.

Effect of different stress factors on IL-6 and leptin expression in HELA cell cultures

International Nuclear Information System (INIS)

Chu Zhenwei; Yang Tao; Wang Luhuan; Hao Xiuhua; Yan Guangtao

2009-01-01

Objective: To study the effect of three stress factors high glucose (HG), lipopolysaccharide (LPS) and hydrogen peroxide (H 2 O 2 ) on the expression of culture supernatant IL-6 (IL-6) and leptin contents of HELA cell line. Methods: HELA cell culture models of severe inflammatory response syndrome were prepared with cultures treated with 50 mmol/L glucose (HG), 4 μg/ ml LPS and 100 μmol/L H 2 O 2 respectively and supernatant contents of IL-6 and leptin were measured with RIA at 1h, 6h and 24h. Results: Generally speaking, the culture supernatant contents of IL-6 gradually increased and leptin contents gradually decreased with significant differences from those in cultures not treated with either stress factor at 6h and 12h (P<0.05). Conclusion: Leptin as a possible anti-inflammatory cytokine might plays an important protective role in severe inflammatory response. (authors)

Immunoproteomic analysis of antibody in lymphocyte supernatant in patients with typhoid fever in Bangladesh.

Science.gov (United States)

Charles, Richelle C; Liang, Li; Khanam, Farhana; Sayeed, M Abu; Hung, Chris; Leung, Daniel T; Baker, Stephen; Ludwig, Albrecht; Harris, Jason B; Larocque, Regina C; Calderwood, Stephen B; Qadri, Firdausi; Felgner, Philip L; Ryan, Edward T

2014-03-01

We have previously shown that an assay based on detection of anti-Salmonella enterica serotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within the S. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique to S. Typhi bacteremic patients, we probed microarrays containing 2,724 S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.

Analysis of metal Bioleaching from thermal power plant fly ash by Aspergillus niger 34770 culture supernatant and reduction of phytotoxicity during the process.

Science.gov (United States)

Jadhav, Umesh U; Hocheng, Hong

2015-01-01

Aspergillus niger culture supernatant is used for bioleaching process. Before starting bioleaching process, fly ash was washed with distilled water. This removed 100 % sodium, 47 % (±0.45) boron, 38.07 % (±0.12) calcium, 29.89 % (±0.78) magnesium, and 11.8 % (±0.05) potassium. The pH was reduced from 10.5 to 8.5 after water washing. During bioleaching process, around 100 % metal removal was achieved in 4 h for all metals except chromium 93 % (±1.18), nickel 83 % (±0.32), arsenic 78 % (±0.52), and lead 70 % (±0.20). The process parameters including temperature, shaking speed, and solid/liquid ratio were optimized for bioleaching process. Experiments were conducted to evaluate effect of fly ash on growth of mung bean (Vigna radiata). At 20 g/100 ml fly ash concentration no germination of V. radiata seeds was observed. With an increasing concentration of untreated fly ash, a gradual decrease in root/shoot length was observed. After bioleaching process 78 % (±0.19) germination of V. radiata was observed with 20 g/100 ml fly ash. This study will help to develop an efficient process to remove the toxic metals from fly ash.

An Exploratory Analysis of American Indian Children's Cultural Engagement, Fluid Cognitive Skills, and Standardized Verbal IQ Scores

Science.gov (United States)

Tsethlikai, Monica

2011-01-01

This exploratory cross-sectional study examined fluid cognitive skills and standardized verbal IQ scores in relation to cultural engagement amongst Tohono O'odham children (N = 99; ages 7 to 12 years). Guardians with higher socioeconomic status engaged their children in more cultural activities, and participation in more cultural activities…

The Effect of Anaerobic and Aerobic Fish Sludge Supernatant on Hydroponic Lettuce

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Simon Goddek

2016-06-01

Full Text Available The mobilization of nutrients from fish sludge (i.e., feces and uneaten feed plays a key role in optimizing the resource utilization and thus in improving the sustainability of aquaponic systems. While several studies have documented the aerobic and anaerobic digestion performance of aquaculture sludge, the impact of the digestate on plant growth has yet to be understood. The present study examines the impact of either an aerobic or an anaerobic digestion effluent on lettuce plant growth, by enriching a mixture of aquaculture and tap water with supernatants from both aerobic and anaerobic batch reactors. The lettuce plants grown in the hydroponic system supplied with supernatant from an anaerobic reactor had significantly better performance with respect to weight gain than both, those in the system where supernatant from the aerobic reactor was added, as well as the control system. It can be hypothesized that this effect was caused by the presence of NH4+ as well as dissolved organic matter, plant growth promoting rhizobacteria and fungi, and humic acid, which are predominantly present in anaerobic effluents. This study should therefore be of value to researchers and practitioners wishing to further develop sludge remineralization in aquaponic systems.

Ultrafiltration and Characterization of AW-101 Supernatant and Entrained Solids

International Nuclear Information System (INIS)

Brooks, K.P.; Bredt, P.R.; Golcar, G.R.; Hartley, S.A.; Urie, M.W.; Tingey, J.M.; Rappe, K.G.; Jagoda, L.K.

1999-01-01

The River Protection Project Waste Treatment Plant (RPP-WTP) (1996) flow sheet uses cross-flow filtration as the solid/liquid separation technique. Unlike traditional dead-end filtration, which has a declining rate caused by the growth of a filter cake on the surface of the filter medium, in cross-flow filtration, the filter cake is swept away by the fluid flowing across it. This filtration method is especially beneficial when there are very fine particles and when system simplicity is required. The objective of this work was to test cross-flow filtration using actual Envelope A Hanford tank waste. Similar to the Phase 1A study, they evaluated the permeability of an Envelope A feed through a single element filter as a function of transmembrane pressure, axial velocity, solids concentration, and time. In addition, the efficiency of back pulse and chemical cleaning on the filter performance was evaluated. The chemical and radiochemical composition of the filtrate and solids was measured to determine efficiency of the filtration process. This report describes the test apparatus, the experimental approach, the results of the tests, and the chemical and radiochemical analysis for supernatants taken from Hanford Tank AW-101. This report also provides a means of transmitting to British Nuclear Fuels, Limited (BNFL) the completed test instruction and raw filtration and analytical data

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

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Harris Dana M

2013-01-01

Full Text Available Abstract Background Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens. Methods Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx, as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH. Results In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5. Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6. Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9% as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1. Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0. Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%. For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS, discontinuation of vancomycin could result in savings of $20.00/day. Conclusions In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to

Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

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Aitana Braza-Boïls

Full Text Available UNLABELLED: Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. METHODS: Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222 by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. RESULTS: Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion , this "in vitro

Long acting β2-agonist and corticosteroid restore airway glandular cell function altered by bacterial supernatant

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Nawrocki-Raby Béatrice

2010-01-01

Full Text Available Abstract Background Staphylococcus aureus releases virulence factors (VF that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting β2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal combined with a corticosteroid (fluticasone propionate, FP was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. Methods A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. Results When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFα. Conclusions Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting β2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of β2 adrenergic receptor agonist and glucocorticoid.

COMPARISON OF CULTURE OF SYNOVIAL FLUID, PERIPROSTHETIC TISSUE AND PROSTHESIS SONICATE FOR THE DIAGNOSIS OF KNEE PROSTHESIS INFECTION

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Andrej Trampuž

2003-03-01

Full Text Available Background. Synovial fluid and periprosthetic tissue specimens are the standard specimens cultured for the diagnosis of prosthetic joint infection (PJI. We hypothesize that ultrasonication of the explanted prosthesis may improve diagnosis of PJI by dislodging biofilm bacteria from the prosthesis surface and improve the sensitivity and specificity of diagnosis of PJI.Methods. Included were patients undergoing knee prosthesis exchange for septic or biomechanical failure and have not received antimicrobial therapy in the last 2 weeks prior specimen collection. Cultures of synovial fluid and periprosthetic tissue specimens were performed per the usual clinical practice. Additionally, explanted joint components were sonicated for 5 minutes at frequency 40 kHz in sterile Ringer’s solution; aliquots of 0.5 ml sonicate were plated onto five aerobic and five anaerobic blood agar plates, and incubated at 37 °C and examined for the next seven days. The number and identity of each colony morphology was recorded.Results. 35 patients undergoing knee replacement have been studied (24 for aseptic biomechanical failure and 11 for suspected PJI. In patients with PJI, coagulase-negative staphylococci (7 cases, Corynebacterium spp. (2 cases, Staphylococcus aureus (1 case, and viridans group streptococcus (1 case were recovered. Culture sensitivity and specificity were for synovial fluid 88% and 100%, for periprosthetic tissue 83% and 81%, and for explant sonicate 91% and 100%, respectively. In sonicate cultures higher numbers of microorganisms than in periprosthetic tissue cultures were consistently detected.Conclusions. Using synovial fluid, periprosthetic tissue, and explant sonicate cultures, 12%, 17% and 9% of PJI were missed, respectively. Explant sonicate cultures were the most sensitive with respect to the diagnosis of PJI, indicating that explant ultrasonication may improve bacterial recovery. In sonicate cultures, infecting organisms were detected in

Experimental data developed to support the selection of a treatment process for West Valley alkaline supernatant

Energy Technology Data Exchange (ETDEWEB)

Bray, L.A.; Holton, L.K.; Myers, T.R.; Richardson, G.M.; Wise, B.M.

1984-01-01

At the request of West Valley Nuclear Services Co., Inc., the Pacific Northwest Laboratory (PNL) has studied alternative treatment processes for the alkaline PUREX waste presently being stored in Tank 8D2 at West Valley, New York. Five tasks were completed during FY 1983: (1) simulation and characterization of the alkaline supernatant and sludge from the tank. The radiochemical and chemical distributions between the aqueous and solid phase were determined, and the efficiency of washing sludge with water to remove ions such as Na/sup +/ and SO/sub 4//sup 2 -/ was investigated; (2) evaluation of a sodium tetraphenylboron (Na-TPB) precipitation process to recover cesium (Cs) and a sodium titanate (Na-TiA) sorption process to recover strontium (Sr) and plutonium (Pu) from the West Valley Alkaline supernatant. These processes were previously developed and tested at the US Department of Energy's Savannah River Plant; (3) evaluation of an organic cation-exchange resin (Duolite CS-100) to recover Cs and Pu from the alkaline supernatant followed by an organic macroreticular cation exchange resin (Amberlite IRC-718) to recover Sr; (4) evaluation of an inorganic ion exchanger (Linde Ionsiv IE-95) to recover Cs, Sr, and Pu from the alkaline supernatant; and (5) evaluation of Dowex-1,X8 organic anion exchange resin to recover technetium (Tc) from alkaline supernatant. The findings of these tasks are reported. 21 references, 36 figures, 34 tables.

Colonic involvement in celiac disease and possible implications of the sigmoid mucosa organ culture in its diagnosis.

Science.gov (United States)

Picarelli, Antonio; Di Tola, Marco; Borghini, Raffaele; Isonne, Claudia; Saponara, Annarita; Marino, Mariacatia; Casale, Rossella; Tiberti, Antonio; Pica, Roberta; Donato, Giuseppe; Frieri, Giuseppe; Corazziari, Enrico

2013-10-01

Celiac disease (CD), a systemic autoimmune disorder that typically involves duodenal mucosa, can also affect other intestinal areas. Duodenal and oral mucosa organ culture has already been demonstrated as a reliable procedure to identify CD. The present study investigated gluten-dependent immunological activation of colonic mucosa in CD patients. We took advantage of the numerous colonoscopies performed for various clinical conditions or only for defensive medicine. Forty-four patients with gastrointestinal symptoms or in need of colorectal cancer screening were divided into patients with serum anti-endomysium (EMA) and anti-tissue transglutaminase (anti-tTG) antibody positive results (Group A), patients with serum antibody negative results (Group B), and patients with inflammatory bowel disease (IBD) (Group C). The autoantibodies EMA and anti-tTG were evaluated in supernatants of cultured sigmoid and duodenal biopsies from patients on a gluten-containing diet. In Group A, EMA and anti-tTG resulted positive in all duodenal culture supernatants. In sigmoid culture supernatants, EMA and anti-tTG were detected in 12/16 (75 %) and 13/16 (81.3 %) patients, respectively. In Group B, none of the 17 patients showed EMA and anti-tTG positive results in both duodenal and sigmoid cultures. In Group C, all 11 patients presented EMA negative results in sigmoid cultures. Only in one patient, anti-tTG were detectable in the sigmoid culture supernatant, as expected in cases of IBD. Data confirm that the gluten-dependent immunological activation affects more intestinal tracts with different degrees of involvement, suggesting that the organ culture of colonic biopsies could represent a new tool to opportunistically detect CD.

Hydrodynamic characteristics and mixing behaviour of Sclerotium glucanicum culture fluids in an airlift reactor with an internal loop used for scleroglucan production.

Science.gov (United States)

Kang, X; Wang, H; Wang, Y; Harvey, L M; McNeil, B

2001-10-01

The filamentous fungus, Sclerotium glucanicum NRRL 3006, was cultivated in a 0.008 m(3) airlift bioreactor with internal recirculation loop (ARL-IL) for production of the biopolymer, scleroglucan. The rheological behaviour of the culture fluid was characterised by measurement of the fluid consistency coefficient (K) and the flow behaviour index (n). Based on these measurements, the culture fluid changed from a low viscosity Newtonian system early in the process, to a viscous non-Newtonian (pseudoplastic) system. In addition, reactor hydrodynamics and mixing behaviour were characterised by measurement of whole mean gas hold-up (epsilon(g)), liquid re-circulation velocity (U(ld)) and mixing time (t(m)). Under identical process conditions, the effects of the viscosity of the culture fluid and air flow rate on epsilon(g), U(ld) and t(m) were examined and empirical correlations for epsilon(g), U(ld) and t(m) with both superficial velocity U(g) and consistency coefficient K were obtained and expressed separately. The correlations obtained are likely to describe the behaviour of real fungal culture fluids more accurately than previous correlations based on Newtonian or simulated non-Newtonian systems.

Studies on Using Cattle and Sheep Hydatid Cyst Fluid Instead of the Fetal Calf Serum in Leishmania Culture

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Hossein Rezvan

2013-12-01

Full Text Available Background: Leishmania is a single cell parasite causing leishmaniasis, which is a common disease between humans and animals. Due to the importance of in-vitro culture of the parasite in leishmania research, developing new methods for in-vitro cultivation of the parasite has always been a goal for leishmania researchers. The main objective of7T 5T7Tthis study was to use sheep and bovine hydatid cyst fluids as alternatives for fetal calf serum (FCS in leishmania in-vitro5T culture5T. Materials and Methods: 7TA total of 5T7T1 million leishmania promastigotes were added to 4 flasks as follow5T7T. A f5T7Tlask containing DMEM medium with 105T7T% 5T7Tfetal bovine serum5T7T, a f5T7Tlask containing DMEM and 10% sheep hydatid cyst fluid5T7T, a f5T7Tlask containing DMEM medium with 105T7T% 5T7Tbovine hydatid cyst fluid and a5T7T f5T7Tlask containing DMEM medium alone. After 2, 45T7T, 5T7T7, 95T7T, 11, 5T7T21 and 24 days, the number of parasites were counted and compared5T7T. Results: The result of this study showed that, DMEM medium enriched with 10% sheep hydatid cyst fluid in 168 hours and medium enriched with 10% bovine hydatid cyst fluid in 96 hours can act as a good alternative for fetal bovine serum in the culture Leishmania major. Conclusion: 5TThe results showed that sheep and bovine hydatid cyst fluid can be used as alternatives to FCS for dense cultivation of leishmania. The results also showed that5T, 5Tthe growth of promastigotes in medium enriched with bovine cyst fluid is more rapid than the medium enriched with sheep5T c5Tyst fluid5T in5T the beginning of cultivation.

Evaluation of Antigen-Specific IgM and IgG Production during an In Vitro Peripheral Blood Mononuclear Cell Culture Assay

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Yoshiko Matsuda

2017-07-01

Full Text Available The recent attention given to diseases associated with memory B-cell (mBC-produced antibodies (Abs suggests the need for a similar in vitro assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs with the intent to collect mBC-derived Abs in vitro and maintain their cell–cell contact-dependent interactions with helper T-cells. PBMCs were cultured with interleukin (IL-21, CpG-oligodeoxynucleotides (ODN, phorbol myristate acetate (PMA, and phytohemagglutinin/leucoagglutinin (PHA-L in 24-well flat-bottom plates (5 × 105 cells/well. A culture supernatant analysis of PBMCs from healthy donors (n = 10 indicated that antigen-specific IgM Ab levels in a PBMC culture supernatant might be better able to demonstrate the antigen sensitization status in a smaller peripheral blood sample, compared to IgG because Epstein–Barr virus-specific IgM mBCs circulate peripherally at a significantly higher frequency once antiviral humoral immunity has stabilized. Thus, our in vitro assay demonstrated the potential significance of antigen-specific IgM Ab production in the culture supernatants. Furthermore, an analysis of cultured PBMCs from allograft kidney recipients (n = 16 sensitized with de novo donor-specific human leukocyte antigen (HLA-specific Abs (DSAs showed that IgM-type HLA-specific Abs were detected mainly from the culture supernatants from PBMCs of patients with stable graft function, whereas IgG isotype HLA Abs were detectable only from patients with biopsy-proven antibody-mediated rejection. In other words, these IgG isotype Abs also represented an activated humoral immune response in vivo. Additionally, IgM- and IgG-expressing mBCs from healthy donors (n = 5 were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19+ B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 105 cells/well, and the resulting in vitro analysis provided some

Comparison of excretory-secretory antigen and positive faecal supernatant antigen in the detection of Echinococcus granulosus infection in dogs by CIEP

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P. R. Prathiush

Full Text Available Coproantigen detection of Echinococcosis in dogs by counter immunoelectrophoresis was standardized. Adult Echinococcus granulosus worms were obtained from intestine of a necropsied positive dog. Excretory-secretory antigen was prepared by culturing adult worms in Medium 199 (pH 7.4. Faeces of positive dog were collected and fecal supernatant was prepared and used for coproantigen detection. CIEP was carried out using tris-borate buffer (pH 8.0 at a constant current of 8mA/slide for 60 minutes. CIEP detected infection with both the antigens. [Vet World 2009; 2(11.000: 421-422

Cement waste form qualification report: WVDP [West Valley Demonstration Project] PUREX decontaminated supernatant

International Nuclear Information System (INIS)

McVay, C.W.; Stimmel, J.R.; Marchetti, S.

1988-08-01

This report provides a summary of work performed to develop a cement-based, low-level waste formulation suitable for the solidification of decontaminated high-level waste liquid produced as a by-product of PUREX spent fuel reprocessing. The resultant waste form is suitable for interim storage and is intended for ultimate disposal as low-level Class C waste; it also meets the stability requirements of the NRC Branch Technical Position on Waste Form Qualification, May 1983 and the requirements of 10 CFR 61. A recipe was developed utilizing only Portland Type I cement based on an inorganic salts simulant of the PUREX supernatant. The qualified recipe was tested full scale in the production facility and was observed to produce a product with entrained air, low density, and lower-than-expected compressive strength. Further laboratory scale testing with actual decontaminated supernatant revealed that set retarders were present in the supernatant, precluding setting of the product and allowing the production of ''bleed water.'' Calcium nitrate and sodium silicate were added to overcome the set retarding effect and produced a final product with improved performance when compared to the original formulation. This report describes the qualification process and qualification test results for the final product formulation. 7 refs., 38 figs., 21 tabs

In Vitro binding capacity of zeolite A to calcium, phosphorus and magnesium in rumen fluid as influenced by changes in pH

DEFF Research Database (Denmark)

Thilsing, Trine; Jørgensen, Rolf Jess; Poulsen, H.D.

2006-01-01

with and without zeolite, as well as varying the content of Ca and/or P. The pH was lowered by addition of HCl so as to mimic abomasal conditions, followed by subsequent HCO3- addition to mimic small intestinal pH. Rumen fluid samples were taken at strategic time points in the experiment. All samples were......An in vitro experiment was designed to mimic the transport of ingested zeolite A in the forestomachs and proximal part of the small intestine so as to evaluate the binding capacity of zeolite A to Ca, P and Mg as influenced by changes in pH. This was done by incubation of rumen fluid solutions...... centrifuged and the supernatant analysed for Ca, P and Mg as indicators of the amount of unbound mineral. The addition of zeolite to rumen fluid solutions reduced the amount of supernatant Ca and Mg at rumen pH, whereas the level of P was not reduced. After adding HCl, a large proportion of the zeolite...

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

Science.gov (United States)

Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard; Schemann, Michael

2018-01-01

The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

Science.gov (United States)

Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

1998-01-01

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

Secular gains in fluid intelligence: evidence from the Culture-Fair intelligence test.

Science.gov (United States)

Colom, Roberto; García-López, Oscar

2003-01-01

There is no doubt about the reality of the secular increase in cognitive test scores. However, there is disagreement about a key issue: does the observed increase reflect a genuine upward trend in intelligence? Evidence from the Raven test is clear, although there are some doubts about its adequacy as a fine-grained measure of fluid intelligence. Evidence from the so-called 'method of correlated vectors' is much less clear. When a crystallized battery is considered, the results leave little doubt: the increase does not reflect gains in general intelligence. However, when a fluid battery is analysed, the increase does reflect gains in general intelligence. The present study uses one of the best available measures of fluid intelligence (the Culture-Fair intelligence test) to provide new evidence for the secular increase in fluid intelligence, beyond the findings from the Raven test and the method of correlated vectors. A total of 4498 Spanish high school students and high school graduates were tested within a time interval of 20 and 23 years, respectively. The results show that there is a clear upward trend in intelligence. Moreover, students show an average increase equivalent to 6 IQ points, while graduates show an average increase of 4 IQ points. Therefore, more selected people (graduates) show a smaller increase than less selected people (students). Some implications are discussed.

Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples.

Science.gov (United States)

Choi, Qute; Kim, Hyun Jin; Kim, Jong Wan; Kwon, Gye Cheol; Koo, Sun Hoe

2018-01-04

The process of plate streaking has been automated to improve routine workflow of clinical microbiology laboratories. Although there were many evaluation reports about the inoculation of various body fluid samples, few evaluations have been reported for blood. In this study, we evaluated the performance of automated inoculating system, Previ Isola for various routine clinical samples including blood. Blood culture, body fluid, and urine samples were collected. All samples were inoculated on both sheep blood agar plate (BAP) and MacConkey agar plate (MCK) using Previ Isola and manual method. We compared two methods in aspect of quality and quantity of cultures, and sample processing time. To ensure objective colony counting, an enumeration reading reference was made through a preliminary experiment. A total of 377 nonduplicate samples (102 blood culture, 203 urine, 72 body fluid) were collected and inoculated. The concordance rate of quality was 100%, 97.0%, and 98.6% in blood, urine, and other body fluids, respectively. In quantitative aspect, it was 98.0%, 97.0%, and 95.8%, respectively. The Previ Isola took a little longer to inoculate the specimen than manual method, but the hands-on time decreased dramatically. The shortened hands-on time using Previ Isola was about 6 minutes per 10 samples. We demonstrated that the Previ Isola showed high concordance with the manual method in the inoculation of various body fluids, especially in blood culture sample. The use of Previ Isola in clinical microbiology laboratories is expected to save considerable time and human resources. © 2018 Wiley Periodicals, Inc.

Culture medium for amylase production by toxigenic fungi

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Figueira Edson Luiz Zangrando

2000-01-01

Full Text Available Mycelial growth and amylase production by a mycotoxigenic strain of Fusarium moniliforme and Aspergillus flavus were evaluated in a culture medium containing starch, glycerol, wheat bran or corn. With emphasis on corn, different fractions composed by germ, degermed seed, starch, milky stage corn and the respective starch or supernatant fraction were analyzed for F. moniliforme growth . The medium composed of milky stage corn supernatant promoted the best mycelial growth (p<0.05, and it was used to prepare amylase production medium in the next step. The medium composed with 2% ground corn in milky stage corn supernatant (350g of milky stage corn blended with 250mL water and centrifuged promoted the highest amylase production, which was at the 10th day of fermentation, both for F. moniliforme (42.32U/mL and A. flavus (4,745.54U/mL.

Cytokine profiles of tumor supernatants in invasive ductal cancer and fibroadenoma of the breast and its relationship with VEGF-A expression in the tumors.

Science.gov (United States)

Autenshlyus, Alexander I; Arkhipov, Sergey A; Kunts, Tatiana A; Marinkin, Igor O; Mikhailova, Elena S; Karpukhina, Xenia V; Varaksin, Nikolay A

2017-03-01

Interrelations between cytokines, produced by invasive ductal carcinoma (IDC) and fibroadenoma (FA) of the breast, and angiogenic growth factor VEGF-A, expressed in IDC and FA, were investigated. The analysis of the cytokine profiles of IDC and FA was performed by cultivation of tumor biopsy specimens in vitro. Testing of the cytokine-producing reserve of the tumors for production of VEGF-A was conducted by culturing samples of IDC and FA in a medium containing polyclonal activator (a complex of phytohemagglutinin, concanavalin A, and lipopolysaccharide). Levels of cytokines and growth factors (IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF-A) and MCP-1 (monocyte chemoattractant protein-1) in tumor supernatants were determined by an ELISA. Expression of VEGF-A was analyzed in tumor biopsy specimens by immunohistochemical analysis. In the IDC supernatants, the concentrations of IL-17, IL-18, and IFN-γ were higher and the concentrations of IL-10 and MCP-1 were lower in comparison with the FA supernatants. We observed negative correlations between the macrophage infiltration and VEGF-A concentration in the IDC supernatants (r = -0.508; P = 0.011) and between VEGF-A expression and the IDC vascularization degree (r = -0.423, P = 0.039). Spontaneous expression of VEGF-A in samples of IDC significantly exceeded the VEGF-A expression in FA. There was no difference between IDC and FA in VEGF-A expression after treatment with the polyclonal activators. Our results indicate that greater malignancy may have a paradoxical effect that is controlled by cytokines and characterized by weakening of tumor angiogenesis during overproduction of VEGF-A. These findings point to complex mechanisms of positive and negative regulation of tumor angiogenesis by cytokines that are produced by the tumor and by cells in its microenvironment, whose cytokine profiles may change at different stages of tumor progression.

Cell mediated lympholysis: CML. A microplate technique requiring few target cells and employing a new method of supernatant collection

International Nuclear Information System (INIS)

Hirschberg, A.; Thorsby, E.

1977-01-01

A micromethod for the 51 Cr release assay is described. Allogeneically induced cytotoxic lymphocytes are generated in mixed lymphocyte microcultures in the wells of microplates. Their cytotoxic capacity is assayed by adding 51 Cr-labelled PHA derived lymphoblasts directly into the microcultures with no pooling or transfer of the cytotoxic effector cells being required. The 51 Cr isotope released into the cell supernatants is collected by inserting a cellulose acetate absorption cartridge into each well. A glass fiber filter attached to the cartridge effectively separates the supernatant from the cellular elemets. This system allows the simultaneous collection of the supernatant from 96 wells, and can be used with either adherent or non-adherent target cells

Periodontitis-associated septic pulmonary embolism caused by Actinomyces species identified by anaerobic culture of bronchoalveolar lavage fluid: a case report.

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Endo, Shun; Mishima, Eikan; Takeuchi, Yoichi; Ohi, Takashi; Ishida, Masatsugu; Yanai, Masaru; Kiyomoto, Hideyasu; Nagasawa, Tasuku; Ito, Sadayoshi

2015-12-01

Periodontal disease is a less common but important cause of septic pulmonary embolism (SPE). However, the pathogens causing periodontal disease-associated SPE (PD-SPE) have been poorly understood. Actinomyces species are resident microbiota in the oral cavity. Here we report a case of PD-SPE caused by Actinomyces species, which was identified by anaerobic culture of bronchoalveolar lavage fluid (BAL). A 64-year-old Asian man, complicated with severe chronic periodontitis, was admitted with chest pain and fever. Chest CT revealed multiple bilateral pulmonary nodules located subpleurally. We diagnosed the case as SPE associated with periodontitis. Although blood cultures were negative for the usual 5-day incubation, anaerobic culture of the BAL fluid sample yielded Actinomyces species. Antibacterial therapy alone did not ameliorate the symptoms; however, additional dental treatment, including tooth extraction, promptly did. The patient was discharged 23 days after admission. The 3-month follow-up revealed no recurrence of the symptoms and complete resolution of the lung lesions. This case demonstrated that Actinomyces species can cause PD-SPE. Additionally, clinicians should consider performing appropriate anaerobic culture of BAL fluid to identify the pathogen of SPE, and to ordering dental treatment, if necessary, in addition to antibiotics for the initial management of PD-SPE.

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

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Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

2013-01-01

Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

Underestimation of phosphorus fraction change in the supernatant after phosphorus adsorption onto iron oxides and iron oxide-natural organic matter complexes.

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Yan, Jinlong; Jiang, Tao; Yao, Ying; Wang, Jun; Cai, Yuanli; Green, Nelson W; Wei, Shiqiang

2017-05-01

The phosphorus (P) fraction distribution and formation mechanism in the supernatant after P adsorption onto iron oxides and iron oxide-humic acid (HA) complexes were analyzed using the ultrafiltration method in this study. With an initial P concentration of 20mg/L (I=0.01mol/L and pH=7), it was shown that the colloid (1kDa-0.45μm) component of P accounted for 10.6%, 11.6%, 6.5%, and 4.0% of remaining total P concentration in the supernatant after P adsorption onto ferrihydrite (FH), goethite (GE), ferrihydrite-humic acid complex (FH-HA), goethite-humic acid complex (GE-HA), respectively. The oxide aggregates was the main mechanism for the formation of the colloid P in the supernatant. And colloidal adsorbent particles co-existing in the supernatant were another important reason for it. Additionally, dissolve organic matter dissolved from iron oxide-HA complexes could occupy large adsorption sites of colloidal iron causing less colloid P in the supernatant. Ultimately, we believe that the findings can provide a new way to deeply interpret the geochemical cycling of P, even when considering other contaminants such as organic pollutants, heavy metal ions, and arsenate at the sediment/soil-water interface in the real environment. Copyright © 2016. Published by Elsevier B.V.

Biological nutrients removal from the supernatant originating from the anaerobic digestion of the organic fraction of municipal solid waste.

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Malamis, S; Katsou, E; Di Fabio, S; Bolzonella, D; Fatone, F

2014-09-01

This study critically evaluates the biological processes and techniques applied to remove nitrogen and phosphorus from the anaerobic supernatant produced from the treatment of the organic fraction of municipal solid waste (OFMSW) and from its co-digestion with other biodegradable organic waste (BOW) streams. The wide application of anaerobic digestion for the treatment of several organic waste streams results in the production of high quantities of anaerobic effluents. Such effluents are characterized by high nutrient content, because organic and particulate nitrogen and phosphorus are hydrolyzed in the anaerobic digestion process. Consequently, adequate post-treatment is required in order to comply with the existing land application and discharge legislation in the European Union countries. This may include physicochemical and biological processes, with the latter being more advantageous due to their lower cost. Nitrogen removal is accomplished through the conventional nitrification/denitrification, nitritation/denitritation and the complete autotrophic nitrogen removal process; the latter is accomplished by nitritation coupled with the anoxic ammonium oxidation process. As anaerobic digestion effluents are characterized by low COD/TKN ratio, conventional denitrification/nitrification is not an attractive option; short-cut nitrogen removal processes are more promising. Both suspended and attached growth processes have been employed to treat the anaerobic supernatant. Specifically, the sequencing batch reactor, the membrane bioreactor, the conventional activated sludge and the moving bed biofilm reactor processes have been investigated. Physicochemical phosphorus removal via struvite precipitation has been extensively examined. Enhanced biological phosphorus removal from the anaerobic supernatant can take place through the sequencing anaerobic/aerobic process. More recently, denitrifying phosphorus removal via nitrite or nitrate has been explored. The removal of

Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

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Amel, Ait Meddour; Farida, Bendali; Djamila, Sadoun

2015-07-01

It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P < 0.05) inhibition potential of the pathogens either on solid media or in broth co-cultures. Characterization of the antibacterial substances indicated their proteinaceous nature which assigned them most probably to bacteriocins group.

Outcomes in culture positive and culture negative ascitic fluid infection in patients with viral cirrhosis: cohort study

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Ali Ailia W

2008-12-01

Full Text Available Abstract Background Ascitic fluid infection (AFI in cirrhotic patients has a high morbidity and mortality. It has two variants namely, spontaneous bacterial peritonitis (SBP and culture negative neutrocytic ascites (CNNA. The aim of this study was to determine the outcome in cirrhotic patients with culture positive (SBP and culture negative neutrocytic ascites. Methods We analyzed 675 consecutive hepatitis B and/or C related cirrhosis patients with ascites admitted in our hospital from November 2005 to December 2007. Of these, 187 patients had AFI; clinical and laboratory parameters of these patients including causes of cirrhosis, Child Turcotte Pugh (CTP score were recorded. Results Out of 187 patients with AFI, 44 (23.5% had SBP while 143 (76.4% had CNNA. Hepatitis C virus (HCV infection was the most common cause of cirrhosis in 139 (74.3% patients. Patients with SBP had high CTP score as compared to CNNA (12.52 ± 1.45 vs. 11.44 ± 1.66; p 9/L as compared to CNNA (132 ± 91 × 109/L, p = 0.005. We found a high creatinine (mg/dl (1.95 ± 1.0 vs. 1.44 ± 0.85, (p = 0.003 and high prothrombin time (PT in seconds (24.8 ± 6.6 vs. 22.4 ± 7.2 (p = 0.04 in SBP as compared to CNNA. More patients with SBP (14/44; 31.8% had blood culture positivity as compare to CNNA (14/143; 9.8%, p = 0.002. Escherichia. Coli was the commonest organism in blood culture in 15/28 (53.5% patients. SBP group had a higher mortality (11/44; 25% as compared to CNNA (12/143; 8.4%, p = 0.003. On multiple logistic regression analysis, creatinine >1.1 mg/dl and positive blood culture were the independent predictors of mortality in patients with SBP. Conclusion Patients with SBP have a higher mortality than CNNA. Independent predictors of mortality in SBP are raised serum creatinine and a positive blood culture.

Selection of the treatment method for the West Valley alkaline supernatant

International Nuclear Information System (INIS)

Carl, D.E.; Leonard, I.M.

1987-02-01

As part of the West Valley Demonstration Project (WVDP), th PUREX supernatant stored in Tank 8d-2 will be partially decontaminated before encapsulation in the final glass form. This report discusses selection of a method for removing Cs-137, the major radioactive ion in the supernatant. Methods considered were: (1) electrodialysis; (2) hyperfiltration; (3) precipitation with ferrocyanide, NaTPB, or PTA; (4) organic ion exchange using Cs-100 or a biologically derived media; (5) chelation using DeVoe/Holbein compostions; and (6) inorganic ion exchange using Durasil, natural zeolities, IE-95 or IE-96 media. Several different methods of using inorganic ion exchange media were also reviewed including (1) four columns with elution, and (2) two, three, or four columns without elution. After the careful evaluation of experimental data with all process constraints taken into account, the inorganic exchange media IE-96 (Linde Ionsiv IE-96 synthetic zeolite) was chosen for WVDP cesium recovery. IE-96 was chosen for the following reasons: high sorption rate, a decontamination factor (DF) over 1000, excellent exchange capacity at WVDP conditions, compatability with the glass formers used for borosilicate glass in direct melter feed applications, and a history of successful application in radio chemical seperation for waste streams. 34 refs., 29 figs., 27 tabs

Culture supernatants of oral cancer cells induce impaired IFN-α production of pDCs partly through the down-regulation of TLR-9 expression.

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Han, Nannan; Zhang, Zun; Jv, Houyu; Hu, Jingzhou; Ruan, Min; Zhang, Chenping

2018-06-05

The aim of the present study was to investigate whether tumor-derived supernatants down-regulate the immune function of plasmacytoid dendritic cells (pDCs) in oral cancer and the potential molecular mechanisms of this effect. Immunohistochemistry (IHC) and flow cytometry were used to detect tumor-infiltrating and peripheral blood pDCs. MTS and flow cytometry were employed to evaluate the immune response of CD4 + T cells. Real-time PCR and ELISA assays were used to identify TLR-7 and TLR-9 expression, IFN-α production and tumor-secreted soluble cytokines. The proportion of pDCs (0.121%±0.043%) was significantly higher in Oral squamous cell carcinoma (OSCC) samples than in normal tissue (0.023%±0.016%) (P = 0.021). TLR9 mRNA was significantly lower in tumor-infiltrating pDCs and positively correlated to low IFN-α production (r = 0.956; Poral cancer cells negatively regulated TLR9 mRNA expression and the subsequent IFN-α production of pDCs, which inhibited the immune response of CD4 + T cells. The neutralizing antibodies blocking assay showed that the specific inhibitory effect of pDC functionality was associated with the soluble fraction of the oral cancer environment, which is mainly mediated by IL-10 and TGF-β cooperation. Tumor-derived supernatants may impair the function of tumor-infiltrating pDCs, which subsequently decreases the immune response of CD4 + T cells in human oral cancer through TGF-β- and IL-10- dependent mechanisms. Careful manipulation of these impaired pDCs may help develop an important alternative immunotherapy for the treatment of oral cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

Elevated levels of the complement activation product C4d in bronchial fluids for the diagnosis of lung cancer.

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Daniel Ajona

Full Text Available Molecular markers in bronchial fluids may contribute to the diagnosis of lung cancer. We previously observed a significant increase of C4d-containing complement degradation fragments in bronchoalveolar lavage (BAL supernatants from lung cancer patients in a cohort of 50 cases and 22 controls (CUN cohort. The present study was designed to determine the diagnostic performance of these complement fragments (hereinafter jointly referred as C4d in bronchial fluids. C4d levels were determined in BAL supernatants from two independent cohorts: the CU cohort (25 cases and 26 controls and the HUVR cohort (60 cases and 98 controls. A series of spontaneous sputum samples from 68 patients with lung cancer and 10 controls was also used (LCCCIO cohort. Total protein content, complement C4, complement C5a, and CYFRA 21-1 were also measured in all cohorts. C4d levels were significantly increased in BAL samples from lung cancer patients. The area under the ROC curve was 0.82 (95%CI = 0.71-0.94 and 0.67 (95%CI = 0.58-0.76 for the CU and HUVR cohorts, respectively. In addition, unlike the other markers, C4d levels in BAL samples were highly consistent across the CUN, CU and HUVR cohorts. Interestingly, C4d test markedly increased the sensitivity of bronchoscopy in the two cohorts in which cytological data were available (CUN and HUVR cohorts. Finally, in the LCCCIO cohort, C4d levels were higher in sputum supernatants from patients with lung cancer (area under the ROC curve: 0.7; 95%CI = 0.56-0.83. In conclusion, C4d is consistently elevated in bronchial fluids from lung cancer patients and may be used to improve the diagnosis of the disease.

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis.

Science.gov (United States)

Ulrich, Martina; Beer, Isabelle; Braitmaier, Peter; Dierkes, Michaela; Kummer, Florian; Krismer, Bernhard; Schumacher, Ulrike; Gräpler-Mainka, Ute; Riethmüller, Joachim; Jensen, Peter Ø; Bjarnsholt, Thomas; Høiby, Niels; Bellon, Gabriel; Döring, Gerd

2010-11-01

Patients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of CF lung disease is unknown. The virulence of Prevotella intermedia and Ps aeruginosa was investigated in vitro and in mice, antibodies against P intermedia in CF sera were assessed and a culture-independent detection method for P intermedia/P nigrescens in CF sputum was tested. P intermedia reached cell numbers of >10(5)->10(7) colony-forming units (CFU)/ml sputum. The majority of patients with CF (16/17; 94.1%) produced antibodies against two immunoreactive antigens of P intermedia. Culture supernatant fluids, collected from 10(9) P intermedia cells, were more cytotoxic to respiratory epithelial cells in vitro and inflammatory in mouse lungs than respective fluids from anaerobically grown Ps aeruginosa, while fluids from aerobically grown Ps aeruginosa had the highest cytotoxicity and inflammation. Both pathological effects were largely reduced when culture supernatant fluids from 10(7) cells of either species were used. P intermedia cells (∼10(6)CFU/lung) did not induce mortality in the agar beads lung infection mouse model, while Ps aeruginosa cells caused death in 30% of mice due to rapid multiplication. A P intermedia/P nigrescens-specific PNA probe was significantly more sensitive than culture-dependent diagnostic assays to detect these strict anaerobes. Ps aeruginosa and P intermedia become significantly virulent in vitro and in vivo when cell numbers exceed 10(8) CFU/lung.

Monitoring Dynamic Interactions between Breast Cancer Cells and Human Bone Tissue in a Co-Culture Model

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Contag, Christopher H.; Lie, Wen-Rong; Bammer, Marie C.; Hardy, Jonathan W.; Schmidt, Tobi L.; Maloney, William J.; King, Bonnie L.

2015-01-01

Purpose Bone is a preferential site of breast cancer metastasis and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. Procedures Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell division and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX® immunoassays. Results BLI demonstrated increased MDA-MB-231-fLuc proliferation (pbones, and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc colonization of bone, and MILLIPLEX® profiles of culture supernatants suggested breast/bone crosstalk. Conclusions Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays. PMID:24008275

Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils--a promising candidate formulation for amnioinfusion fluid.

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Uchida, Toshiyuki; Itoh, Hiroaki; Nakamura, Yuki; Kobayashi, Yukiko; Hirai, Kyuya; Suzuki, Kazunao; Sugihara, Kazuhiro; Kanayama, Naohiro; Hiramatsu, Mitsuo

2010-06-01

Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Immunity to Schistosoma mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae. T-cell activation of macrophages for larval killing

International Nuclear Information System (INIS)

Gordon, J.R.; McLaren, D.J.

1988-01-01

This study addresses macrophage activation in guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosom mansoni. Peritoneal exudate macrophages elicited in vaccinated animals by mineral oil injection were activated to kill larval schistosomes in vitro. Killing efficiency is dependent upon the cell:target ratio employed and is enhanced by, but is not strictly dependent on, the presence of specific antibodies. Macrophages co-cultured with parasites release superoxide radicals and hydrogen peroxide, but the use of inhibitors has shown that neither of these reactive oxygen intermediates are the causal agents of cellular cytotoxicity in this system. Oil-elicited macrophages from naive guinea-pigs do not show comparable activation; they can, however, be activated in vitro by incubation with culture supernatant fluids from schistosome antigen-stimulated spleen, or lymph node cells harvested from vaccinated guinea-pigs. Naive macrophages activated in this way kill schistosomula in vitro and release the activation markers IL-l and superoxide anion. The macrophage-activating factor (MAF) present in spleen cell culture supernatant fluids has a MW of 35,000-55,000, but does not have the chemical characteristics of gamma-interferon. (author)

Electrolytic destruction of oxalate ions in plutonium oxalate supernatant

International Nuclear Information System (INIS)

Michael, K.M.; Talnikar, S.G.; Jambunathan, U.; Kapoor, S.C.; Ramanujam, A.; Venkataraman, N.

1996-01-01

A simple and efficient electrolytic method is described for the destruction of the oxalate ions present in plutonium oxalate supernatant. Using platinum electrode and very little KMnO 4 , in situ generation of Mn 3+ ions is achieved which in turn destroys the oxalate. The use of lower current density helps in achieving maximum current efficiency. The end point is easily detectable by the pink colour of permanganate. By reversing the current, this slight excess of permanganate can be destroyed, thus avoiding the use of hydrogen peroxide. By this simple electrolytic method, the corrosive oxalate ion is completely destroyed and the salt content of the waste solution is considerably reduced. (author). 4 refs., 1 fig., 6 tabs

Rapid Biosynthesis of Silver Nanoparticles Using Culture Supernatant of Bacteria with Microwave Irradiation

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N. Saifuddin

2009-01-01

Full Text Available The development of rapid and reliable processes for the synthesis of nanosized materials is of great importance in the field of nanotechnology. Synthesis of silver nanoparticles using microorganism have been reported, but the process is rather slow. In this paper, we describe a novel combinatorial synthesis approach which is rapid, simple and “green” for the synthesis of metallic nanostructures of noble metals such as silver (Ag, by using a combination of culture supernatanant of Bacillus subtilis and microwave (MW irradiation in water in absence of a surfactant or soft template. It was found that exposure of culture supernatanant of Bacillus subtilis and microwave irradiation to silver ion lead to the formation of silver nanoparticles. The silver nanoparticles were in the range of 5-60 nm in dimension. The nanoparticles were examined using UV-Visible Spectroscopy, and Transmission Electron Microscopy (TEM analyses. The formation of nanoparticles by this method is extremely rapid, requires no toxic chemicals and the nanoparticles are stable for several months. The main conclusion is that the bio-reduction method to produce nanoparticles is a good alternative to the electrochemical methods.

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

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Ale, Cesar E; Farías, Marta E; Strasser de Saad, Ana M; Pasteris, Sergio E

2014-07-01

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L-malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end-product. 8.0 mM glycerol was found in 6-day culture supernatants. In simultaneous cultures, total sugars and L-malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diagnosis of spontaneous bacterial peritonitis: Role of tween 80 and triton X in ascitic fluid cultures

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Iyer R

2009-01-01

Full Text Available A patient with alcoholic cirrhosis of the liver, portal hypertension with hepatic encephalopathy and spontaneous bacterial peritonitis (SBP was admitted in an obtunded condition. Attempts at delineating the aetiology of the SBP using conventional cultures as well as automated systems were not successful. The use of non-anionic surfactant agents such as Tween 80-incorporated blood agar and Triton X treatment of the specimens facilitated the growth of Klebsiella pneumoniae from the ascitic fluid, which otherwise would have been concluded to represent culture-negative neutrocytic ascites. Thus, the use of the aforementioned agents could be explored in elucidating the aetiology of body cavity infections when conventional methods fail.

[Effect of Pseudomonas aeruginosa exometabolites on planktonic and biofilm cultures of Escherichia coli].

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Kuznetsova, M V; Karpunina, T I; Maslennikova, I L; Nesterova, L Iu; Demakov, V A

2012-01-01

Study the effect of P. aeruginosa exometabolites on planktonic and biofilm cultures of bioluminescent E. coli strain. E. coli K12 TG1 (pF1 lux+ Ap(r)) recombinant bioluminescent strain, P. aeruginosa ATCC 27853 reference strain and 2 nosocomial isolates were used. Pyocyanin and pyoverdin content in supernatant of P. aeruginosa over-night cultures was evaluated according to E. Deziel et al. (2001). Planktonic and biofilm cultures of E. coli were obtained in 96-well plates (LB, statically, 37 degrees C), optical density of plankton, film biomass (OD600, OD580) and bioluminescence in plankton and biofilm were evaluated in microplate reader Infiniti M200 (Tecan, Austria). P. aeruginosa exometabolites increased the duration of lag-phase in E. coli, and short term exposition inhibited luminescence of planktonic cells. These effects are determined by bactericidal action ofpyocyanin and pyoverdin. Supernatants ofover-night cultures of P. aeruginosa inhibit formation of biofilm and disrupt the formed biofilm of E. coli. Effect of pyocyanin and pyoverdin on these processes is not established, other factors may have higher significance. Bioluminescence of E. coli K12 TGI that reflects the energetic status of the cell allows to evaluate and prognose the character of coexistence of P. aeruginosa in combined with E. coli planktonic and biofilm culture.

Bactericidal activity of culture fluid components of Lactobacillus fermentum strain 90 TS-4 (21) clone 3, and their capacity to modulate adhesion of Candida albicans yeast-like fungi to vaginal epithelial cells.

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Anokhina, I V; Kravtsov, E G; Protsenko, A V; Yashina, N V; Yermolaev, A V; Chesnokova, V L; Dalin, M V

2007-03-01

Antagonistic activities of L. fermentum strain 90 TS-4 (21), L. casei ATCC 27216, and L. acidophilus ATCC 4356 and bactericidal activity of lactobacillus culture fluid towards E. coli strain K12, S. aureus, and S. epidermidis test cultures were studied. The bactericidal effect of L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation (pH 6.0) on the test cultures was dose-dependent. Adhesion of C. albicans yeast-like fungi to vaginal epitheliocytes was more pronounced for strains isolated from women with asymptomatic infection than for strains isolated from women with manifest forms. L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation modulated adhesion of yeast-like fungi only if the fungal strain was initially highly adherent.

Field assessment of the enzyme linked immunosorbent assay for foot-and-mouth disease virus diagnosis and typing

International Nuclear Information System (INIS)

Smitsaart, E.; Fondevila, N.; Compaired, D.; Maradei, E.; Fernandez, E.

1998-01-01

The objective of the present study was to evaluate the enzyme linked immunosorbent assay (ELISA) in comparison with the complement fixation test (CFT) for the diagnosis and typing of foot-and-mouth disease (FMD) virus (FMDV). Diagnostic material was epithelium from either suspected cases of FMD or from animals experimentally infected with FMDV. Epithelial suspensions and supernatant fluids from cell culture passage were assayed by CFT and ELISA. The superiority of the ELISA over the CFT was demonstrated: 1) the detection rate was 23% higher than that of CFT on original (epithelial) suspensions (OS) submissions of all sample (positive and negative) and 30% higher on supernatant fluids from cell culture passage, 2) the detection rate of ELISA on OS of confirmed positive samples was 28% higher than that of CFT, 3) no significant differences were observed in the detection and typing rates between the PANAFTOSA and FAO/IAEA ELISA kits (P<0.05) and 4) the sensitivity of the ELISA was 16 to 85 times higher than that of CFT when serial dilutions of sample homogenates were examined. (author)

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

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Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

2014-05-01

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gastric fluid versus amniotic fluid analysis for the identification of intra-amniotic infection due to Ureaplasma species.

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Kim, Sun Min; Romero, Roberto; Lee, JoonHo; Chaemsaithong, Piya; Docheva, Nikolina; Yoon, Bo Hyun

2016-01-01

Early neonatal sepsis is often due to intra-amniotic infection. The stomach of the neonate contains fluid swallowed before and during delivery. The presence of bacteria as well as neutrophils detected by culture or Gram stain of the gastric fluid during the first day of life is suggestive of exposure to bacteria or inflammation. We undertook this study to determine the relationship between gastric fluid analysis and amniotic fluid obtained by transabdominal amniocentesis in the detection of Ureaplasma species, the most frequent microorganisms responsible for intra-amniotic infection. The study population consisted of 100 singleton pregnant women who delivered preterm neonates (Ureaplasma species was performed. Intra-amniotic inflammation was defined as an elevated amniotic fluid matrix metalloproteinase-8 concentration (>23 ng/mL). (1) Ureaplasma species were detected by culture or PCR in 18% (18/100) of amniotic fluid samples and in 5% (5/100) of gastric fluid samples; (2) among the amniotic fluid cases positive for Ureaplasma species, these microorganisms were identified in 27.8% (5/18) of gastric fluid samples; (3) none of the cases negative for Ureaplasma species in the amniotic fluid were found to be positive for these microorganisms in the gastric fluid; (4) patients with amniotic fluid positive for Ureaplasma species but with gastric fluid negative for these microorganisms had a significantly higher rate of intra-amniotic inflammation, acute histologic chorioamnionitis, and neonatal death than those with both amniotic fluid and gastric fluid negative for Ureaplasma species; and (5) no significant differences were observed in the rate of intra-amniotic inflammation, acute histologic chorioamnionitis, and neonatal death between patients with amniotic fluid positive for Ureaplasma species but with gastric fluid negative for these microorganisms and those with both amniotic fluid and gastric fluid positive for Ureaplasma species. Gastric fluid analysis has 100

Sampling and analysis of high level waste tank supernatant: an overview

International Nuclear Information System (INIS)

Goergen, C.R.

1981-01-01

The Savannah River Plant routinely samples its high level radioactive waste tank supernatants for analysis of major components. These results are important in maintaining proper levels of corrosion inhibiters for protection of the tank walls. Because the tank ambient temperature is elevated, the sample is heated to 70 0 C prior to removing aliquots for use in a variety of analytical methods. Typical analyses include density, pH, OH - , NO 3 - , and NO 2 - , with occasional requests for Al(OH) 4 - , CO 3 /sup =/, PO 4 /sup =/, SO 4 /sup =/, and various radionuclides

Fate of cyanobacteria in drinking water treatment plant lagoon supernatant and sludge.

Science.gov (United States)

Pestana, Carlos J; Reeve, Petra J; Sawade, Emma; Voldoire, Camille F; Newton, Kelly; Praptiwi, Radisti; Collingnon, Lea; Dreyfus, Jennifer; Hobson, Peter; Gaget, Virginie; Newcombe, Gayle

2016-09-15

In conventional water treatment processes, where the coagulation and flocculation steps are designed to remove particles from drinking water, cyanobacteria are also concentrated into the resultant sludge. As a consequence, cyanobacteria-laden sludge can act as a reservoir for metabolites such as taste and odour compounds and cyanotoxins. This can pose a significant risk to water quality where supernatant from the sludge treatment facility is returned to the inlet to the plant. In this study the complex processes that can take place in a sludge treatment lagoon were investigated. It was shown that cyanobacteria can proliferate in the conditions manifest in a sludge treatment lagoon, and that cyanobacteria can survive and produce metabolites for at least 10days in sludge. The major processes of metabolite release and degradation are very dependent on the physical, chemical and biological environment in the sludge treatment facility and it was not possible to accurately model the net effect. For the first time evidence is provided to suggest that there is a greater risk associated with recycling sludge supernatant than can be estimated from the raw water quality, as metabolite concentrations increased by up to 500% over several days after coagulation, attributed to increased metabolite production and/or cell proliferation in the sludge. Copyright © 2016 Elsevier B.V. All rights reserved.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

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Ricardo Chaves Vilela

2013-02-01

Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

Gastric cancer cell supernatant causes apoptosis and fibrosis in the peritoneal tissues and results in an environment favorable to peritoneal metastases, in vitro and in vivo

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Na Di

2012-04-01

Full Text Available Abstract Background In this study, we examined effects of soluble factors released by gastric cancer cells on peritoneal mesothelial cells in vitro and in vivo. Methods HMrSV5, a human peritoneal mesothelial cell line, was incubated with supernatants from gastric cancer cells. Morphological changes of HMrSV5 cells were observed. Apoptosis of HMrSV5 cells was observed under a transmission electron microscope and quantitatively determined by MTT assay and flow cytometry. Expressions of apoptosis-related proteins (caspase-3, caspase-8, Bax, bcl-2 were immunochemically evaluated. Results Conspicuous morphological changes indicating apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. In vivo, peritoneal tissues treated with gastric cancer cell supernatant were substantially thickened and contained extensive fibrosis. Conclusions These findings demonstrate that supernatants of gastric cancer cells can induce apoptosis and fibrosis in HMrSV5 human peritoneal mesothelial cells through supernatants in the early peritoneal metastasis, in a time-dependent manner, and indicate that soluble factors in the peritoneal cavity affect the morphology and function of mesothelial cells so that the resulting environment can become favorable to peritoneal metastases.

Human Tear Fluid Reduces Culturability of Contact Lens Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence Associated Type III Secretion System

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Wu, Yvonne T.; Tam, Connie; Zhu, Lucia S.; Evans, David J.; Fleiszig, Suzanne M. J.

2017-01-01

Purpose The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens (CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. Methods P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. Results With or without tear fluid, biofilms grew to ~108 cfu viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ~180-fold (pbacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (p=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS (p=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (p = 0.04) and 1.89 ± 0.26-fold (p<.001), respectively. Conclusions Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis. PMID:27670247

Cytologic examination of hemorrhagic fluid by capillary centrifugation: a new technique.

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Agarwal, Padam Kumari

2009-01-01

To develop a simplified technique for processing hemorrhagic body fluids, allowing elimination of red blood cells (RBCs) to obtain tumor cell-rich cytosmears for accurate diagnosis of malignancy. Hemorrhagic fluid is collected with ethylenediamine tetraacetic acid anticoagulant. The cells are separated by centrifugation in glass capillaries into 3 layers, with the uppermost containing supernatant, middle buffy coat and lowermost dark layer of RBCs. The smears of the buffy coat are prepared by breaking the capillaries at the junction of buffy coat and RBC layer. The procedure is named capillary centrifugation technique. This procedure was developed in the cytology laboratory at King George's Medical College, Lucknow, India in 1974. Cell yield is good and cellular details are well preserved. No cell debris is present on the slides. The background of the smears is absolutely clear. Any number of slides may be prepared for special study for exact typing of tumor cells This is a simple, economical procedure and a good substitute for a cytocentrifuge machine for preparing smears from small amount of fluids. Technicians learn it quickly and are quite comfortable with the procedure.

Effects of peritoneal fluid from endometriosis patients on the release of monocyte-specific chemokines by leukocytes.

Science.gov (United States)

Na, Yong-Jin; Lee, Dong-Hyung; Kim, Seung-Chul; Joo, Jong-Kil; Wang, Ji-Won; Jin, Jun-O; Kwak, Jong-Young; Lee, Kyu-Sup

2011-06-01

Chemokines have been implicated in the pathological process of endometriosis. We compared the effects of peritoneal fluid obtained from patients with endometriosis (ePF) and controls without endometriosis (cPF) on the release of monocyte-specific CC chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) by neutrophils, monocytes, and T cells. Moreover, we evaluated the correlation between the levels of chemokines in ePF and their release by these cells. Cells were obtained from healthy young volunteers and cultured with ePF (n = 12) or cPF (n = 8). The chemokine levels in the ePF and the supernatants of cultured cells with ePF were then measured by ELISA. There was a positive correlation between the levels of MCP-1 and MIP-1α in ePF. The addition of ePF to the cell cultures failed to increase the release of MCP-1, RANTES, and MIP-1α when compared to cPF, but the levels of RANTES in ePF were positively correlated with the release of RANTES by ePF-treated monocytes and T cells. Moreover, there was a positive correlation between the levels of RANTES and MIP-1α released by neutrophils and between the levels of MCP-1 and MIP-1α released by T cells. Finally, the levels of RANTES released by monocyte-derived macrophages and monocytes cultured with ePF were positively correlated. These findings suggest that monocytes, neutrophils, and T cells release differential levels of MCP-1, RANTES, and MIP-1α in response to stimulation with ePF.

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity

NARCIS (Netherlands)

Buhner, Sabine; Braak, Breg; Li, Qin; Kugler, Eva Maria; Klooker, Tamira; Wouters, Mira; Donovan, Jemma; Vignali, Sheila; Mazzuoli-Weber, Gemma; Grundy, David; Boeckxstaens, Guy; Schemann, Michael

2014-01-01

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that

Evaluation of BioFM liquid medium for culture of cerebrospinal fluid in tuberculous meningitis to identify Mycobacterium tuberculosis.

Science.gov (United States)

Kashyap, R S; Ramteke, S S; Gaherwar, H M; Deshpande, P S; Purohit, H J; Taori, G M; Daginawala, H

2010-01-01

The present study was designed to evaluate the sensitivity and specificity of liquid culture medium (BioFM broth) for the diagnosis of tuberculous meningitis (TBM) in cerebrospinal fluid (CSF). CSF samples from 200 patients (TBM group = 150 and non-TBM group = 50) were tested for culture of Mycobacterium tuberculosis in BioFM liquid culture medium. Out of 150 TBM cases, 120 were found to be culture positive, indicating a sensitivity of 80% in BioFM broth within 2-3 weeks of inoculation. Positive cultures were also observed for CSF from 32 (64%) out of 50 non-TBM patients in BioFM liquid culture medium within 4 days of sample inoculation. Therefore, according to our study, BioFM broth system yielded 80% sensitivity [95% confidence interval (CI): 67-93%] and 36% specificity (95% CI: 57-98%) for TBM diagnosis. Our results indicate that although BioFM broth allows the detection of positive cultures within a shorter time, it has a high potential for contamination or for the coexistence of M. tuberculosis and non-tuberculous meningitis (NTM). This coexistence may go undetected or potentially lead to erroneous reporting of results.

Evaluation of BioFM liquid medium for culture of cerebrospinal fluid in tuberculous meningitis to identify Mycobacterium tuberculosis

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Kashyap R

2010-01-01

Full Text Available The present study was designed to evaluate the sensitivity and specificity of liquid culture medium (BioFM broth for the diagnosis of tuberculous meningitis (TBM in cerebrospinal fluid (CSF. CSF samples from 200 patients (TBM group = 150 and non-TBM group = 50 were tested for culture of Mycobacterium tuberculosis in BioFM liquid culture medium. Out of 150 TBM cases, 120 were found to be culture positive, indicating a sensitivity of 80% in BioFM broth within 2-3 weeks of inoculation. Positive cultures were also observed for CSF from 32 (64% out of 50 non-TBM patients in BioFM liquid culture medium within 4 days of sample inoculation. Therefore, according to our study, BioFM broth system yielded 80% sensitivity [95% confidence interval (CI: 67-93%] and 36% specificity (95% CI: 57-98% for TBM diagnosis. Our results indicate that although BioFM broth allows the detection of positive cultures within a shorter time, it has a high potential for contamination or for the coexistence of M. tuberculosis and non-tuberculous meningitis (NTM. This coexistence may go undetected or potentially lead to erroneous reporting of results.

Particulate metal bioaccessibility in physiological fluids and cell culture media: Toxicological perspectives.

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Leclercq, Bérénice; Alleman, Laurent Yves; Perdrix, Esperanza; Riffault, Véronique; Happillon, Mélanie; Strecker, Alain; Lo-Guidice, Jean-Marc; Garçon, Guillaume; Coddeville, Patrice

2017-07-01

According to the literature, tiny amounts of transition metals in airborne fine particles (PM 2.5 ) may induce proinflammatory cell response through reactive oxygen species production. The solubility of particle-bound metals in physiological fluids, i.e. the metal bioaccessibility is driven by factors such as the solution chemical composition, the contact time with the particles, and the solid-to-liquid phase ratio (S/L). In this work, PM 2.5 -bound metal bioaccessibility was assessed in various physiological-like solutions including cell culture media in order to evidence the potential impact on normal human bronchial epithelial cells (NHBE) when studying the cytotoxicity and inflammatory responses of PM 2.5 towards the target bronchial compartment. Different fluids (H 2 O, PBS, LHC-9 culture medium, Gamble and human respiratory mucus collected from COPD patients), various S/L conditions (from 1/6000 to 1/100,000) and exposure times (6, 24 and 72h) were tested on urban PM 2.5 samples. In addition, metals' total, soluble and insoluble fractions from PM 2.5 in LHC-9 were deposited on NHBE cells (BEAS-2B) to measure their cytotoxicity and inflammatory potential (i.e., G6PDH activity, secretion of IL-6 and IL-8). The bioaccessibility is solution-dependent. A higher salinity or organic content may increase or inhibit the bioaccessibiliy according to the element, as observed in the complex mucus matrix. Decreasing the S/L ratio also affect the bioaccessibility depending on the solution tested while the exposure time appears less critical. The LHC-9 culture medium appears to be a good physiological proxy as it induces metal bioaccessibilities close to the mucus values and is little affected by S/L ratios or exposure time. Only the insoluble fraction can be linked to the PM 2.5 -induced cytotoxicity. By contrast, both soluble and insoluble fractions can be related to the secretion of cytokines. The metal bioaccessibility in LHC-9 of the total, soluble, and insoluble

Influence of exogenous leptin on redox homeostasis in neutrophils and lymphocytes cultured in synovial fluid isolated from patients with rheumatoid arthritis

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Michał Gajewski

2016-07-01

Full Text Available Objectives : Leptin is an adipose cells derived hormone that regulates energy homeostasis within the body. Energy metabolism of immune cells influences their activity within numerous pathological states, but the effect of leptin on these cells in unclear. On the one hand, it was observed that leptin induces neutrophils chemotaxis and modulates phagocytosis. On the other hand, neutrophils exposed to leptin did not display detectable Ca 2+ ions mobilization or β 2 -integrin upregulation. In this study, we investigated the effect of leptin on the redox homeostasis in lymphocytes and neutrophils. Material and methods : Neutrophils and lymphocytes were isolated by density-gradient centrifugation of blood from healthy volunteers. Cells were cultured with or without leptin (100 ng/ml for lymphocytes and 500 ng/ml for neutrophils or with or without synovial fluid (85% for 0–72 h. Culture media were not changed during incubation. Cells were homogenized and homogenate was frozen until laboratory measurements. Redox homeostasis was assessed by the reduced glutathione (GSH vs. oxidized glutathione (GSSG ratio and membrane lipid peroxidation evaluation. Results : Lymphocytes cultured with leptin and synovial fluid showed a significant increase of the GSSG level. The GSSG/GSH ratio increased by 184 ±37%. In neutrophils incubated in a similar environment, the GSSG/GSH ratio increased by just 21 ±7%, and the effect was observed irrespectively of whether they were exposed to leptin or synovial fluid or both together. Neither leptin nor synovial fluid influenced lipid peroxidation in neutrophils, but in lymphocytes leptin intensified lipid peroxidation. Conclusions : Leptin altered the lymphocytes, but not neutrophils redox state. Because firstly neutrophils are anaerobic cells and have just a few mitochondria and secondly lymphocytes have typical aerobic metabolism, the divergence of our data supports the hypothesis that leptin induces oxidative stress by

Radiation inactivation of animal viruses in culture fluid and sewage; a case study

International Nuclear Information System (INIS)

Groneman, A.F.; Frenkel, S.; Terpstra, C.

1977-01-01

Inactivation studies of different animal viruses were performed with gamma irradiation from a 60 Co-source to evaluate the technical and economic feasibility of sterilization of sewage of a veterinary institute involved in research on virus diseases and the production of virus vaccines. The D 10 values for swine fever virus, foot-and-mouth disease virus (FMDV) and swine vesicular disease virus (SVDV) irradiated in culture medium at 0degC were 1.8, 4.5, and 5.9 kGy (0.18, 0.45, and 0.59 Mrad), respectively. Suspensions of SVDV and FMDV were mixed with raw sludge and irradiated at 8degC. Raw sludge had a protecting effect on FMDV, if compared to culture fluid, increasing the D 10 value significantly to 6.5 kGy (0.65 Mrad). No similar protective effect was observed in the case of SVDV. Addition of 0.2 M NaBr did not significantly increase the radiosensitivity of these two viruses. The technical and economic feasibility for sterilization of sewage and sludge by 60 Co-gamma irradiation are discussed

Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

Science.gov (United States)

Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

2015-01-01

The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

Bile culture

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Culture - bile ... is placed in a special dish called a culture medium to see if bacteria, viruses, or fungi ... Chernecky CC, Berger BJ. Body fluid - anaerobic culture. In: ... . 6th ed. St Louis, MO: Elsevier Saunders; 2013:225-226. Kim AY, ...

The assessment of cytokines in Quantiferon supernatants for the diagnosis of latent TB infection in a tribal population of Melghat, India.

Science.gov (United States)

Bapat, Prachi R; Husain, Aliabbas A; Daginawala, Hatim F; Agrawal, Neha P; Panchbhai, Milind S; Satav, Ashish R; Taori, Girdhar M; Kashyap, Rajpal S

2015-01-01

The tuberculin skin test (TST) and interferon-gamma release assays (IGRA), namely, the QuantiFERON-TB Gold test (QFT), remain the standard immunological diagnostic tools for latent tuberculosis (TB) infection (LTBI). However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma (IFN-γ) as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: (i) QFT+/TST+ (n = 26), (ii) QFT+/TST- (n = 12), (iii) QFT-/TST- (n = 35) and (iv) QFT-/TST+ (n = 1) groups. A panel of cytokines (IL-6, IL-10, TNF-α and IL-2R) was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT-/TST- group. Correlation analysis identified a positive correlation between IL-6 and the QFT response (r = 0.6723, P < 0.0001), while a negative correlation was seen between QFT and IL-10 expression (r = -0.3271, P = 0.0044). Similarly, IL-6 was positively correlated with TST levels (r = 0.6631, P <0 .0001), and conversely, a negative correlation was found between TST and IL-10 expression (r = -0.5698, P < 0.0001). The positive and negative predictive values of IL-6 were found to be 92.59 and 93.33%, respectively, and the positive and negative predictive values of IL-10 were 96.55 and 91.18%, respectively. No significant impact of the demographic characteristics on cytokine positivity was observed. Our preliminary results suggest that the evaluation of additional cytokines in QFT cell-free culture supernatants may be valuable for the identification of LTBI. Combining IL-6 and IL-10 with QFT and/or TST could markedly improve the detection

Supernatants from oral epithelial cells and gingival fibroblasts modulate human immunodeficiency virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages.

Science.gov (United States)

González, O A; Ebersole, J L; Huang, C B

2010-04-01

Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes

The Etiology, Incidence, and Impact of Preservation Fluid Contamination during Liver Transplantation.

Science.gov (United States)

Oriol, Isabel; Lladó, Laura; Vila, Marina; Baliellas, Carme; Tubau, Fe; Sabé, Núria; Fabregat, Joan; Carratalà, Jordi

2016-01-01

The role of contaminated preservation fluid in the development of infection after liver transplantation has not been fully elucidated. To assess the incidence and etiology of contaminated preservation fluid and determine its impact on the subsequent development of infection after liver transplantation, we prospectively studied 50 consecutive liver transplants, and cultured the following samples in each instance: preservation fluid (immediately before and at the end of the back-table procedure, and just before implantation), blood, and bile from the donor, and ascitic fluid from the recipient. When any culture was positive, blood cultures were obtained and targeted antimicrobial therapy was started. We found that the incidence of contaminated preservation fluid was 92% (46 of 50 cases of liver transplantation per year), but only 28% (14/50) were contaminated by recognized pathogens. Blood and bile cultures from the donor were positive in 28% and 6% respectively, whereas ascitic fluid was positive in 22%. The most frequently isolated microorganisms were coagulase-negative staphylococci. In nine cases, the microorganisms isolated from the preservation fluid concurred with those grown from the donor blood cultures, and in one case, the isolate matched with the one obtained from bile culture. No liver transplant recipient developed an infection due to the transmission of an organism isolated from the preservation fluid. Our findings indicate that contamination of the preservation fluid is frequent in liver transplantation, and it is mainly caused by saprophytic skin flora. Transmission of infection is low, particularly among those recipients given targeted antimicrobial treatment for organisms isolated in the preservation fluid.

Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

DEFF Research Database (Denmark)

Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

2011-01-01

in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

Comparison of MGIT and Myco/F lytic liquid-based blood culture systems for recovery of Mycobacterium tuberculosis from pleural fluid.

Science.gov (United States)

Harausz, Elizabeth; Lusiba, John Kafuluma; Nsereko, Mary; Johnson, John L; Toossi, Zahra; Ogwang, Sam; Boom, W Henry; Joloba, Moses L

2015-04-01

The specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery of Mycobacterium tuberculosis from pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively; P=0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Selection for autochthonous bifidobacteial isolates adapted to simulated gastrointestinal fluid

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H Jamalifar

2010-03-01

Full Text Available "nBackground and the purpose of the study: Bifidobacterial strains are excessively sensitive to acidic conditions and this can affect their living ability in the stomach and fermented foods, and as a result, restrict their use as live probiotic cultures. The aim of the present study was to obtain bifidobacterial isolates with augmented tolerance to simulated gastrointestinal condition using cross-protection method. "nMethods: Individual bifidobacterial strains were treated in acidic environment and also in media containing bile salts and NaCl. Viability of the acid and acid-bile-NaCl tolerant isolates was further examined in simulated gastric and small intestine by subsequent incubation of the probiotic bacteria in the corresponding media for 120 min. Antipathogenic activities of the adapted isolates were compared with those of the original strains. "nResults and major conclusion: The acid and acid-bile-NaCl adapted isolates showed improved viabilities significantly (p<0.05 in simulated gastric fluid compared to their parent strains. The levels of reduction in bacterial count (Log cfu/ml of the acid and acid-bile-NaCl adapted isolates obtained in simulated gastric fluid ranged from 0.64-3.06 and 0.36-2.43 logarithmic units after 120 min of incubation. There was no significant difference between the viability of the acid-bile-NaCl-tolerant isolates and the original strains in simulated small intestinal condition except for Bifidobacterium adolescentis (p<0.05. The presence of 15 ml of supernatants of acid-bile-NaCl-adapted isolates and also those of the initial Bifidobacterium strains inhibited pathogenic bacterial growth for 24 hrs. Probiotic bacteria with improved ability to survive in harsh gastrointestinal environment could be obtained by subsequent treatment of the strains in acid, bile salts and NaCl environments.

Modelling antagonic effect of lactic acid eacteria supernatants on some pathogenic bacteria

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Augustus Caeser Franke Portella

2009-11-01

Full Text Available This work presents a statistical model of survival analysis for three pathogenic bacterial strains (Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, when treated with neutralized and non-neutralized filtered supernatants broth from cultures of Lactobacillus acidhophilus, Lactobacillus rhamnosus and Lactobacillus sake. Survival analysis is a method employed to determine the period of time from an initial stage up to the occurrence of a particular event of interest, as death or a particular culture growth failure. In order to evaluate the potential efficacy of the ahead mentioned lactic acid bacteria when used as bioprotective starters in foods, experimental data were statistically treated and expressed by simple representative curves. Following the methodology of Cox and Kaplan-Meier, it was possible to make the selection of the best bioprotective lactic starter, as a predictive tool for evaluation of shelf life and prevention of eventual risks in fresh sausages and other similar food products.Este trabalho apresenta um modelo estatístico de análise de sobrevivência para três bactérias patogénicas (Escherichia coli, Listeria monocytogenes e Staphylococcus aureus, quando tratados com sobrenadantes filtrados neutralizado e não neutralizado provenientes de culturas de Lactobacillus acidhophilus, Lactobacillus rhamnosus e Lactobacillus sake. A Análise de sobrevivência é um método utilizado para determinar o período de tempo a partir de uma fase inicial até a ocorrência de um determinado evento de interesse, como a morte ou a inibição de uma particular cultura, a fim de avaliar a eficácia potencial das referidas bactérias lácticas quando usadas como bioproteção em alimentos. Os dados experimentais foram tratados estatisticamente, seguindo a metodologia de Cox e Kaplan-Meier e foi possível fazer a seleção dos melhores fermentos láticos bioprotectivos, como uma ferramenta para avaliação preditiva, vida de

Application of FTA technology to extraction of sperm DNA from mixed body fluids containing semen.

Science.gov (United States)

Fujita, Yoshihiko; Kubo, Shin-ichi

2006-01-01

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA card is available for application to routine work.

The study of the peptide composition of the supernatants from mealworm Tenebrio molitor larvae and goldfish Carassius auratus during cold acclimation

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А. К. Гулевский

2016-11-01

Full Text Available The molecular-mass distribution of peptides from supernatants, obtained from the tissues of larvae Tenebrio molitor and goldfish Carassius auratus during cold acclimation, has been determined by chromatography. The results showed that peptide spectrum of the supernatants from larvae T. molitor and C. auratus varied during cold acclimation. The supernatants from non-acclimated larvae of T. molitor and deacclimated fish possessed the highest number of peptide fractions. Furthermore, the cold-acclimated larvae of T. molitor had the peptide fractions of the low molecular weight (ca. 5.4×102 ÷22.6×102 Da, and non-acclimated insects had the peptides of the high molecular weight (ca. 46.8×102÷66×102 Da. Next, the organ-specific changes of the peptide composition of the goldfish during winter deacclimation have been revealed. Specifically, the low molecular weight peptides (ca. (14.1 ± 0.3×102 and (6.75 ± 0.25×102 Da, have been detected in the C. auratus muscles, and both the high (ca. (67.83 ± 0.21×102 ( ca. 64.16 ± 0.26×102 Da and low (ca. (34.1 ± 1.0×102 and (14.29 ± 0.15×102 Da molecular weight peptides have been detected in the liver. Quantitative and qualitative changes in the peptide spectra from supernatants of the T. molitor and C. auratus during cold acclimation could be one of the mechanisms of their natural adaptation to low temperatures.

Porphyromonas gingivalis displays a competitive advantage over Aggregatibacter actinomycetemcomitans in co-cultured biofilm.

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Takasaki, K; Fujise, O; Miura, M; Hamachi, T; Maeda, K

2013-06-01

Biofilm formation occurs through the events of cooperative growth and competitive survival among multiple species. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are important periodontal pathogens. The aim of this study was to demonstrate competitive or cooperative interactions between these two species in co-cultured biofilm. P. gingivalis strains and gingipain mutants were cultured with or without A. actinomycetemcomitans. Biofilms formed on glass surfaces were analyzed by crystal violet staining and colony counting. Preformed A. actinomycetemcomitans biofilms were treated with P. gingivalis culture supernatants. Growth and proteolytic activities of gingipains were also determined. Monocultured P. gingivalis strains exhibited a range of biofilm-formation abilities and proteolytic activities. The ATCC33277 strain, noted for its high biofilm-formation ability and proteolytic activity, was found to be dominant in biofilm co-cultured with A. actinomycetemcomitans. In a time-resolved assay, A. actinomycetemcomitans was primarily the dominant colonizer on a glass surface and subsequently detached in the presence of increasing numbers of ATCC33277. Detachment of preformed A. actinomycetemcomitans biofilm was observed by incubation with culture supernatants from highly proteolytic strains. These results suggest that P. gingivalis possesses a competitive advantage over A. actinomycetemcomitans. As the required biofilm-formation abilities and proteolytic activities vary among P. gingivalis strains, the diversity of the competitive advantage is likely to affect disease recurrence during periodontal maintenance. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Struvite Crystallization of Anaerobic Digestive Fluid of Swine Manure Containing Highly Concentrated Nitrogen

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Eun Young Lee

2015-07-01

Full Text Available In this study, the optimal operation factors for struvite crystallization for removing and recovering nitrogen and phosphorus from anaerobic digestive fluid of swine manure containing highly concentrated nitrogen was determined. Every experiment for the struvite crystallization reaction was conducted by placing 1,000 mL of digestion fluid in a 2,000 mL Erlenmeyer flask at various temperatures, pH, and mixing speed. Except for special circumstances, the digestion fluid was centrifuged (10,000 rpm, 10 min and then the supernatant was used for the experiment at room temperature and 100 rpm. The optimal mole ratio of PO43−:Mg2+ was 1:1.5, and the pH effect ranging from 9 to 11 was similar, when mixed for 1 hour. Under this condition, the removal efficiency of NH4+-N and PO43−-P was 40% and 88.6%, respectively. X-shaped crystal was observed by light and scanning electron microscopy. In addition, struvite crystal structure was confirmed through X-ray diffraction analysis.

Phosphate removal from digested sludge supernatant using modified fly ash.

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Xu, Ke; Deng, Tong; Liu, Juntan; Peng, Weigong

2012-05-01

The removal of phosphate in digested sludge supernatant by modified coal fly ash was investigated in this study. Modification of the fly ash by the addition of sulfuric acid could significantly enhance its immobilization ability. The experimental results also showed that adsorption of phosphate by the modified fly ash was rapid with the removal percentage of phosphate reaching an equilibrium of 98.62% in less than 5 minutes. The optimum pH for phosphate removal was 9 and the removal percentage increased with increasing adsorbent dosage. The effect of temperature on phosphate removal efficiency was not significant from 20 to 40 degrees C. X-ray diffraction and scanning electron microscope analyses showed that phosphate formed an amorphous precipitate with water-soluble calcium, aluminum, and iron ions in the modified fly ash.

Dynamics of the Content of Lactobacilli, Microbial Metabolites and Antimicrobial Activity of Growing Culture of Lactobacillus Plantarum 8P-A3

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I. Yu. Chicherin

2013-01-01

Full Text Available The dynamics of the content of lactobacilli, microbial metabolites and antimicrobial activity of growing cultures of Lactobacillus plantarum 8Р-А3 was studied. Lactobacilli L. plantarum 8Р-А3 and test microorganisms isolated from the intestinal contents of patients with dysbacteriosis were used in experiments. Study of the component composition of growing culture supernatant of lactobacilli was carried out by gas liquid chromatography with mass selective detection. By 54 h of cultivation the content of viable microbial cells in the native culture of Lactobacillus achieves 3,0·109 in 1 mL without further increase during the cultivation. The principal component of lactobacilli culture medium possessing antibacterial activity is lactic acid. In addition to lactic acid, which accounts for 70% of the total metabolites, the culture medium and the supernatant contain salts of phosphoric acid (14% as well as amino acids, carboxylic acids, fatty acids, sugars and polyhydric alcohol constituting of up to 16% of the total metabolites. It is found that during the cultivation in liquid medium lactobacilli produce metabolites which possess antibacterial activity against pathogenic bacteria that cause intestinal infections.

Phenotypic and Genetic Evaluation of the Influence of Pseudomonas aeruginosa Culture Fractions on the Human Mesenchymal Stem Cells Viability, Apoptotic Pathways and Cytokine Profile.

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Holban, Alina Maria; Bleotu, Coralia; Chifiriuc, Mariana Carmen; Lazar, Veronica

2017-01-01

The objective of this study was to investigate the effects of P. aeruginosa PAO1 cellular and soluble culture fractions on human mesenchymal stem cells (MSCs) death signaling pathways and cytokine profile. The bone marrow isolated MSCs, incubated for different periods of time with one of the three P. aeruginosa PAO1 culture fractions, i.e. low density whole cultures, heat inactivated bacterial cultures sediments and sterile supernatants, were submitted to the following assays: i) fluorescence microscopy evaluation of cellular morphology and viability; ii) bax, caspase 9, relA and bcl-2 genes expression analysis by qRT-PCR; and iii) quantification of the level of IL-1β, IL-6, IL-8 and IL-10 cytokines released in the MSCs supernatants determined by ELISA. Results were statistically analyzed using the GraphPad In Stat software. The PAO1 whole cultures exhibited the most relevant influences, impacting on MSCs morphology and viability, interfering with apoptotic pathways and significantly stimulating the production of IL-1β and IL-10, while decreasing the production of IL-6 and IL-8. The culture supernatants increased the production of IL-1β and reduced the secretion of all other tested cytokines, while heat-inactivated bacterial cells significantly stimulated both IL-1β and IL-10 production. These data could suggest that in vivo, the fate of P. aeruginosa infection depends on the proportion between different bacterial culture fractions (i.e. the number of viable bacterial cells, the number of dead cells and the amount of bacterial soluble products accumulated locally) that could be influenced by the initial infective dose, by the host defense mechanisms, and also by the administered antimicrobial treatment that may thus interfere with the evolution and magnitude of the induced lesions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

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Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

2017-01-01

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

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W. P. Lokapirnasari

2015-03-01

Full Text Available Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4-β-D-glucanase, exo-(1,4-β-Dglucanase, and β-glucosidase, at optimum temperature and optimum pH of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase and β-glucosidase.

Cellular Biotechnology Operations Support System Fluid Dynamics Investigation

Science.gov (United States)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Medium (TCM) is the bioreactor vessel in which cell cultures are grown. With its two syringe ports, it is much like a bag used to administer intravenous fluid, except it allows gas exchange needed for life. The TCM contains cell culture medium, and when frozen cells are flown to the ISS, they are thawed and introduced to the TCM through the syringe ports. In the Cellular Biotechnology Operations Support System-Fluid Dynamics Investigation (CBOSS-FDI) experiment, several mixing procedures are being assessed to determine which method achieves the most uniform mixing of growing cells and culture medium.

Comparison of BacT/Alert FAN and FAN Plus Bottles with Conventional Medium for Culturing Cerebrospinal Fluid.

Science.gov (United States)

Yoo, In Young; Chun, Sejong; Song, Dong Joon; Huh, Hee Jae; Lee, Nam Yong

2016-11-01

We compared the BacT/Alert system FAN and FAN Plus media to conventional media for culturing cerebrospinal fluid (CSF) with 2,545 samples. FAN/FAN Plus bottles showed better performance for isolating microorganisms in CSF than conventional media (positive rate, 7.2% [182/2,545] versus 3.1% [80/2,545]). The incremental recovery rate of Cryptococcus neoformans from FAN Plus bottles was higher than that from FAN bottles. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The assessment of cytokines in Quantiferon supernatants for the diagnosis of latent TB infection in a tribal population of Melghat, India

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Prachi R. Bapat

2015-07-01

Full Text Available Summary: The tuberculin skin test (TST and interferon-gamma release assays (IGRA, namely, the QuantiFERON-TB Gold test (QFT, remain the standard immunological diagnostic tools for latent tuberculosis (TB infection (LTBI. However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma (IFN-γ as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: (i QFT+/TST+ (n = 26, (ii QFT+/TST− (n = 12, (iii QFT−/TST− (n = 35 and (iv QFT−/TST+ (n = 1 groups. A panel of cytokines (IL-6, IL-10, TNF-α and IL-2R was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT−/TST− group. Correlation analysis identified a positive correlation between IL-6 and the QFT response (r = 0.6723, P < 0.0001, while a negative correlation was seen between QFT and IL-10 expression (r = −0.3271, P = 0.0044. Similarly, IL-6 was positively correlated with TST levels (r = 0.6631, P < 0.0001, and conversely, a negative correlation was found between TST and IL-10 expression (r = −0.5698, P < 0.0001. The positive and negative predictive values of IL-6 were found to be 92.59 and 93.33%, respectively, and the positive and negative predictive values of IL-10 were 96.55 and 91.18%, respectively. No significant impact of the demographic characteristics on cytokine positivity was observed. Our preliminary results suggest that the evaluation of additional cytokines in QFT cell-free culture supernatants may be valuable for the identification of LTBI. Combining IL-6 and IL-10 with QFT and

Food waste co-digestion with slaughterhouse waste and sewage sludge: Digestate conditioning and supernatant quality.

Science.gov (United States)

Borowski, Sebastian; Boniecki, Paweł; Kubacki, Przemysław; Czyżowska, Agata

2018-04-01

In this study, the anaerobic mesophilic co-digestion of food waste (FW) with municipal sewage sludge (MSS) and slaughterhouse waste (SHW) was undertaken in 3-dm 3 laboratory reactors as well as in 50-dm 3 reactors operated in semi-continuous conditions. The highest methane yield of around 0.63 m 3 CH 4 /kgVS fed was achieved for the mixture of FW and SHW treated in the laboratory digester operated at solids retention time (SRT) of 30 days, whereas the co-digestion of FW with MSS under similar operating conditions produced 0.46 m 3 of methane from 1 kgVS fed . No significant differences between methane yields from laboratory digesters and large-scale reactors were reported. The conditioning tests with the digestates from reactor experiments revealed the highest efficiency of inorganic coagulants among all investigated chemicals, which applied in a dose of 10 g/kg allowed to reduce capiliary suction time (CST) of the digestate below 20 s. The combined conditioning with coagulants and bentonite did not further reduce the CST value but improved the quality of the digestate supernatant. In particular, the concentrations of suspended solids, COD as well as metals in the supernatant were considerably lowered. Copyright © 2017. Published by Elsevier Ltd.

The application of cell cultures, body fluids and tissues in oncoproteomics

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Kamila Duś-Szachniewicz

2014-11-01

Full Text Available Mass spectrometry (MS-based proteomics is a rapidly developing technology for the large scale analysis of proteins, their interactions and subcellular localization. In recent years proteomics has attracted much attention in medicine. Since a single biomarker might not have sufficient sensitivity and specificity in clinical practice, the identification of biomarker panels that comprise several proteins would improve the detection and clinical management of cancer patients. Additionally, the characteristics of protein profiles of most severe human malignancies certainly contribute to the understanding of the biology of cancer and fill the gap in our knowledge of carcinogenesis. This knowledge also is likely to result in the discovery of novel potential cancer markers and targets for molecular therapeutics. It is believed that the novel biomarkers will help in the development of personalized therapy tailored to the individual patient and will thereby reduce the mortality rate from cancer. In this review, the use of different types of human clinical samples (cell cultures, tissues and body fluids in oncoproteomics is explained and the latest advances in mass spectrometry-based proteomics biomarker discovery are discussed.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

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Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

2018-02-01

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

Validity of antibodies in lymphocyte supernatant in diagnosing tuberculosis in severely malnourished children presenting with pneumonia.

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Mohammod Jobayer Chisti

Full Text Available The diagnosis of tuberculosis (TB in young children can be challenging, especially in severely malnourished children. There is a critical need for improved diagnostics for children. Thus, we sought to evaluate the performance of a technique that measures antibodies in lymphocyte supernatant (ALS for the diagnosis of TB in severely malnourished children presenting with suspected pneumonia.Children less than 5 years with severe acute malnutrition and radiological features of pneumonia admitted to the Dhaka Hospital of International Centre for Diarrhoeal Disease Research, Bangladesh, were enrolled consecutively following informed written consent. In addition to clinical and radiological assessment, samples taken for TB diagnosis included gastric lavage fluid and induced sputum for microbiological confirmation. ALS was measured from venous blood, and results were evaluated in children classified as "confirmed", "non-confirmed TB" or "not TB".Among 224 children who had ALS analysis, 12 (5.4% children had microbiologically "confirmed TB", a further 41 (18% had clinically diagnosed "non-confirmed TB" and the remaining 168 (75% were considered not to have TB. ALS was positive in 89 (40% and negative in 85 (39% of children, with a large number (47 or 21% reported as "borderline". These proportions were similar between the three diagnostic groups. The sensitivity and specificity of ALS when comparing "Confirmed TB" to "Not TB" was only 67% (95% CI: 31-91% and 51% (95% CI: 42-60%, respectively.Our data suggest that ALS is not sufficiently accurate to improve the diagnosis of TB in children with severe malnutrition.

Clinical impact of culture-positive preservation fluid on solid organ transplantation: A systematic review and meta-analysis.

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Oriol, Isabel; Sabé, N; Tebé, C; Veroux, M; Boin, I F S F; Carratalà, J

2018-04-01

Contamination of the preservation fluid (PF) used for donated organs is a potential source of post-transplant infection. However, the information on this issue is scarce. We therefore conducted a systematic review and meta-analysis to assess the incidence of culture-positive PF and its impact on solid organ transplant (SOT) recipients. Seventeen studies were identified and included. The overall incidence of culture-positive PF was 37% (95% CI: 27% to 49%), and the incidence of PF-related infections among SOT recipients with PF cultures that grew pathogenic microorganisms was 10% (95% CI: 7% to 15%). There were differences in the rates of infections due to pathogenic microorganisms between SOT recipients who received pre-emptive treatment and those who did not, but without statistical significance. The mortality rate among SOT recipients with PF-related infection was 35% (95% CI: 21% to 53%). In conclusion, although contamination of the PF of donated organs is frequent, the incidence of PF-related infection is relatively low. A closely clinical and microbiologic monitoring of the SOT recipient in case of culture-positive PF, regardless of the type of microorganism isolated might be do in order to establish a prompt diagnosis of PF-related infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and localization of a soluble antigen, Ag2, of 136 kDa from Plasmodium falciparum in vitro cultures

DEFF Research Database (Denmark)

Jakobsen, P H; Grellier, P; Theander, T G

1991-01-01

The soluble antigens, antigen 2 (Ag2) and antigen 6 (Ag6), were copurified from supernatants of P. falciparum in vitro cultures by affinity chromatography and Fast Protein Liquid Chromatography. Rabbit antibodies to Ag2 were raised and characterized by crossed immunoelectrophoresis. Ag2 appeared...

Cerebral spinal fluid (CSF) collection

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... Ventricular puncture; Lumbar puncture; Cisternal puncture; Cerebrospinal fluid culture ... the meaning of your specific test results. The examples above show the common measurements for results for ...

[Detection of metals and metalloids in the lavage fluid of whole-lung lavage of the cases of pneumoconiosis].

Science.gov (United States)

Wen, K; Ding, C G; Chen, G; Ma, G X; Wang, H Q

2017-11-20

Objective: To detect of the components and concentration of the metals and metalloids in the lavage fluid of whole-lung lavage (WLL) of the cases of pneumoconiosis, and analyze the characteristics, and explore the method to sample and process the samples of bronchoalveolar lavage fluid (BAL) . Methods: The samples of urine and serum of three cases of pneumoconiosis were collected before WLL, and the samples of BAL were collected during the WLL from the left and right lungs according to the sequence of four pressured gas flow and five negative pressure drainage. Each of 10ml original samples of WLL was collected firstly, and the left was centrifuged to acquire all the sediment samples and each of 10 ml samples from the centrifuge clear liquids, The components and concentration of the metals and metalloids in the samples were measured by Inductively Coupled Plasma mass spectrometer (ICP-MS) . Results: The average volume of BAL from unilat-eral lung for 3 patients was 10 758.3±1518 ml, and the average recovery rate was 89.7%. The average dry weight of sediment samples of BAL of three cases of pneumoconiosis was 0.292 gram with the right lung sam-ples slightly higher than the left lung samples. The detectable elements from the samples included Barium (Ba) , Strontium (Sr) , Calcium (Ca) , Magnesium (Mg) , Manganum (Mn) , Ferrum (Fe) , Cuprum (Cu) , Zinc (Zn) , Kalium (K) , Natrium (Na) , Selenium (Se) , Silicon (Si) and Uranium (U) . Each of concentration dis-tributions of these elements were not normal. Except for Cuprum, Selenium and Uranium, the concentrations of the other ten elements in the supernatant samples, mixture samples and sediments samples were statistical-ly different with the nonparametric test of Kruskal-Wallis. The concentrations of Natrium, Kalium and Barium in supernatant samples were higher, while the others in precipitation samples were higher. The concentration of elements in the sample from the right lung was slightly higher than that from the

Diagnosing feline infectious peritonitis using the Sysmex XT-2000iV based on frozen supernatants from cavitary effusions.

Science.gov (United States)

Stranieri, Angelica; Paltrinieri, Saverio; Giordano, Alessia

2017-05-01

The delta total nucleated cells (ΔTNC) measurement with the Sysmex XT-2000iV (Sysmex Europe, Norderstedt, Germany) has high diagnostic accuracy on effusions in feline infectious peritonitis (FIP) cases, but the test can be performed only on fresh samples. We evaluated whether supernatants from effusions retain the ability to induce cell clumping and assessed the diagnostic accuracy of this modified ΔTNC method. Effusions were collected from FIP cats ( n = 19) and from cats with other diseases ( n = 15). ΔTNC was measured on fresh samples and on frozen-thawed supernatants after the addition of feline blood at 1:10 dilution. Diagnostic accuracy was assessed at the cutoffs of suggestive of FIP (ΔTNC = 1.7) and consistent with FIP (ΔTNC = 3.4). The influence of the protein content, number of added cells, and magnitude of dilution were also investigated. Specificity and positive predictive value were 100% for both the methods. Sensitivity and negative predictive value were higher for the modified ΔTNC (84.2% and 83.3%, respectively, at the cutoff of 1.7; 78.9% and 78.9%, respectively, at the cutoff of 3.4) than for the ΔTNC on fresh samples (78.6% and 81.3%, respectively, at the cutoff of 1.7; 57.1% and 68.4%, respectively, at the cutoff of 3.4). Protein content, total cell count of the added blood, and magnitude of dilutions did not influence the results. Supernatants of frozen effusions from FIP cats retain the ability to induce cell clumping, thus the modified ΔTNC measurement is a reliable tool to diagnose FIP on samples that cannot be analyzed immediately.

Recovery of urinary nanovesicles from ultracentrifugation supernatants.

Science.gov (United States)

Musante, Luca; Saraswat, Mayank; Ravidà, Alessandra; Byrne, Barry; Holthofer, Harry

2013-06-01

Urinary vesicles represent a newly established source of biological material, widely considered to faithfully represent pathological events in the kidneys and the urogenital epithelium. The majority of currently applied isolation protocols involve cumbersome centrifugation steps to enrich vesicles from urine. To date, the efficiency of these approaches has not been investigated with respect to performing quantitative and qualitative analyses of vesicle populations in the pellet and supernatant (SN) fractions. After the series of differential centrifugations, the final SN was reduced to one-twentieth of the original volume by ammonium sulphate precipitation, with the precipitate pellet subjected to another round of differential centrifugations. Electron microscopy, dynamic light scattering and western blot analysis were used to characterize the vesicles present in individual fractions of interest. Pellets obtained after the second set of centrifugations at 200 000 g revealed the presence of vesicles which share a common marker profile, but with distinct differences from those seen in the initial 200 000 g pellet used as the reference. This suggests an enrichment of previously uncharacterized urinary vesicles still in solution after the initial centrifugation steps. Analysis of protein yields recovered post-ultracentrifugation revealed an additional 40% of vesicles retained from the SN. Moreover, these structures showed a formidable resistance to harsh treatments (e.g. 95% ammonium sulphate saturation, hypotonic dialysis, 0.3 M sodium hydroxide). Methods which employ differential centrifugations of native urine are remarkably ineffective and may lose a substantial population of biologically important vesicle species.

Recovery of plutonium from oxalate supernatant using 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone

International Nuclear Information System (INIS)

Mohapatra, P.K.; Manchanda, V.K.; Gupta, K.K.; Singh, R.K.

1997-01-01

Extraction of Pu(IV) from oxalate supernatant was carried out employing varying concentrations of 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP). Near quantitative extraction of Pu(IV) from an aqueous solution of 0.2M oxalic acid and 3M HNO 3 was possible employing 0.05M PMBP solution in xylene. Extraction studies at different uranium loading conditions were carried out and conditions for quantitative stripping were arrived at. (author). 2 refs., 4 tabs

Microfluidic Cell Culture Device

Science.gov (United States)

Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

2014-01-01

Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

Ion exchange flowsheet for recovery of cesium from purex sludge supernatant at B Plant

International Nuclear Information System (INIS)

Carlstrom, R.F.

1977-01-01

Purex Sludge Supernatant (PSS) contains significant amounts of 137 Cs left after removal of strontium from fission product bearing Purex wastes. To remove cesium from PSS, an Ion Exchange Recovery system has been set up in Cells 17-21 at B Plant. The cesium that is recovered is stored within B Plant for eventual purification through the Cesium Purification process in Cell 38 and eventual encapsulation and storage in a powdered form at the Waste Encapsulation Storage Facility. Cesium depleted waste streams from the Ion Exchange processes are transferred to underground storage

Cell culture supernatants for detection perforin ELISA

African Journals Online (AJOL)

Najwa

2014-02-19

Feb 19, 2014 ... filtration chromatography using Sephadex G-150, and the study utilized an in vitro ... 1.73 U/mg in crude extract to 2.29 U/mg after ion-exchange and 10.5 U/mg ..... survival: application to proliferation and cytotoxicity assays .J.

Cell culture supernatants for detection perforin ELISA

African Journals Online (AJOL)

Najwa

2014-02-19

Feb 19, 2014 ... Leukemia is a cancer originating in any of hematopoietic cell that tends to ... treatment of children (Borek and Jaskolski, 2001). The current study was .... which led to the best results at 48 h of exposure than after 72 h of cells ...

A fast and simple dot-immunobinding assay for quantifiction of mouse immunoglobulins in hybridoma culture supernatants

Czech Academy of Sciences Publication Activity Database

Sulimenko, Tetyana; Dráber, Pavel

2004-01-01

Roč. 289, - (2004), s. 89-95 ISSN 0022-1759 R&D Projects: GA AV ČR IBS5052301; GA MŠk LN00A026; GA MŠk 1P04OE158 Keywords : dot-immunobinding assay * hybridoma culture superntatants * mouse immunoglobulins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.464, year: 2004

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

DEFF Research Database (Denmark)

Wang, Bijue; Chen, Pingping; Jensen, Anne-Christine Bay

2009-01-01

BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggreca......BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP......- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were...... cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo...

Performance of automated multiplex PCR using sonication fluid for diagnosis of periprosthetic joint infection: a prospective cohort.

Science.gov (United States)

Renz, Nora; Feihl, Susanne; Cabric, Sabrina; Trampuz, Andrej

2017-12-01

Sonication of explanted prostheses improved the microbiological diagnosis of periprosthetic joint infections (PJI). We evaluated the performance of automated multiplex polymerase chain reaction (PCR) using sonication fluid for the microbiological diagnosis of PJI. In a prospective cohort using uniform definition criteria for PJI, explanted joint prostheses were investigated by sonication and the resulting sonication fluid was analyzed by culture and multiplex PCR. McNemar's Chi-squared test was used to compare the performance of diagnostic tests. Among 111 patients, PJI was diagnosed in 78 (70%) and aseptic failure in 33 (30%). For the diagnosis of PJI, the sensitivity and specificity of periprosthetic tissue culture was 51 and 100%, of sonication fluid culture 58 and 100%, and of sonication fluid PCR 51 and 94%, respectively. Among 70 microorganisms, periprosthetic tissue culture grew 52 (74%), sonication fluid culture grew 50 (71%) and sonication fluid PCR detected 37 pathogens (53%). If only organisms are considered, for which primers are included in the test panel, PCR detected 37 of 58 pathogens (64%). The sonication fluid PCR missed 19 pathogens (predominantly oral streptococci and anaerobes), whereas 7 additional microorganisms were detected only by PCR (including Cutibacterium spp. and coagulase-negative staphylococci). The performance of multiplex PCR using sonication fluid is comparable to culture of periprosthetic tissue or sonication fluid. The advantages of PCR are short processing time (PCR, especially of low-virulent organisms.

Removal of hydrocarbon from refinery tank bottom sludge employing microbial culture.

Science.gov (United States)

Saikia, Rashmi Rekha; Deka, Suresh

2013-12-01

Accumulation of oily sludge is becoming a serious environmental threat, and there has not been much work reported for the removal of hydrocarbon from refinery tank bottom sludge. Effort has been made in this study to investigate the removal of hydrocarbon from refinery sludge by isolated biosurfactant-producing Pseudomonas aeruginosa RS29 strain and explore the biosurfactant for its composition and stability. Laboratory investigation was carried out with this strain to observe its efficacy of removing hydrocarbon from refinery sludge employing whole bacterial culture and culture supernatant to various concentrations of sand-sludge mixture. Removal of hydrocarbon was recorded after 20 days. Analysis of the produced biosurfactant was carried out to get the idea about its stability and composition. The strain could remove up to 85 ± 3 and 55 ± 4.5 % of hydrocarbon from refinery sludge when whole bacterial culture and culture supernatant were used, respectively. Maximum surface tension reduction (26.3 mN m(-1)) was achieved with the strain in just 24 h of time. Emulsification index (E24) was recorded as 100 and 80 % with crude oil and n-hexadecane, respectively. The biosurfactant was confirmed as rhamnolipid containing C8 and C10 fatty acid components and having more mono-rhamnolipid congeners than the di-rhamnolipid ones. The biosurfactant was stable up to 121 °C, pH 2-10, and up to a salinity value of 2-10 % w/v. To our knowledge, this is the first report showing the potentiality of a native strain from the northeast region of India for the efficient removal of hydrocarbon from refinery sludge.

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures.

Science.gov (United States)

Montville, T J; Parris, N; Conway, L K

1985-01-01

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs. PMID:4004207

Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response.

Directory of Open Access Journals (Sweden)

Emily C Moorefield

Full Text Available Amniotic fluid stem (AFS cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO and monocyte chemotactic protein (MCP family members as well as interleukin-6 (IL-6. AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α, MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and

Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

International Nuclear Information System (INIS)

Haubner, Frank; Leyh, Michaela; Ohmann, Elisabeth; Pohl, Fabian; Prantl, Lukas; Gassner, Holger G

2013-01-01

The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

Cellular changes in motor neuron cell culture produced by cytotoxic cerebrospinal fluid from patients with amyotrophic lateral sclerosis.

Science.gov (United States)

Gomez-Pinedo, U; Yáñez, M; Matías-Guiu, J; Galán, L; Guerrero-Sola, A; Benito-Martin, M S; Vela, A; Arranz-Tagarro, J A; García, A G

2014-01-01

The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Copyright © 2013 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

Determination of methylglyoxal-bis(guanylhydrazone) in body fluids by ion-pair chromatography.

Science.gov (United States)

Roboz, J; Wu, K T; Hart, R D

1980-01-01

Methylglyoxal-bis(guanylhydrazone), Methyl-G, is a potent antineoplastic agent currently undergoing Phase l clinical trials. Serum, ascitic and pleural fluids, and urine are deproteinized with methanol, supernatant is evaporated, residue is redissolved in the eluent, lipids are removed with carbon tetrachloride, and an aliquot of the aqueous layer injected into the chromatograph. Ethylglyoxal-bis(guanylhydrazone) (Ethyl-G) is the internal standard. The mobile phase is a mixture of an aqueous buffer (containing 0.004 M heptane and pentane sulfonic acid, 90%:10%, buffered to pH 3.5) and methanol (68%:32%). The ion-pair complex is retained on a micro Bondapak C18 column, eluted with a flow of 2.0 mL/min. Absorbance is measured at 280 nm. Detectability: 30 ng/mL (0.11 micro M) in serum, ascitic and pleural fluids, 300 ng/mL (1.1 micro M) in urine. Calibration curves (peak height ratios of Methyl-G/Ethyl-G plotted against known drug concentrations) were linear in the 0.1-30 microg/mL range. Correlation coefficinets were 0.999; coefficients of variation for reproducibility were less than 5%. Residual blood levels of Methyl-G persist for several days. Methyl-G was found to pass into ascitic fluid.

Mechanical stimulation of bone cells using fluid flow

NARCIS (Netherlands)

Huesa, C.; Bakker, A.D.

2012-01-01

This chapter describes several methods suitable for mechanically stimulating monolayers of bone cells by fluid shear stress (FSS) in vitro. Fluid flow is generated by pumping culture medium through two parallel plates, one of which contains a monolayer of cells. Methods for measuring nitric oxide

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

International Nuclear Information System (INIS)

Aoki, Shigehisa; Ikeda, Satoshi; Takezawa, Toshiaki; Kishi, Tomoya; Makino, Junichi; Uchihashi, Kazuyoshi; Matsunobu, Aki; Noguchi, Mitsuru; Sugihara, Hajime; Toda, Shuji

2011-01-01

Highlights: ► Late-onset peritoneal fibrosis leading to EPS remains to be elucidated. ► Fluid streaming is a potent factor for peritoneal fibrosis in PD. ► We focused on the prolonged effect of fluid streaming on mesothelial cell kinetics. ► A history of fluid streaming exposure promoted mesothelial proliferative activity. ► We have thus identified a potent new factor for late-onset peritoneal fibrosis. -- Abstract: Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial–mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination

Investigations on the mechanism of chlorpromazine phototoxicity: effects on lysosomes of cultured human fibroblasts

International Nuclear Information System (INIS)

Hasei, K.; Ichihashi, M.; Mojamdar, M.

1984-01-01

The effect of chlorpromazine (CPZ) and UVA on lysosomes of cultured normal human fibroblasts has been investigated. Acid phosphatase (ACPase) activity in 12000 g pellet of cells treated with CPZ (10 μg/ml) and UVA (6 x 10 4 J/m 2 ) was found to be decreased as compared with non-treated, CPZ or UVA treated control cells. This decrease, however, was not accompanied by a concomitant increase in ACPase activity in the 12000 g supernatant. The addition of Triton X-100 to cells pre-treated with CPZ + UVA resulted in only a moderate increase in ACPase activity of the 12000 g supernatant. ACPase activity of the cells incubated in media containing pre-irradiated CPZ was also found to be decreased. These results indicate that CPZ + UVA directly inactivate lysosomal enzymes, possibly without affecting the membrane. (author)

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids

Czech Academy of Sciences Publication Activity Database

Tishchenko, Galina; Šimůnek, Jiří; Bartoňová, Hana; Dušková, Jarmila; Dohnálek, Jan; Ponomareva, Evgenia; Tennikova, T.

2011-01-01

Roč. 879, č. 22 (2011), s. 2175-2178 ISSN 1570-0232 R&D Projects: GA ČR GA310/09/1407; GA ČR GA525/05/2584 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515 Keywords : sample preparation * culture fluids C. paraputrificum J4 * chitinases Subject RIV: EE - Microbiology, Virology Impact factor: 2.888, year: 2011

Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

DEFF Research Database (Denmark)

Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

2011-01-01

Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

Antifungal activity of streptomycetes isolated bentonite clay

Directory of Open Access Journals (Sweden)

V. P. Shirobokov

2016-12-01

Full Text Available Aim. To investigate the biological activity of streptomycetes, isolated from Ukrainian bentonite clay. Methods. For identification of the investigated microorganisms there were used generally accepted methods for study of morpho-cultural and biochemical properties and sequencing of 16Ѕ rRNA producer. Antagonistic activity of the strain was determined by agar diffusion and agar block method using gram-positive, gram-negative microorganisms and fungi. Results. Research of autochthonous flora from bentonite clay of Ukrainian various deposits proved the existence of stable politaxonomic prokaryotic-eukaryotic consortia there. It was particularly interesting that the isolated microorganisms had demonstrated clearly expressed antagonistic properties against fungi. During bacteriological investigation this bacterial culture was identified like representative of the genus Streptomyces. Bentonite streptomycetes, named as Streptomyces SVP-71, inagar mediums (agar block method inhibited the growth of fungi (yeast and mold; zones of growth retardation constituted of 11-36 mm, and did not affect the growth of bacteria. There were investigated the inhibitory effects of supernatant culture fluid, ethanol and butanol extracts of biomass streptomycetes on museum and clinical strains of fungi that are pathogenic for humans (Candida albicans, C. krusei, C. utilis, C. parapsilosis, C. tropicalis, C. kefir, S. glabrata, C. lusitaniae, Aspergillus niger, Mucor pusillus, Fusarium sporotrichioides. It has been shown that research antifungal factor had 100% of inhibitory effect against all fungi used in experiments in vitro. In parallel, it was found that alcohol extracts hadn’t influence to the growth of gram-positive and gram-negative bacteria absolutely. It was shown that the cultural fluid supernatant and alcoholic extracts of biomass had the same antagonistic effect, but with different manifestation. This evidenced about identity of antifungal substances

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

Directory of Open Access Journals (Sweden)

Sondergaard Bodil-Cecilie

2009-12-01

Full Text Available Abstract Background Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. Findings Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM and tumor necrosis factor alpha (TNFα. In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1 sandwich 342FFGVG-G2 ELISA, (2 competition NITEGE373ELISA (3 sandwich G1-NITEGE373 ELISA (4 competition 374ARGSV ELISA, and (5 sandwich 374ARGSV-G2 ELISA all detecting aggrecan fragments, and (6 sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1 aggrecanase-derived aggrecan fragments are released in the early (day 2-7 and mid phase (day 9-14 into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2 the release of NITEGE373 neo-epitopes are delayed compared to the corresponding 374ARGSV fragments, (3 the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4 the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21 only. Conclusion Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if

Effects of zirconium and nitrogen plasma immersion ion implantation on the electrochemical corrosion behavior of Mg–Y–RE alloy in simulated body fluid and cell culture medium

International Nuclear Information System (INIS)

Jamesh, Mohammed Ibrahim; Wu, Guosong; Zhao, Ying; Jin, Weihong; McKenzie, David R.; Bilek, Marcela M.M.; Chu, Paul K.

2014-01-01

Highlights: • Dual Zr and N plasma ion implantation are conducted on WE43Mg alloy. • Zr and N implanted WE43 (ZrN-WE43) enhanced corrosion resistance in cell culture medium. • ZrN-WE43 enhanced corrosion resistance in simulated body fluid (SBF). • ZrN-WE43 shows near capacitive impedance spectra in cell culture medium. • Calcium phosphate is formed on the corrosion product. - Abstract: The effects of dual Zr and N plasma immersion ion implantation (PIII) on the corrosion behavior of WE43Mg alloy are evaluated in simulated body fluid (SBF) and cell culture medium (cDMEM). Zr and N PIII improves the corrosion resistance of WE43 which exhibits smaller i corr , larger R 1 and R 2 , smaller CPE 2 , and larger phase angle maxima in SBF and cDMEM. The Zr and N PIII WE43 samples exhibit 12-folds decrease in i corr in SBF and 71-folds decrease in i corr with near capacitive EIS in cDMEM. Analysis of the corrosion products reveals calcium phosphate

Clinical significance of histologic chorioamnionitis with a negative amniotic fluid culture in patients with preterm labor and premature membrane rupture.

Directory of Open Access Journals (Sweden)

Jeong Woo Park

Full Text Available To evaluate the effect of histological chorioamnionitis (HCA with a negative amniotic fluid (AF culture on adverse pregnancy and neonatal outcomes and inflammatory status in the AF compartment in women with preterm labor or preterm premature rupture of membranes (PPROM.This is a retrospective cohort study of 153 women diagnosed as having a preterm labor or PPROM (20-34 weeks who delivered singleton gestations within 48 hours of amniocentesis. AF obtained through amniocentesis was cultured, and interleukin (IL-6, IL-8, and metalloproteinase-9 (MMP-9 levels were determined. The placentas were examined histologically.The prevalence of HCA with negative AF culture was 23.5% (36/153. The women with HCA but with a negative AF culture (group 2 and those with a positive AF culture (group 3 had a significantly lower mean gestational age at amniocentesis and delivery than those with a negative AF culture and without HCA (group 1. Women in group 3 had the highest levels of AF IL-6, IL-8, and MMP-9, followed by those in group 2, and those in group 1. Composite neonatal morbidity was significantly higher in groups 2 and 3 than in group 1, but this was no longer significant after adjusting for confounders caused mainly by the impact of gestational age.In the women who delivered preterm neonates, HCA with a negative AF culture was associated with increased risks of preterm birth, intense intra-amniotic inflammatory response, and prematurity-associated composite neonatal morbidity, and its risks are similar to the risk posed by positive AF culture.

Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

DEFF Research Database (Denmark)

Gräs, S; Ovesen, Per Glud; Andersen, A N

1994-01-01

Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicula...

Expression of Pluripotency and Oocyte-Related Genes in Single Putative Stem Cells from Human Adult Ovarian Surface Epithelium Cultured In Vitro in the Presence of Follicular Fluid

Directory of Open Access Journals (Sweden)

Irma Virant-Klun

2013-01-01

Full Text Available The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The majority of primitive oocyte-like cells was mononuclear and expressed several genes related to pluripotency and oocytes, including genes related to meiosis, although they did not express some important oocyte-specific genes. Our work reveals the presence of putative stem cells in the ovarian surface epithelium of women with premature ovarian failure.

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer.

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Motohiko Tokuhisa

Full Text Available Peritoneal metastasis is the most frequent type of recurrence in patients with gastric cancer (GC and is associated with poor prognosis. Peritoneal lavage cytology, used to evaluate the risk of peritoneal metastasis, has low sensitivity. Here, we assessed the diagnostic potential of exosomal miRNA profiles in peritoneal fluid for the prediction of peritoneal dissemination in GC. Total RNA was extracted from exosomes isolated from six gastric malignant ascites (MA samples, 24 peritoneal lavage fluid (PLF samples, and culture supernatants (CM of two human gastric carcinoma cell lines that differ in their potential for peritoneal metastasis. Expression of exosomal miRNAs was evaluated with Agilent Human miRNA microarrays and quantitative reverse transcription polymerase chain reaction (qRT-PCR. The microarray analysis indicated a low variability in the number and signal intensity of miRNAs detected among the samples. In the six MA fluids, miR-21 showed the highest signal intensity. We identified five miRNAs (miR-1225-5p, miR-320c, miR-1202, miR-1207-5p, and miR-4270 with high expression in MA samples, the PLF of serosa-invasive GC, and the CM of a highly metastatic GC cell line; these candidate miRNA species appear to be related to peritoneal dissemination. Differential expression of miR-21, miR-320c, and miR-1225-5p was validated in the PLF of serosa-invasive and non-invasive GC by qRT-PCR and miR-21 and miR-1225-5p were confirmed to be associated with serosal invasion in GC. PLF can be used to profile the expression of exosomal miRNAs. Our findings suggest that miR-21 and miR-1225-5p may serve as biomarkers of peritoneal recurrence after curative GC resection, thus providing a novel approach to early diagnosis of peritoneal dissemination of GC.

Proteomic analysis of human blastocoel fluid and blastocyst cells

DEFF Research Database (Denmark)

Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

2013-01-01

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required......, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...... from the ICM of the human blastocyst are exposed to via the blastocoel fluid. These results can be an inspiration for the development of improved culture conditions for hESCs....

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition

OpenAIRE

Liu, Jing; Saponjian, Yero; Mahoney, Mark M.; Staley, Kevin J.; Berdichevsky, Yevgeny

2017-01-01

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic ...

Hip Synovial Fluid Cell Counts in Children From a Lyme Disease Endemic Area.

Science.gov (United States)

Dart, Arianna H; Michelson, Kenneth A; Aronson, Paul L; Garro, Aris C; Lee, Thomas J; Glerum, Kimberly M; Nigrovic, Peter A; Kocher, Mininder S; Bachur, Richard G; Nigrovic, Lise E

2018-05-01

Patients with septic hip arthritis require surgical drainage, but they can be difficult to distinguish from patients with Lyme arthritis. The ability of synovial fluid white blood cell (WBC) counts to help discriminate between septic and Lyme arthritis of the hip has not been investigated. We assembled a retrospective cohort of patients ≤21 years of age with hip monoarticular arthritis and a synovial fluid culture obtained who presented to 1 of 3 emergency departments located in Lyme disease endemic areas. Septic arthritis was defined as a positive synovial fluid culture result or synovial fluid pleocytosis (WBC count ≥50 000 cells per µL) with a positive blood culture result. Lyme arthritis was defined as positive 2-tiered Lyme disease serology results and negative synovial fluid bacterial culture results. All other patients were classified as having other arthritis. We compared median synovial fluid WBC counts by arthritis type. Of the 238 eligible patients, 26 (11%) had septic arthritis, 32 (13%) had Lyme arthritis, and 180 (76%) had other arthritis. Patients with septic arthritis had a higher median synovial fluid WBC count (126 130 cells per µL; interquartile range 83 303-209 332 cells per µL) than patients with Lyme arthritis (53 955 cells per µL; interquartile range 33 789-73 375 cells per µL). Eighteen patients (56%) with Lyme arthritis had synovial fluid WBC counts ≥50 000 cells per µL. Of the 94 patients who underwent surgical drainage, 13 were later diagnosed with Lyme arthritis. In Lyme disease endemic areas, synovial fluid WBC counts cannot always help differentiate septic from Lyme arthritis. Rapid Lyme diagnostics could help avoid unnecessary operative procedures in patients with Lyme arthritis. Copyright © 2018 by the American Academy of Pediatrics.

Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

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Alaín González Pose

2014-09-01

Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa

OpenAIRE

Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

2015-01-01

Background: The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. Objectives: In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) aga...

Making Material and Cultural Connections: the fluid meaning of ‘Living Electrically’ in Japan and Canada 1920–1960

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Heather Chappells

2018-05-01

Full Text Available This article explores how material and cultural ‘connections’ to households were crucial in defining historical pathways of domestic electrification in Canada and Japan. Though relatively overlooked in the history of energy, we contend that the intricacies of household connections are important in understanding convergence and divergence in electrified ways of life. Electricity’s arrival in urban and rural homes was an uneven process in part due to geographical diversity and the variable connection policies of public or private providers. Selective insights from our two countries show that spatial irregularities in grid expansion and social inequities in the meaning of electrical service further arose out of misalignments between developers’ visions and the diverse material cultures of households. This disparity manifested in the form of disconnections between non-standard wiring and equipment, as well as in the form of consumer resistance, protests and even illicit acts as people fought to get the electrical service they desired. Instead of seeing electrification as a single, universal ideal around which all countries eventually converged, we conclude that it has always been an open-ended, negotiable and culturally fluid process.

Host markers in Quantiferon supernatants differentiate active TB from latent TB infection: preliminary report

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Walzl Gerhard

2009-05-01

Full Text Available Abstract Background Interferon gamma release assays, including the QuantiFERON® TB Gold In Tube (QFT have been shown to be accurate in diagnosing Mycobacterium tuberculosis infection. These assays however, do not discriminate between latent TB infection (LTBI and active TB disease. Methods We recruited twenty-three pulmonary TB patients and 34 household contacts from Cape Town, South Africa and performed the QFT test. To investigate the ability of new host markers to differentiate between LTBI and active TB, levels of 29 biomarkers in QFT supernatants were evaluated using a Luminex multiplex cytokine assay. Results Eight out of 29 biomarkers distinguished active TB from LTBI in a pilot study. Baseline levels of epidermal growth factor (EGF soluble CD40 ligand (sCD40L, antigen stimulated levels of EGF, and the background corrected antigen stimulated levels of EGF and macrophage inflammatory protein (MIP-1β were the most informative single markers for differentiation between TB disease and LTBI, with AUCs of 0.88, 0.84, 0.87, 0.90 and 0.79 respectively. The combination of EGF and MIP-1β predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-α and IL-1α also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-α and sCD40L levels were higher in TB patients. Conclusion These preliminary data suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of EGF, sCD40L, MIP-1β, VEGF, TGF-α or IL-1α in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

Science.gov (United States)

Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

2014-09-01

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

A dense cell retention culture system using stirred ceramic membrane reactor.

Science.gov (United States)

Suzuki, T; Sato, T; Kominami, M

1994-11-20

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.

Purity and stability of online-prepared hemodiafiltration fluid after storage.

Science.gov (United States)

Corradi, V; Cruz, D; Vázquez-Rangel, A; Furlan, F; Grillone, R; Bonaccorsi, A; Cazzavillan, S; de Cal, M; Frisone, P; Morea, A; Brendolan, A; Rassu, M; Ronco, C

2013-01-01

Previous studies have suggested that online hemodiafiltration (OL-HDF) fluid can be used as dialysate for continuous renal replacement therapies, and thus HDF costs can be reduced. The aims of this study were to determine the purity of OL-HDF fluid and to verify the stability of the electrolyte composition and acid-base balance during its storage. OL-HDF fluid was collected in 70 individual bags and stored for up to 7 days. The following tests were performed daily in 10 bags: natural visible precipitation (macrocrystallization), sample collection for chemical analysis and fluid culture, limulus amebocyte lysate endotoxin test, standard culture of NALGENE® filters after passing of the fluid, and molecular analysis of bacterial DNA. The values of pH and pCO(2) showed a significant change starting at 24 h (p values were beyond the measurable range. Coefficient of variation for pCO(2) was as high as 25.7%. Electrolyte composition (Na(+), K(+), Cl(-), Ca(2+) and glucose) showed a statistically significant difference over time (p bags in future studies. Copyright © 2013 S. Karger AG, Basel.

Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus.

OpenAIRE

Smith, J S; Sumner, J W; Roumillat, L F

1984-01-01

A rapid and sensitive enzyme immunoassay is described for detecting rabies antibody in hybridoma culture fluids. Glass fiber filter disks were used to immobilize gamma-irradiated mouse neuroblastoma cells infected with street or laboratory strains of rabies virus. Bound rabies-specific antibody was detected by reaction with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G. The assay was performed in a 96-well filtration device developed by Cleveland et al. (J. Clin. Microbiol. ...

Immunoglobulin production in human mixed lymphocyte cultures: implications for co-cultures of cells from patients and healthy donors

International Nuclear Information System (INIS)

Ruemke, H.C.; Terpstra, F.G.; Huis, B.; Out, T.A.; Zeijlemaker, W.P.

1982-01-01

When human peripheral blood lymphocytes (PBL) are cultured in the presence of irradiated allogeneic lymphocytes, the resulting mixed lymphocyte reaction (MLR) leads to the secretion into the supernatant of substantial amounts of IgM and IgG, derived from nonirradiated responder B lymphocytes. Our data indicate that stimulation to Ig production by responder B cells may result from different types of of interactions. First, B cells and monocytes among the irradiated stimulator cells activate T responder B cells to produce Ig; second, ''responder'' B cells activate irradiated ''stimulator'' T cells, leading to a ''helper'' signal, back to the responder B cells and leading to Ig production. The latter system is radiosensitive, because allogeneic T cells, irradiated at a dose of 4000 rad or more, failed to induce Ig production by responder B cells. In some combinations of human allogeneic lymphocytes, the co-culture of the cells leads to inhibition of Ig production, both in the presence and in the absence of PWM. Thus, co-culture of allogeneic cells may cause ''positive'' as well as ''negative'' allogeneic effects. The implications of these findings for the interpretation of co-cultures that are aimed at establishing defects in lymphocytes from patients with, for example, immunodeficiencies, who fail to produce Ig in the presence of PWM are discussed

Isotope inequilibrium of glucose metabolites in intact cells and particlefree supernatants of Ehrlich ascites tumor

International Nuclear Information System (INIS)

Daehnfeldt, J.L.; Winge, P.

1975-01-01

With an enzyme degradative technique, isotope inequilibrium of glucose metabolites was demonstrated in intact cells and particle-free supernatants of Ehrlich ascites tumor using I- 14 C-glucose as tracer. Inequilibrium was found between glucose and glucose-6-phosphate, glucose and fructose-6-phosphate, glucose and 6-phosphogluconate, while glucose-6-phosphate and fructose-6-phosphate were found to be in near equilibrium within the incubation time investigated. Glucose and lactate were found to be in near equilibrium after 8 min in intact cells. Calculations based on the equilibrium levels found, showed that these inequilibria could not be explained by the effects of the pentose cycle. (U.S.)

Simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

Energy Technology Data Exchange (ETDEWEB)

Lemon, S M; Jansen, R W

1985-06-01

Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

International Nuclear Information System (INIS)

Lemon, S.M.; Jansen, R.W.

1985-01-01

Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus. (Auth.)

Comparison between traditional laboratory tests, permeability measurements and CT-based fluid flow modelling for cultural heritage applications

Energy Technology Data Exchange (ETDEWEB)

De Boever, Wesley, E-mail: Wesley.deboever@ugent.be [UGCT/PProGRess, Dept. of Geology, Ghent University, Krijgslaan 281, 9000 Ghent (Belgium); Bultreys, Tom; Derluyn, Hannelore [UGCT/PProGRess, Dept. of Geology, Ghent University, Krijgslaan 281, 9000 Ghent (Belgium); Van Hoorebeke, Luc [UGCT/Radiation Physics, Dept. of Physics & Astronomy, Ghent University, Proeftuinstraat 86, 9000 Ghent (Belgium); Cnudde, Veerle [UGCT/PProGRess, Dept. of Geology, Ghent University, Krijgslaan 281, 9000 Ghent (Belgium)

2016-06-01

In this paper, we examine the possibility to use on-site permeability measurements for cultural heritage applications as an alternative for traditional laboratory tests such as determination of the capillary absorption coefficient. These on-site measurements, performed with a portable air permeameter, were correlated with the pore network properties of eight sandstones and one granular limestone that are discussed in this paper. The network properties of the 9 materials tested in this study were obtained from micro-computed tomography (μCT) and compared to measurements and calculations of permeability and the capillary absorption rate of the stones under investigation, in order to find the correlation between pore network characteristics and fluid management characteristics of these sandstones. Results show a good correlation between capillary absorption, permeability and network properties, opening the possibility of using on-site permeability measurements as a standard method in cultural heritage applications. - Highlights: • Measurements of capillary absorption are compared to in-situ permeability. • We obtain pore size distribution and connectivity by using micro-CT. • These properties explain correlation between permeability and capillarity. • Correlation between both methods is good to excellent. • Permeability measurements could be a good alternative to capillarity measurement.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

Energy Technology Data Exchange (ETDEWEB)

Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

2012-04-05

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

International Nuclear Information System (INIS)

Sun, Dao-Cai; Li, De-Hua; Ji, Hui-Cang; Rao, Guo-Zhou; Liang, Li-Hua; Ma, Ai-Jie; Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang

2012-01-01

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones

Effect of Bifidobacterium upon Clostridium difficile growth and toxicity when co-cultured in different prebiotic substrates

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Lorena Valdés Varela

2016-05-01

Full Text Available The intestinal overgrowth of Clostridium difficile, often after disturbance of the gut microbiota by antibiotic treatment, leads to C. difficile infection (CDI which manifestation ranges from mild diarrhoea to life-threatening conditions. The increasing CDI incidence, not only in compromised subjects but also in traditionally considered low-risk populations, together with the frequent relapses of the disease, has attracted the interest for prevention/therapeutic options. Among these, probiotics, prebiotics or synbiotics constitute a promising approach. In this study we determined the potential of selected Bifidobacterium strains for the inhibition of C. difficile growth and toxicity in different carbon sources. We conducted co-cultures of the toxigenic strain C. difficile LMG21717 with four Bifidobacterium strains (Bifidobacterium longum IPLA20022, Bifidobacterium breve IPLA20006, Bifidobacterium bifidum IPLA20015, and Bifidobacterium animalis subsp. lactis Bb12 in the presence of various prebiotic substrates (Inulin, Synergy and Actilight or glucose, and compared the results with those obtained for the corresponding mono-cultures. C. difficile and bifidobacteria levels were quantified by qPCR; the pH and the production of short chain fatty acids was also determined. Moreover, supernatants of the cultures were collected to evaluate their toxicity using a recently developed model. Results showed that co-culture with B. longum IPLA20022 and B. breve IPLA20006 in the presence of short-chain fructooligosaccharides, but not of Inulin, as carbon source significantly reduced the growth of the pathogen. With the sole exception of B. animalis Bb12, whose growth was enhanced, the presence of C. difficile did not show major effects upon the growth of the bifidobacteria. In accordance with the growth data, B. longum and B. breve were the strains showing higher reduction in the toxicity of the co-culture supernatants.

Efficacy of nasopharyngeal culture in identification of pathogens in middle ear fluid in chronic otitis media with effusion

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Eser O

2009-01-01

Full Text Available Purpose: Chronic otitis media with effusion (OME is the leading cause of hearing loss during childhood. In bacterial etiology of OME, the most frequent pathogens responsible are Haemophilus influenzae followed by Streptococcus pneumoniae and Moraxella catarrhalis . This study aimed at evaluating the accuracy of nasopharyngeal (NP specimens in the identification of pathogens in the middle ear fluid (MEF in patients with OME. Materials and Methods: In this cross sectional, case-control study, 95 MEFs and 53 NP secretion specimens were obtained from 53 children. As a control group, 102 NP specimens were taken from children having an operation other than an otological disease. Conventional culture methods and multiplex-PCR method have been used to determine the etiology of OME; NP carriage between cases and control groups were compared using conventional culture methods. Pearson Chi-Square and Fisher′s Exact tests were used in statistical analysis. Results : Bacteria were isolated by culture in 37.9% of MEF specimens, 14.7% of which belonged to the group H. influenzae , S. pneumoniae and M. catarrhalis. PCR was positive in 30.5% specimens targeting the same pathogens. There was a two-fold increase in carriage rate of S. pneumoniae and H. influenzae in patients than controls for each pathogen. Conclusion: PCR is a more reliable method to detect middle ear pathogens in MEF in comparison with the conventional culture methods. The NP colonization wasn′t found to be an indicator of the pathogen in MEF although middle ear pathogens colonize more in nasopharynx of diseased children.

In vitro culture and characterization of human umbilical cord blood-derived plasmacytoid dendritic cell subsets

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PENG Jianping

2015-11-01

Full Text Available ObjectiveTo establish a method for in vitro culture of plasmacytoid dendritic cell (pDC. MethodsUmbilical cord blood (40 ml was collected from healthy parturients in the First Affiliated Hospital of Hunan University of Chinese Medicine, and cord blood mononuclear cell (CBMC were isolated. The CBMC were cultured for 7 days with RPMI 1640 complete medium containing rh Flt3-ligand (Flt3-L (100 ng/ml and rh interleukin (IL-3 (10 ng/ml, and the medium was half changed every 2 days. On the eighth day, CpG ODN (2 μg/ml was added to the cells, and the attached cells and supernatant were collected 24 h later for flow cytometry and interferon (IFNα measurement, respectively. On days 1, 3, 5, 7, and 8 of cell culture, the morphological changes of pDC were observed. Results After 2 h of culture, the CBMC showed circular, flat morphology. Twenty-four hours later, the cells began to adhere to the wall, with extended cytoplasm and increased volumes, and they became round and translucent, with scattered small colonies. On days 3-4 of culture, the cell volume continued increasing; most cells were round, and some had small protrusions; few cells were spindle-, tadpole-, star- or irregularly shaped; the number and volumes of colonies increased substantially. On days 5-8 of culture, the number of colonies and the number of cells in colonies gradually decreased, and suspended cells that were round or had small protrusions gradually increased in the medium. The cells expressing CD123, BDCA-2, and BDCA-4, which were considered pDC, were detected by flow cytometry. Flow cytometry revealed that the proportion of pDC in CBMC increased during the culture: increasing from 1.08% at the beginning of culture to 5.32% on day 4, and finally reaching a peak of 19.8% on day 8. On day 8, the level of IFNα in pDC culture supernatant was(11 302.61±1745.31 pg/ml. ConclusionpDC can be successfully induced in vitro by rh Flt3-L combined with IL-3 from human umbilical CBMC.

Comparison of bedside inoculation of culture media with ...

African Journals Online (AJOL)

Background: The yield of bacterial cultures from cerebrospinal fluid (CSF) at Kenyatta National Hospital (KNH) is very low. Bedside inoculation of culture media with CSF may improve yields. Objective: To compare the culture yield of CSF inoculated onto culture medium at the bedside to that of CSF inoculated onto culture ...

Automobile windshield washer fluid: A potential source of transmission for Legionella

International Nuclear Information System (INIS)

Schwake, David Otto; Alum, Absar; Abbaszadegan, Morteza

2015-01-01

Epidemiological evidence suggesting driving cars to be a risk factor for legionellosis has prompted public health studies to investigate vehicle windshield washer fluid as a novel transmission source of this disease. The goal of the current study was to investigate whether or not windshield washer fluid could serve as a potential source of transmission for Legionella. A wide variation in the survival of L. pneumophila was observed when incubated in different washer fluids at 25 and 37 °C, however, one brand tested supported Legionella survival similar to or greater than sterilized deionized water. In addition, 1 L of tap water contained in a washer fluid reservoir was able to support population growth and survival of Legionella for several months. In a field study examining the windshield washer fluid of 12 elementary school buses, Legionella were detected from 84% of samples at a high concentration of 8.1 × 10 4 CFU/mL. Culturable cells were also detected in aerosolized washer fluid during washer fluid spray. By demonstrating survival in certain windshield washer fluids, growth within washer fluid reservoirs, and the presence of viable cells in bus washer fluid spray, we have provided evidence suggesting the potential for a novel route of Legionella exposure. - Highlights: • L. pneumophila survival in one brand of washer fluid and sterilized deionized water were similar. • Legionella population was maintained in tap water for several months. • Culturable Legionella were detected in 10 of 12 school bus washer fluid reservoirs sampled. • Legionella concentrations up to 8.1 × 104 CFU/mL were detected in school bus washer fluid. • L. pneumophila was detected in washer fluid reservoirs and aerosolized washer fluid

Automobile windshield washer fluid: A potential source of transmission for Legionella

Energy Technology Data Exchange (ETDEWEB)

Schwake, David Otto [School of Life Sciences, Arizona State University, P.O. Box 875306, Tempe, AZ 85287 (United States); Alum, Absar [School of Sustainable Engineering and the Built Environment, Arizona State University, P.O. Box 875306, Tempe, AZ 85287 (United States); Abbaszadegan, Morteza, E-mail: abbaszadegan@asu.edu [School of Sustainable Engineering and the Built Environment, Arizona State University, P.O. Box 875306, Tempe, AZ 85287 (United States)

2015-09-01

Epidemiological evidence suggesting driving cars to be a risk factor for legionellosis has prompted public health studies to investigate vehicle windshield washer fluid as a novel transmission source of this disease. The goal of the current study was to investigate whether or not windshield washer fluid could serve as a potential source of transmission for Legionella. A wide variation in the survival of L. pneumophila was observed when incubated in different washer fluids at 25 and 37 °C, however, one brand tested supported Legionella survival similar to or greater than sterilized deionized water. In addition, 1 L of tap water contained in a washer fluid reservoir was able to support population growth and survival of Legionella for several months. In a field study examining the windshield washer fluid of 12 elementary school buses, Legionella were detected from 84% of samples at a high concentration of 8.1 × 10{sup 4} CFU/mL. Culturable cells were also detected in aerosolized washer fluid during washer fluid spray. By demonstrating survival in certain windshield washer fluids, growth within washer fluid reservoirs, and the presence of viable cells in bus washer fluid spray, we have provided evidence suggesting the potential for a novel route of Legionella exposure. - Highlights: • L. pneumophila survival in one brand of washer fluid and sterilized deionized water were similar. • Legionella population was maintained in tap water for several months. • Culturable Legionella were detected in 10 of 12 school bus washer fluid reservoirs sampled. • Legionella concentrations up to 8.1 × 104 CFU/mL were detected in school bus washer fluid. • L. pneumophila was detected in washer fluid reservoirs and aerosolized washer fluid.

Use of Bacillus thuringiensis supernatant from a fermentation process to improve bioremediation of chlorpyrifos in contaminated soils.

Science.gov (United States)

Aceves-Diez, Angel E; Estrada-Castañeda, Kelly J; Castañeda-Sandoval, Laura M

2015-07-01

The aim of this research was to investigate the potential of a nutrient-rich organic waste, namely the cell-free supernatant of Bacillus thuringiensis (BtS) gathered from fermentation, as a biostimulating agent to improve and sustain microbial populations and their enzymatic activities, thereby assisting in the bioremediation of chlorpyrifos-contaminated soil at a high dose (70 mg kg(-1)). Experiments were performed for up to 80 d. Chlorpyrifos degradation and its major metabolic product, 3,5,6-trichloro-2-pyridinol (TCP), were quantified by high-performance liquid chromatography (HPLC); total microbial populations were enumerated by direct counts in specific medium; and fluorescein diacetate (FDA) hydrolysis was measured as an index of soil microbial activity. Throughout the experiment, there was higher chlorpyrifos degradation in soil supplemented with BtS (83.1%) as compared to non-supplemented soil. TCP formation and degradation occurred in all soils, but the greatest degradation (30.34%) was observed in soil supplemented with BtS. The total microbial populations were significantly improved by supplementation with BtS. The application of chlorpyrifos to soil inhibited the enzymatic activity; however, this negative effect was counteracted by BtS, inducing an increase of approximately 16% in FDA hydrolysis. These results demonstrate the potential of B. thuringiensis supernatant as a suitable biostimulation agent for enhancing chlorpyrifos and TCP biodegradation in chlorpyrifos-contaminated soils. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enriched cultures of lactic acid bacteria from selected Zimbabwean fermented food and medicinal products with potential as therapy or prophylaxis against yeast infections

Directory of Open Access Journals (Sweden)

Alec Chabwinja

2017-10-01

Full Text Available Objective: To investigate the antifungal activity of crude cultures of putative strains of lactic acid bacteria (LAB from a selection of Zimbabwean traditional and commercial food/ medicinal products against yeasts (strains of environmental isolates of Candida albicans and Rhodotorula spp.. Methods: Cultures of putative LAB from our selection of fermented products were enriched in de Man, Rogosa and Sharpe and isolated on de Man, Rogosa and Sharpe agar. Results: The crude microbial cultures from the products that showed high antifungal activities (zone of inhibition, mm were as follows: supernatant-free microbial pellet (SFMP from an extract of Melia azedarach leaves [(27.0 ± 2.5 mm] > cell-free culture supernatants (CFCS from Maaz Dairy sour milk and Mnandi sour milk [approximately (26.0 ± 1.8/2.5 mm] > CFCS and SFMP from Amansi hodzeko [(25.0 ± 1.5 mm] > CFCS from Parinari curatellifolia fruit [(24.0 ± 1.5 mm], SFMP from Parinari curatellifolia fruit [(24.0 ± 1.4 mm] and SFMP from mahewu [(20.0 ± 1.5 mm]. These cultures also showed high tolerance to acidic conditions (pH 4.0 and pH 5.0. However, culture from WAYA LGG (shown elsewhere to harbour antimicrobial activities showed no antifungal activity. The LAB could have inhibited yeasts by either competitive exclusion or the release of antimicrobial metabolites. Conclusions: Our cultures of LAB from a selection of Zimbabwean fermented products, especially Ziziphus mauritiana and fermented milk products have great potential for use as antifungal probiotics against yeast infections. Studies are ongoing to determine the exact mechanisms that are employed by the putative LAB to inhibit Candida albicans.

Low dose radiation induced hormesis and its mechanism of free radicals

International Nuclear Information System (INIS)

Zhang Liyuan; Huo Hongmei; Zhang Yusong; Zhao Peifeng; Li Wei; Jiang Jiagui

2008-01-01

Objective: To investigate whether the supernatant (the stimulating fluid) centrifuged from myeloid cells suspension after low dose radiation in vitro can produce hormesis on the normal or radiation damage cells. The mechanism of free radical was probed. Methods: Mouse myeloid cell suspension was irradiated respectively by 0, 2 and 5 Gy, and cultured in vitro. MTT method was used to measure the reproductive activity of cells. Meanwhile, Cytochrome C reduction method was used to determine the concentration of O 2 - . Lastly, the concentration of O 2 - was decreased or increased by adding DPI or PMA, and the effect of such changes on 'the stimulating fluid' was observed. Results: Co-cultured with 'the stimulating fluid', the reproductive activity of the myeloid cells after large dose radiation or the normal myeloid cells were enhanced. Decreasing the concentration of O 2 - ; may degrade the proliferation of the cells after radiation damage; while increasing it may lead to the opposite result. Conclusions: The stimulating fluid can enhance the proliferation of the myeloid cells after radiation damage and also the normal ones. The mechanism of above-mentioned phenomena might be related with the changes of O 2 - concentration. (authors)

Significance of the interleukin-1 receptor antagonist/interleukin-1 beta ratio as a prognostic factor in patients with pulmonary sarcoidosis.

Science.gov (United States)

Mikuniya, T; Nagai, S; Takeuchi, M; Mio, T; Hoshino, Y; Miki, H; Shigematsu, M; Hamada, K; Izumi, T

2000-01-01

Various factors such as serum angiotensin-converting enzyme (sACE) activity, bronchoalveolar lavage (BAL) fluid lymphocyte percent, CD4/CD8 ratio, and shadows on chest radiograph have been identified as indexes of disease activity in patients with sarcoidosis. However, it remains to be confirmed whether these factors can predict clinical outcomes. To examine whether the interleukin-1 receptor antagonist (IL-1ra)/IL-1 beta ratio can predict the clinical course, we prospectively followed the clinical courses of 30 patients with pulmonary sarcoidosis 4 years after measurement of immunoreactive amounts of IL-1ra or IL-1 beta in the culture supernatants obtained from BAL fluid macrophages. Immunoreactive amounts of IL-1ra or IL-1 beta were measured using ELISA. Changes in pulmonary function, sACE activity, and shadows on chest radiographs during observation periods were evaluated as markers of changes in disease activity. We found that the patients whose shadows on chest radiographs showed improvement had a higher molar IL-1ra/IL-1 beta ratio than the patients whose shadows persistently remained 4 years after BAL examination (p sACE activity at the time of the last observation to sACE activity at the time of BAL (sACE(LAST)/sACE(BAL), p sACE(LAST)/sACE(BAL) ratio was significantly lower in patients whose shadows on chest radiographs decreased than in those whose shadows remained unchanged (p < 0.005). The IL-1ra/IL-1 beta ratio in the BAL fluid macrophage culture supernatants in patients with pulmonary sarcoidosis could be a useful marker in predicting the persistence of granulomatous lesions (chronicity). Copyright 2000 S. Karger AG, Basel

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Biorelevant media resistant co-culture model mimicking permeability of human intestine.

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Antoine, Delphine; Pellequer, Yann; Tempesta, Camille; Lorscheidt, Stefan; Kettel, Bernadette; Tamaddon, Lana; Jannin, Vincent; Demarne, Frédéric; Lamprecht, Alf; Béduneau, Arnaud

2015-03-15

Cell culture models are currently used to predict absorption pattern of new compounds and formulations in the human gastro-intestinal tract (GIT). One major drawback is the lack of relevant apical incubation fluids allowing mimicking luminal conditions in the GIT. Here, we suggest a culture model compatible with biorelevant media, namely Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF). Co-culture was set up from Caco-2 and mucus-secreting HT29-MTX cells using an original seeding procedure. Viability and cytotoxicity assays were performed following incubation of FeSSIF and FaSSIF with co-culture. Influence of biorelevant fluids on paracellular permeability or transporter proteins were also evaluated. Results were compared with Caco-2 and HT29-MTX monocultures. While Caco-2 viability was strongly affected with FeSSIF, no toxic effect was detected for the co-cultures in terms of viability and lactate dehydrogenase release. The addition of FeSSIF to the basolateral compartment of the co-culture induced cytotoxic effects which suggested the apical mucus barrier being cell protective. In contrast to FeSSIF, FaSSIF induced a slight increase of the paracellular transport and both tested media inhibited partially the P-gp-mediated efflux in the co-culture. Additionally, the absorptive transport of propranolol hydrochloride, a lipophilic β-blocker, was strongly affected by biorelevant fluids. This study demonstrated the compatibility of the Caco-2/HT29-MTX model with some of the current biorelevant media. Combining biorelevant intestinal fluids with features such as mucus secretion, adjustable paracellular and P-gp mediated transports, is a step forward to more realistic in-vitro models of the human intestine. Copyright © 2015. Published by Elsevier B.V.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

Science.gov (United States)

Makino, K; Takeichi, O; Imai, K; Inoue, H; Hatori, K; Himi, K; Saito, I; Ochiai, K; Ogiso, B

2018-06-01

To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P=0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. n-butyric acid produced by P. endodontalis reactivated latent EBV. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Batch test equilibration studies examining the removal of Cs, Sr, and Tc from supernatants from ORNL underground storage tanks by selected ion exchangers

International Nuclear Information System (INIS)

Collins, J.L.; Egan, B.Z.; Anderson, K.K.; Chase, C.W.; Bell, J.T.

1995-01-01

Bench-scale batch equilibration tests have been conducted with supernatants from two underground tanks at the Melton Valley Storage Tank (MVST) Facility at Oak Ridge National Laboratory (ORNL) to determine the effectiveness of selected ion exchangers in removing cesium, strontium, and technetium. Seven sorbents were evaluated for cesium removal, nine for strontium removal, and four for technetium removal. The results indicate that granular potassium cobalt hexacyanoferrate was the most effective of the exchangers evaluated for removing cesium from the supernatants. The powdered forms of sodium titanate (NaTiO) and cystalline silicotitanate (CST) were superior in removing the strontium; however, for the sorbents of suitable particle size for column use, titanium monohydrogen phosphate (TiHP φ), sodium titanate/polyacrylonitrile (NaTiO-PAN), and titanium monohydrogen phosphate/polyacrylonitrile (TiP-PAN) gave the best results and were about equally effective. Reillex trademark 402 was the most effective exchanger in removing the technetium; however, it was only slightly more satisfactory than Reillex trademark HPQ

Transcriptional and metabolic flux profiling of triadimefon effects on cultured hepatocytes

International Nuclear Information System (INIS)

Iyer, Vidya V.; Ovacik, Meric A.; Androulakis, Ioannis P.; Roth, Charles M.; Ierapetritou, Marianthi G.

2010-01-01

Conazoles are a class of azole fungicides used to prevent fungal growth in agriculture, for treatment of fungal infections, and are found to be tumorigenic in rats and/or mice. In this study, cultured primary rat hepatocytes were treated to two different concentrations (0.3 and 0.15 mM) of triadimefon, which is a tumorigenic conazole in rat and mouse liver, on a temporal basis with daily media change. Following treatment, cells were harvested for microarray data ranging from 6 to 72 h. Supernatant was collected daily for three days, and the concentrations of various metabolites in the media and supernatant were quantified. Gene expression changes were most significant following exposure to 0.3 mM triadimefon and were characterized mainly by metabolic pathways related to carbohydrate, lipid and amino acid metabolism. Correspondingly, metabolic network flexibility analysis demonstrated a switch from fatty acid synthesis to fatty acid oxidation in cells exposed to triadimefon. It is likely that fatty acid oxidation is active in order to supply energy required for triadimefon detoxification. In 0.15 mM triadimefon treatment, the hepatocytes are able to detoxify the relatively low concentration of triadimefon with less pronounced changes in hepatic metabolism.

Effects of peritoneal fluid from endometriosis patients on interferon-gamma-induced protein-10 (CXCL10) and interleukin-8 (CXCL8) released by neutrophils and CD4+ T cells.

Science.gov (United States)

Kim, Ji-Yeon; Lee, Dong-Hyung; Joo, Jong-Kil; Jin, Jun-O; Wang, Ji-Won; Hong, Young-Seoub; Kwak, Jong-Young; Lee, Kyu-Sup

2009-09-01

Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-gamma-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4(+) T cells, and monocytes. Neutrophils, CD4(+) T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay. The addition of ePF to cultures of CD4(+) T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF (R = 0.89, P = 0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4(+) T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils (R = 0.89, P ePF significantly enhanced the interferon-gamma-induced release of IP-10 by nuetrophils and CD4(+) T cells. These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.

Neurotoxicity of cerebro-spinal fluid from patients with Parkinson's disease on mesencephalic primary cultures as an in vitro model of dopaminergic neurons.

Science.gov (United States)

Kong, Ping; Zhang, Ben-Shu; Lei, Ping; Kong, Xiao-Dong; Zhang, Shi-Shuang; Li, Dai; Zhang, Yun

2015-08-01

Parkinson's disease is a degenerative disorder of the central nervous system. In spite of extensive research, neither the cause nor the mechanisms have been firmly established thus far. One assumption is that certain toxic substances may exist in the cerebro-spinal fluid (CSF) of Parkinson's disease patients. To confirm the neurotoxicity of CSF and study the potential correlation between neurotoxicity and the severity of Parkinson's disease, CSF was added to cultured cells. By observation of cell morphology, changes in the levels of lactate dehydrogenase, the ratio of tyrosine hydroxylase-positive cells, and the expression of tyrosine hydroxylase mRNA and protein, the differences between the two groups were shown. The created in vitro model of dopaminergic neurons using primary culture of mouse embryonic mesencephalic tissue is suitable for the study of neurotoxicity. The observations of the present study indicated that CSF from Parkinson's disease patients contains factors that can cause specific injury to cultured dopaminergic neurons. However, no obvious correlation was found between the neurotoxicity of CSF and the severity of Parkinson's disease.

Chronic subdural hematoma fluid and its computerized tomographic density

International Nuclear Information System (INIS)

Masuzawa, Hideaki; Sato, Jinichi; Kamitani, Hiroshi; Yamashita, Midori

1983-01-01

Laboratory and in vivo CT analysis were performed on 19 chronic subdural hematomas and five subdural hygromas. In these 25 hematoma samples, red blood cells (RBC), hematocrit, and hemoglobin (Hgb) varied greatly, though, these values correlated well with the CT densities. Plasma protein content was fairly constant with an average of 7.1+-0.8g/dl. There were four hematoma samples with RBC of less than 20x10 4 μl or Hgb of less than 2.0g/dl. Their CT values ranged between 18 and 23 H.U., which were considered close to the in vivo serum level CT density. Five hygroma fluid showed no RBC and very little protein content of less than 0.4g/dl. CT density ranged between -2 and 13 H.U. The edge effect of the skull was experimentally studied using a phantom skull filled with water. This revealed a remarkable overshoot of the CT values within ten pixels from the inner wall of the skull. Visual observation of the original CT pictures revealed four low density hematomas and seven mixed density ones. When compared to the density of the ventricular cavity, all of the low density hematomas and the supernatant part of the mixed density ones were clearly higher in density. All five hygromas appeared CSF dense or lower. In conclusion, because of the edge effect by the skull, thin subdural fluids could not be diagnosed by CT alone. Thick subdural fluids could be differentiated as either hematoma or hygroma by their CT densities. Subdural hematomas had in vivo CT densities of at least serum level or approximately 20 H.U., while subdural hygromas had densities close to CSF. These characteristics were best appreciated by visual observation of the CT scan films. (J.P.N.)

Optimization of high-rate TN removal in a novel constructed wetland integrated with microelectrolysis system treating high-strength digestate supernatant.

Science.gov (United States)

Guo, Luchen; He, Keli; Wu, Shubiao; Sun, Hao; Wang, Yanfei; Huang, Xu; Dong, Renjie

2016-08-01

The potential of high-rate TN removal in three aerated horizontal subsurface-flow constructed wetlands to treat high-strength anaerobic digestate supernatant was evaluated. Different strategies of intermittent aeration and effluent recirculation were applied to compare their effect on nitrogen depuration performance. Additional glucose supply and iron-activated carbon based post-treatment systems were established and examined, respectively, to further remove nitrate that accumulated in the effluents from aerated wetlands. The results showed that intermittent aeration (1 h on:1 h off) significantly improved nitrification with ammonium removal efficiency of 90% (18.1 g/(m(2)·d)), but limited TN removal efficiency (53%). Even though effluent recirculation (a ratio of 1:1) increased TN removal from 53% to 71%, the effluent nitrate concentration was still high. Additional glucose was used as a post-treatment option and further increased the TN removal to 82%; however, this implementation caused additional organic pollution. Furthermore, the iron-activated carbon system stimulated with a microelectrolysis process achieved greater than 85% effluent nitrate removal and resulted in 86% TN removal. Considering the high TN removal rate, aerated constructed wetlands integrated with a microelectrolysis-driven system show great potential for treating high-strength digestate supernatant. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oral bacterial DNA findings in pericardial fluid

Directory of Open Access Journals (Sweden)

Anne-Mari Louhelainen

2014-11-01

Full Text Available Background: We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. Objective: To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic–anatomic findings linked to cardiovascular diseases. Methods: Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009–2010. Of the autopsies, 10 (45.5% were free of coronary artery disease (CAD, 7 (31.8% had mild and 5 (22.7% had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes. Results: Of 22 cases, 14 (63.6% were positive for endodontic and 8 (36.4% for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035. Conclusions: Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

Ultrastructural Histopathology of Vervet Monkey Colonic Epithelium After In Vitro Exposure to Cell-free Supernatants of Shigella Cultures

OpenAIRE

Hill, R. R.; Collins, N. E.; Cowley, H. M.

2011-01-01

The full dysentery syndrome of human shigellosis is often preceded by a transient diarrhoea that may be induced by bacterial extracellular products before invasion of the colonic mucosa and development of subsequent pathology. To examine this hypothesis, we studied the effects of cell-free cultures of Shigella sp. on the ultrastructure of monkey colonic epithelium in vitro. Clinical isolates of shigella strains were grown in a niche-simulating medium. Sheets of colon wall collected from verve...

Identification of Fusarium oxysporum in the fluid of hydroponic culture using DNA analysis; DNA kaiseki ni yoru suiko saibai yoekichu no horenso ichobyokin

Energy Technology Data Exchange (ETDEWEB)

Oyama, K.; Kawabata, T. [Chugoku Electric Power Co. Inc., Hiroshima (Japan)

1999-09-24

In the case of circularly using a fluid of the hydroponic culture for crops such as vegetable, the destructive damage is caused when specified harmful germs multiply in the growing crop. therefore, it is necessary to find the germs in question out and deal with them as quickly as possible. In this paper, studies were made on a sure identification technology of F. oxysporum using DNA analysis and the measures to prevent crop damages caused by diseases. The results of the study are reported. (translated by NEDO)

Production of cellulolytic enzymes by fungal cultures. [Aspergillus, Trichoderma, Chaetomium, Stachybotrys, and Hypocrea

Energy Technology Data Exchange (ETDEWEB)

Pyc, R; Fiechter, A. Galas, E.

1977-01-01

Twelve fungal cultures belonging to the genera of Aspergillus, Trichoderma, Chaetomium, Stachybotrys, and Hypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose. More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity. The growth of Aspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity. Culture supernatant from Aspergillus wentii was found to hydrolyze from 30-32% of Solka-Floc and from 2-10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of Solka-Floc indicates that with suitable treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.

The influence of fluid shear stress on the expression of Cbfa1 in MG-63 cells cultured under different gravitational conditions

Science.gov (United States)

Zhang, S.; Wang, B.; Cao, X. S.; Yang, Z.; Sun, X. Q.

2008-12-01

AuthorPurposeThis study was aimed to explore the effect of flow shear stress on the expression of Cbfa1 in human osteosarcoma cells and to survey its functional alteration in simulated microgravity. After culture for 48 h in two different gravitational environments, i.e. 1 G terrestrial gravitational condition and simulated microgravity condition, human osteosarcoma cells (MG-63) were treated with 0.5 or 1.5 Pa fluid shear stress (FSS) in a flow chamber for 15, 30, and 60 min, respectively. The total RNA in cells was isolated. RT-PCR analysis was made to examine the gene expression of Cbfa1. The total protein of cells was extracted and the expression of Cbfa1 protein was detected by means of Western blotting. ResultsMG-63 cells cultured in 1 G condition reacted to FSS treatment with an enhanced expression of Cbfa1. Compared with no-FSS control group, Cbfa1 mRNA expression increased significantly at 30 and 60 min with the treatment of FSS ( P cells cultured in simulated microgravity by using clinostat, the expression of Cbfa1 was significantly different between 1 G and simulated microgravity conditions at each test time ( P cultured in simulated microgravity, Cbfa1 mRNA expression increased significantly at 30 and 60 min with the treatment of FSS ( P osteosarcoma cells. And this inducible function of FSS was adversely affected by simulated microgravity.

Recovery of the proliferative and functional integrity of mouse bone marrow in long-term cultures established after whole-body irradiation at different doses and dose rates

International Nuclear Information System (INIS)

Bierkens, J.G.; Hendry, J.H.; Testa, N.G.

1991-01-01

Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

Science.gov (United States)

Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

2000-01-01

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

Membrane damage induced in cultured human skin fibroblasts by UVA irradiation

International Nuclear Information System (INIS)

Gaboriau, F.; Morliere, P.; Marquis, I.; Moysan, A.; Geze, M.; Dubertret, L.

1993-01-01

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells. (Author)

Cultural neuroscience and psychopathology: prospects for cultural psychiatry.

Science.gov (United States)

Choudhury, Suparna; Kirmayer, Laurence J

2009-01-01

There is a long tradition that seeks to understand the impact of culture on the causes, form, treatment, and outcome of psychiatric disorders. An early, colonialist literature attributed cultural characteristics and variations in psychopathology and behavior to deficiencies in the brains of colonized peoples. Contemporary research in social and cultural neuroscience holds the promise of moving beyond these invidious comparisons to a more sophisticated understanding of cultural variations in brain function relevant to psychiatry. To achieve this, however, we need better models of the nature of psychopathology and of culture itself. Culture is not simply a set of traits or characteristics shared by people with a common geographic, historical, or ethnic background. Current anthropology understands culture as fluid, flexible systems of discourse, institutions, and practices, which individuals actively use for self-fashioning and social positioning. Globalization introduces new cultural dynamics and demands that we rethink culture in relation to a wider domain of evolving identities, knowledge, and practice. Psychopathology is not reducible to brain dysfunction in either its causes, mechanisms, or expression. In addition to neuropsychiatric disorders, the problems that people bring to psychiatrists may result from disorders in cognition, the personal and social meanings of experience, and the dynamics of interpersonal interactions or social systems and institutions. The shifting meanings of culture and psychopathology have implications for efforts to apply cultural neuroscience to psychiatry. We consider how cultural neuroscience can refine use of culture and its role in psychopathology using the example of adolescent aggression as a symptom of conduct disorder.

Extracellular toxins of pseudomonas aeruginosa. Pt. 4

International Nuclear Information System (INIS)

Obernesser, H.J.; Doering, G.

1982-01-01

A sensitive and specific solid phase radioimmunoassay (RIA) for detection of the elastase (Ela) of Pseudomonas aeruginosa (PA) was developed and the RIA was used to assay 10 PA strains of various origin and serotype. A great strain variability of Ela production was found which different from 94.1 to 0.1 μg per ml of culture supernatant fluid (CSF). The Ela and alkaline protease (AP) concentrations were converted to proteolytic activity and combined. The sum of the calculated enzymatic values of Ela and AP correlated well with the experimentally determined values of total proteolytic activity of the CSF. (orig.) [de

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

Czech Academy of Sciences Publication Activity Database

Ujčíková, Hana; Eckhardt, Adam; Kagan, Dmytro; Roubalová, Lenka; Svoboda, Petr

2014-01-01

Roč. 12, Feb 14 (2014), s. 11 ISSN 1477-5956 R&D Projects: GA ČR(CZ) GAP207/12/0919; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : morphine * long-term exposure * rat brain cortex * isolated plasma membranes * post-nuclear supernatant * 2D electrophoresis Subject RIV: CE - Biochemistry Impact factor: 1.725, year: 2014

Inhibition of oncostatin M in osteoarthritic synovial fluid enhances GAG production in osteoarthritic cartilage repair

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M Beekhuizen

2013-09-01

Full Text Available Mediators in the synovial fluid are thought to play a major role in osteoarthritic cartilage turnover. The purpose of the current study was to investigate the role of oncostatin M (OSM in osteoarthritis (OA by evaluating the presence of the cytokine and its receptors in the OA joint and interfering with its activity in synovial fluid co-cultured with cartilage explants. OSM levels were increased in the synovial fluid of osteoarthritic patients compared to healthy donors. Immunohistochemistry confirmed the presence of both the leukaemia inhibitory factor (LIF and OSM receptors for OSM throughout the whole depth of osteoarthritic cartilage and synovial tissue, whereas in healthy cartilage their presence seemed more restricted to the superficial zone. Blocking OSM activity, using an activity inhibiting antibody, in 25 % osteoarthritic synovial fluid added to OA cartilage explant cultures increased glycosaminoglycan (GAG content from 18.6 mg/g to 24.3 mg/g (P < 0.03 and total production from 7.0 mg/g to 11.9 mg/g (P < 0.003. However, OSM exogenously added to cartilage explant cultures reflecting low and high concentrations in the synovial fluid (5 and 50 pg/mL did not affect cartilage matrix turnover, suggesting that factors present in the synovial fluid act in concert with OSM to inhibit GAG production. The current study indicates the potential to enhance cartilage repair in osteoarthritis by modulating the joint environment by interfering with OSM activity.

Value of PCR in sonication fluid for the diagnosis of orthopedic hardware-associated infections: Has the molecular era arrived?

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Renz, Nora; Cabric, Sabrina; Morgenstern, Christian; Schuetz, Michael A; Trampuz, Andrej

2018-04-01

Bone healing disturbance following fracture fixation represents a continuing challenge. We evaluated a novel fully automated polymerase chain reaction (PCR) assay using sonication fluid from retrieved orthopedic hardware to diagnose infection. In this prospective diagnostic cohort study, explanted orthopedic hardware materials from consecutive patients were investigated by sonication and the resulting sonication fluid was analyzed by culture (standard procedure) and multiplex PCR (investigational procedure). Hardware-associated infection was defined as visible purulence, presence of a sinus tract, implant on view, inflammation in peri-implant tissue or positive culture. McNemar's chi-squared test was used to compare the performance of diagnostic tests. For the clinical performance all pathogens were considered, whereas for analytical performance only microorganisms were considered for which primers are included in the PCR assay. Among 51 patients, hardware-associated infection was diagnosed in 38 cases (75%) and non-infectious causes in 13 patients (25%). The sensitivity for diagnosing infection was 66% for peri-implant tissue culture, 84% for sonication fluid culture, 71% (clinical performance) and 77% (analytical performance) for sonication fluid PCR, the specificity of all tests was >90%. The analytical sensitivity of PCR was higher for gram-negative bacilli (100%), coagulase-negative staphylococci (89%) and Staphylococcus aureus (75%) than for Cutibacterium (formerly Propionibacterium) acnes (57%), enterococci (50%) and Candida spp. (25%). The performance of sonication fluid PCR for diagnosis of orthopedic hardware-associated infection was comparable to culture tests. The additional advantage of PCR was short processing time (PCR has the potential to complement conventional cultures. Copyright © 2018 Elsevier Ltd. All rights reserved.

Use of a solid-phase 3H-radioimmunoassay for the measurement of immunoglobulin produced in short-term cultures of antibody-secreting cells

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Mongini, P.K.A.; Heber-Katz, E.

1982-01-01

A solid-phase radioimmunoassay (RIA) for assaying immunoglobulin produced from antibody-secreting myeloma, hybridoma and immune spleen cells is described. Specific antibody is detected by culturing antibody-producing cells on antigen-coated flexible polyvinylchloride microtiter wells, washing away the cells, and measuring the bound specific antibody with tritiated affinity-purified anti-isotype reagents. Antibody produced from 10 3 myeloma cells can be detected with as little as 4 h of incubation. With 24-48 h of incubation, antibody from as few as approximately 3-15 myeloma, hybridoma or immune spleen plaque-forming cells (PFC) can be detected. This culture-well RIA has certain distinct advantages over the hemolytic PFC assay and RIA assays in which antibody in culture supernatants is measured. (Auth.)

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

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Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer.

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Ramakrishnan, Divakar; Curtis, Wayne R

2004-10-20

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.

[Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

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Zhang, Hua-Dong; Lu, Zhi; Feng, Yi; Liu, Xiao-Li; Hou, Hui-Ming

2014-03-01

To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

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Jing Liu

Full Text Available Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium, which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF. Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH levels (biomarkers of ictal activity and cell death, respectively in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

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Liu, Jing; Saponjian, Yero; Mahoney, Mark M; Staley, Kevin J; Berdichevsky, Yevgeny

2017-01-01

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

Investigation of the proinflammatory activity of bronchoalveolar lavage fluid in lung cancer and inflammatory diseases of respiratory organs

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O. P. Makarova

2014-01-01

Full Text Available The proinflammatory activity of bronchoalveolar lavage fluid (BALF was investigated using the biological test system (donor blood in 71 patients with lung cancer and inflammatory disease of respiratory organs (lung abscess, infiltrative tuberculosis, tuberculoma, fibrocavernous tuberculosis. The supernatant of BALF was purified by filtration through millipores and added to blood, and a nitroblue tetrazolium recovery test (HBT was carried out. Hank’s balanced salt solution was used in the control. The proinflammatory effect of BALF was defined as a diformazan-positive neutrophil count ratio in the experimental and control samples (stimulation index. The inflammatory process developing in lung cancer was active, as suggested by the accumulation of neutrophils in the lung and by the high proinflammatory activity of BALF, which were comparable with the similar indicators in lung abscess and fibrocavernous tuberculosis.

Influence of in vitro irradiation upon LIF production by ConA stimulated mononuclear cells

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Sandru, G.; Veraguth, P.

1981-01-01

Leukocyte migration inhibitory factor (LIF) activity of culture supernatants of in vitro irradiated Concanavalin A (ConA) stimulated lymphocytes was tested by measuring granulocyte migration from clotted plasma droplets placed in flat bottom microplates. The specificity of inhibition was assured by pretreating the assay supernatants with anti-LIF antibodies which abrogated granulocyte migration inhibition but did not impair guinea pig Peritoneal Exudate Cells (PEC) migration inhibition. In vitro irradiation (150-1200 rads) of MNC cultures either before or after ConA stimulation did not impair lymphokine production and sometimes significantly improved the supernatants' LIF activity as compared with that of unirradiated cultures. The existence of radiosensitive suppressor cells regulating LIF production by ConA stimulated mononuclear cells is suggested

Interleukin-6 in cerebrospinal fluid as a biomarker of acute meningitis.

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García-Hernández, Pablo; Prieto, Belén; Martínez-Morillo, Eduardo; Rodríguez, Verónica; Álvarez, Francisco V

2016-01-01

Microbiological culture of cerebrospinal fluid is the gold standard to differentiate between aseptic and bacterial meningitis, but this method has low sensitivity. A fast and reliable new marker would be of interest in clinical practice. Interleukin-6, secreted by T cells in response to meningeal pathogens and quickly delivered into cerebrospinal fluid, was evaluated as a marker of acute meningitis. A total of 150 cerebrospinal fluid samples were analysed by an electrochemiluminescence method, selected according to patient diagnosis: (a) bacterial meningitis confirmed by positive culture (n = 26); (b) bacterial meningitis with negative culture or not performed (n = 15); (c) viral meningitis confirmed by polymerase chain reaction or immunoglobulin G determination (n = 23); (d) viral meningitis with polymerase chain reaction negative or not performed (n = 42); and (e) controls (n = 44). Cerebrospinal fluid interleukin-6 concentration showed significant differences between all pathologic groups and the control group (P meningitis, interleukin-6 showed an area under the curve of 0.937 (95% confidence intervals: 0.895-0.978), significantly higher than those of classical biomarkers. An interleukin-6 cutoff of 1418 pg/mL showed 95.5% sensitivity and 77.5% specificity, whereas a value of 15,060 pg/mL showed 63.6% sensitivity and 96.7% specificity, for diagnosis of bacterial meningitis. Interleukin-6 measured by electrochemiluminescence method is a promising marker for early differentiation between aseptic and bacterial meningitis. More studies are needed to validate clinical implications for future practice in an emergency laboratory. © The Author(s) 2015.

Biosynthesis, characterization and antibacterial activity of silver nanoparticles using an endophytic fungal supernatant of Raphanus sativus

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Tej Singh

2017-06-01

Full Text Available In this study, biological synthesis of silver nanoparticles (AgNPs from supernatant of endophytic fungus Alternaria sp. isolated from the healthy leaves of Raphanus sativus is studied. The synthesized AgNPs are characterized using UV-vis spectroscopy and Fourier transform-infrared spectroscopy (FTIR. The structural analysis is done by powder X-ray diffraction (XRD method. The stability of AgNPs is studied by dynamic light scattering (DLS method. The size and shape of AgNPs are observed by transmission electron microscopy (TEM and atomic force microscopy (AFM and found to be spherical with an average particles size of 4–30 nm. Further, these AgNPs have been found to be highly toxic against human pathogenic bacteria, suggesting the possibility of using AgNPs as efficient antibacterial agents.

Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease.

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Capik, Sarah F; White, Brad J; Lubbers, Brian V; Apley, Michael D; DeDonder, Keith D; Larson, Robert L; Harhay, Greg P; Chitko-McKown, Carol G; Harhay, Dayna M; Kalbfleisch, Ted S; Schuller, Gennie; Clawson, Michael L

2017-03-01

OBJECTIVE To compare predictive values, extent of agreement, and gamithromycin susceptibility between bacterial culture results of nasopharyngeal swab (NPS) and bronchoalveolar lavage fluid (BALF) samples obtained from calves with bovine respiratory disease (BRD). ANIMALS 28 beef calves with clinical BRD. PROCEDURES Pooled bilateral NPS samples and BALF samples were obtained for bacterial culture from calves immediately before and at various times during the 5 days after gamithromycin (6 mg/kg, SC, once) administration. For each culture-positive sample, up to 12 Mannheimia haemolytica, 6 Pasteurella multocida, and 6 Histophilus somni colonies underwent gamithromycin susceptibility testing. Whole-genome sequencing was performed on all M haemolytica isolates. For paired NPS and BALF samples collected 5 days after gamithromycin administration, the positive and negative predictive values for culture results of NPS samples relative to those of BALF samples and the extent of agreement between the sampling methods were determined. RESULTS Positive and negative predictive values of NPS samples were 67% and 100% for M haemolytica, 75% and 100% for P multocida, and 100% and 96% for H somni. Extent of agreement between results for NPS and BALF samples was substantial for M haemolytica (κ, 0.71) and H somni (κ, 0.78) and almost perfect for P multocida (κ, 0.81). Gamithromycin susceptibility varied within the same sample and between paired NPS and BALF samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated culture results of NPS and BALF samples from calves with BRD should be interpreted cautiously considering disease prevalence within the population, sample collection relative to antimicrobial administration, and limitations of diagnostic testing methods.

Oscillating Cell Culture Bioreactor

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Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

2010-01-01

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

Bions: a family of biomimetic mineralo-organic complexes derived from biological fluids.

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Cheng-Yeu Wu

Full Text Available Mineralo-organic nanoparticles form spontaneously in human body fluids when the concentrations of calcium and phosphate ions exceed saturation. We have shown previously that these mineralo-organic nanoparticles possess biomimetic properties and can reproduce the whole phenomenology of the so-called nanobacteria-mineralized entities initially described as the smallest microorganisms on earth. Here, we examine the possibility that various charged elements and ions may form mineral nanoparticles with similar properties in biological fluids. Remarkably, all the elements tested, including sodium, magnesium, aluminum, calcium, manganese, iron, cobalt, nickel, copper, zinc, strontium, and barium form mineralo-organic particles with bacteria-like morphologies and other complex shapes following precipitation with phosphate in body fluids. Upon formation, these mineralo-organic particles, which we term bions, invariably accumulate carbonate apatite during incubation in biological fluids; yet, the particles also incorporate additional elements and thus reflect the ionic milieu in which they form. Bions initially harbor an amorphous mineral phase that gradually converts to crystals in culture. Our results show that serum produces a dual inhibition-seeding effect on bion formation. Using a comprehensive proteomic analysis, we identify a wide range of proteins that bind to these mineral particles during incubation in medium containing serum. The two main binding proteins identified, albumin and fetuin-A, act as both inhibitors and seeders of bions in culture. Notably, bions possess several biomimetic properties, including the possibility to increase in size and number and to be sub-cultured in fresh culture medium. Based on these results, we propose that bions represent biological, mineralo-organic particles that may form in the body under both physiological and pathological homeostasis conditions. These mineralo-organic particles may be part of a

Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

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Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

2018-01-01

The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

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Karina Sánchez-Reyes

2014-01-01

Full Text Available Cervical cancer (CC is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated and M2 (alternatively activated. Macrophage polarization exerts profound effects on the Toll-like receptor (TLR profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

Enhanced biofilm formation in dual-species culture of Listeria monocytogenes and Ralstonia insidiosa

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Yunfeng Xu

2017-09-01

Full Text Available In the natural environments microorganisms coexist in communities as biofilms. Since foodborne pathogens have varying abilities to form biofilms, investigation of bacterial interactions in biofilm formation may enhance our understanding of the persistence of these foodborne pathogens in the environment. Thus the objective of this study was to investigate the interactions between Listeria monocytogenes and Ralstonia insidiosa in dual species biofilms. Biofilm development after 24 h was measured using crystal violet in 96-well microtiter plate. Scanning electron microscopy and cell enumeration were employed after growth on stainless steel coupons. When compared with their single species counterparts, the dual species biofilms exhibited a significant increase in biofilm biomass. The number of L. monocytogenes in co-culture biofilms on stainless steel also increased significantly. However, there was no effect on the biofilm formation of L. monocytogenes when cultured with R. insidiosa separated by a semi-permeable membrane-linked compartment or cultured in R. insidiosa cell-free supernatant, indicating that direct cell-cell contact is critical for this interaction.

BIOTECHNOLOGICAL ASPECTS OF INTERFACIAL TENSIOMETRY AND RHEOMETRY OF XYLOTROPHIC BASIDIOMYCETES CULTURE FLUID

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Chaika A. V.

2013-12-01

Full Text Available The tensio-rheometric characteristics of 63 strains belonging to 19 basidiomycetes species submerged culture filtrate were investigated by the axisymmetric pendent drop profile analysis. The method showed required high sensitivity with mycological material. It was found that the interfacial tensiometric and rheometric parameters depend significantly on culture species, hence it is proposed to use ones complex for systematic identification of cultures and as a selection criterion for biosurfactantsproducing strains of basidiomycetes. Correlations of tensio-rheometric characteristics both among themselves and with the culture growth and lipid peroxidation rates were found. This provides an integrated indicator of the submerged culture metabolic state. By the results of the study several strains of basidiomycetes — potential producers of biosurfactants with a high growth rate and intensity of lipid peroxidation were selected for biotechnological manufacture.

Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boilign vs. conventional RNA extraction

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Fereidouni, S.R.; Starick, E.; Ziller, M.; Harder, T.C.; Unger, H.; Hamilton, K.; Globig, A.

2016-01-01

Full text: RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commerical lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTaPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing material materials, including diluted virus positive allontoic fluid or cell culture supernatnat, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. (author)

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa

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McCurtain, Jennifer L.; Gilbertsen, Adam J.; Kalstabakken, Kyle A.; Williams, Bryan J.

2018-01-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled 13C5,15N4-agmatine (synthesized by decarboxylation of uniformly labeled 13C6,15N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,l,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1 % (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients. PMID:25957842

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

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Dalluge, Joseph J; McCurtain, Jennifer L; Gilbertsen, Adam J; Kalstabakken, Kyle A; Williams, Bryan J

2015-07-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

Quantitative culture of endotracheal aspirate and BAL fluid samples in the management of patients with ventilator-associated pneumonia: a randomized clinical trial

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Ricardo de Amorim Corrêa

2014-12-01

Full Text Available OBJECTIVE: To compare 28-day mortality rates and clinical outcomes in ICU patients with ventilator-associated pneumonia according to the diagnostic strategy used. METHODS: This was a prospective randomized clinical trial. Of the 73 patients included in the study, 36 and 37 were randomized to undergo BAL or endotracheal aspiration (EA, respectively. Antibiotic therapy was based on guidelines and was adjusted according to the results of quantitative cultures. RESULTS: The 28-day mortality rate was similar in the BAL and EA groups (25.0% and 37.8%, respectively; p = 0.353. There were no differences between the groups regarding the duration of mechanical ventilation, antibiotic therapy, secondary complications, VAP recurrence, or length of ICU and hospital stay. Initial antibiotic therapy was deemed appropriate in 28 (77.8% and 30 (83.3% of the patients in the BAL and EA groups, respectively (p = 0.551. The 28-day mortality rate was not associated with the appropriateness of initial therapy in the BAL and EA groups (appropriate therapy: 35.7% vs. 43.3%; p = 0.553; and inappropriate therapy: 62.5% vs. 50.0%; p = 1.000. Previous use of antibiotics did not affect the culture yield in the EA or BAL group (p = 0.130 and p = 0.484, respectively. CONCLUSIONS: In the context of this study, the management of VAP patients, based on the results of quantitative endotracheal aspirate cultures, led to similar clinical outcomes to those obtained with the results of quantitative BAL fluid cultures.

Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy

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Jūratė Savickienė

2016-01-01

Full Text Available Human amniotic-fluid-derived mesenchymal stem cells (AF-MSCs are interesting for their multilineage differentiation potential and wide range of therapeutic applications due to the ease of culture expansion. However, MSCs undergo replicative senescence. So far, the molecular mechanisms that underlie fetal diseases and cell senescence are still poorly understood. Here, we analyzed senescence-associated morphologic, molecular, and epigenetic characteristics during propagation of MSCs derived from AF of normal and fetus-affected pregnancy. AF-MSCs cultures from both cell sources displayed quite similar morphology and expression of specific cell surface (CD44, CD90, and CD105 and stemness (Oct4, Nanog, Sox2, and Rex1 markers but had interindividual variability in proliferation capability and time to reach senescence. Within passages 4 and 8, senescent cultures exhibited typical morphological features, senescence-associated β-galactosidase activity, increased levels of p16, and decreased levels of miR-17 and miR-21 but showed differential expression of p21, p53, and ATM dependently on the onset of cell senescence. These differences correlated with changes in the level of chromatin modifiers (DNMT1 and HDAC1 and polycomb group proteins (EZH2, SUZ12, and BMI1 paralleling with changes in the expression of repressive histone marks (H3K9me3 and H3K27me3 and stemness markers (Oct4, Nanog, Sox2, and Rex1. Therefore epigenetic factors are important for AF-MSCs senescence process that may be related with individuality of donor or a fetus malignancy status.

Studies on the interaction between the Ehrlich ascites tumor cell and its fluid environment

International Nuclear Information System (INIS)

Magnani, B.

1984-01-01

In this dissertation, the glycolytic nature of the Ehrlich ascites tumor (EAT) cell is disclosed both in vivo and in vitro by experiments challenging it with glucose. It is demonstrated that EAT cells can cause the extracellular pH to drop to values sufficiently acidic so as to inhibit EAT glycolysis. However, the extracellular fluid or the Ascites Supernatant Fluid (ASF) reduced the extent to which the pH dropped during EAT cell glycolysis. A comparison of the activities of the sera from tumor-bearing mice and normal mice revealed that the serumfrom the tumor-bearing mice reduced the pH fall generated by the EAT cell in the same way as did ASF; normal mouse serum had no such effect. The metabolic pathways utilized during glucose catabolism were examined by radio-respirometry and the results demonstrated that the high percentage of the glucose conversion to lactate occurred because of partial blockade of the TCA cycle. The databolism of glutamine, glutamic acid, asparagine, aspartic acid, and alanine was enhanced by ASF as determined by measuring 14 CO 2 from 14 C-labelled amino acids, with glutamine catabolism enhanced about three-fold. Fractionation experiments revealed that ASF contained a factor(s) responsible for this enhancement that had a molecular weight greater than 300,000 daltons and was heat-labile

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients.

Science.gov (United States)

Maggi, Laura; Margheri, Francesca; Luciani, Cristina; Capone, Manuela; Rossi, Maria Caterina; Chillà, Anastasia; Santarlasci, Veronica; Mazzoni, Alessio; Cimaz, Rolando; Liotta, Francesco; Maggi, Enrico; Cosmi, Lorenzo; Del Rosso, Mario; Annunziato, Francesco

2016-01-01

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

In Vitro Culture Conditions for Maintaining a Complex Population of Human Gastrointestinal Tract Microbiota

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Bong-Soo Kim

2011-01-01

Full Text Available A stable intestinal microbiota is important in maintaining human physiology and health. Although there have been a number of studies using in vitro and in vivo approaches to determine the impact of diet and xenobiotics on intestinal microbiota, there is no consensus for the best in vitro culture conditions for growth of the human gastrointestinal microbiota. To investigate the dynamics and activities of intestinal microbiota, it is important for the culture conditions to support the growth of a wide range of intestinal bacteria and maintain a complex microbial community representative of the human gastrointestinal tract. Here, we compared the bacterial community in three culture media: brain heart infusion broth and high- and low-carbohydrate medium with different growth supplements. The bacterial community was analyzed using denaturing gradient gel electrophoresis (DGGE, pyrosequencing and real-time PCR. Based on the molecular analysis, this study indicated that the 3% fecal inoculum in low-concentration carbohydrate medium with 1% autoclaved fecal supernatant provided enhanced growth conditions to conduct in vitro studies representative of the human intestinal microbiota.

Conversion of Squid Pen to Homogentisic Acid via Paenibacillus sp. TKU036 and the Antioxidant and Anti-Inflammatory Activities of Homogentisic Acid

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San-Lang Wang

2016-10-01

Full Text Available The culture supernatant of Paenibacillus sp. TKU036, a bacterium isolated from Taiwanese soils, showed high antioxidant activity (85% when cultured in a squid pen powder (SPP-containing medium at 37 °C for three days. Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA was isolated and found to be the major antioxidant in the culture supernatant of the SPP-containing medium fermented by Paenibacillus sp. TKU036. Tryptophan was also present in the culture supernatant. The results of high-performance liquid chromatography (HPLC fingerprinting showed that HGA and tryptophan were produced via fermentation but did not pre-exist in the unfermented SPP-containing medium. Neither HGA nor tryptophan was found in the culture supernatants obtained from the fermentation of nutrient broth or other chitinous material, i.e., medium containing shrimp head powder, by Paenibacillus sp. TKU036. The production of HGA via microorganisms has rarely been reported. In this study, we found that squid pen was a potential carbon and nitrogen source for Paenibacillus sp. Tryptophan (105 mg/L and HGA (60 mg/L were recovered from the culture supernatant. The isolated HGA was found to have higher antioxidant activity (IC50 = 6.9 μg/mL than α-tocopherol (IC50 = 17.6 μg/mL. The anti-inflammatory activity of the isolated HGA (IC50 = 10.14 μg/mL was lower than that of quercetin (IC50 = 1.14 μg/mL. As a result, squid pen, a fishery processing byproduct, is a valuable material for the production of tryptophan and the antioxidant and anti-inflammatory HGA via microbial conversion.

Antimicrobial Activity of Cell Free Supernatant of Irradiated Lactic Acid Bacteria Isolates

International Nuclear Information System (INIS)

Abdelaleem, M.A.; AL-Hagar, O.E.Aa.

2015-01-01

Attempts were made to isolate bio preservatives using food wastes with no value and low cost. Whey is the raw material achieved that value. Whey and many other food wastes are used in our study to isolate Lactic acid bacteria (LAB). Cell free supernatants (CFS) of isolates are used to evaluate their antimicrobial activity against indicator pathogenic bacterial strains. CFS-9 isolate from whey has the highest inhibitory activity compared to all other isolates. The inhibitory activity of CFS-9, Nisin (400 IU / ml) and the standard Lactococcus Lactis Subsp. Lactis ATCC 11454 (Lacto) were determined. Furthermore, isolate-9 and Lacto strains were exposed to irradiation at different doses. The inhibition zones of; control isolate-9 (non-irradiated) showed the highest values against all indicator strains, CFS of irradiated Lacto at dose 250 Gy was the highest value against Bacillus cereus and Escherichia coli compared to other irradiation treatments, CFS of irradiated Lacto at dose 100 Gy was the highest value against Staph aureus, while the inhibition zone was in the highest value in CFS of irradiated Lacto at dose 500 Gy against Salmonella typhimurium. Nisin (400 IU / ml) was significantly higher than all CFS of irradiated isolate-9 while, the inhibition zones of all CFS-Lacto (irradiated and nonirradiated) are better and higher than nisin-400

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

Science.gov (United States)

Uteng, Marianne; Hauge, Håvard Hildeng; Brondz, Ilia; Nissen-Meyer, Jon; Fimland, Gunnar

2002-01-01

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis. PMID:11823243

Diagnosis Of Persistent Infection In Prosthetic Two-Stage Exchange: PCR analysis of Sonication fluid From Bone Cement Spacers.

Science.gov (United States)

Mariaux, Sandrine; Tafin, Ulrika Furustrand; Borens, Olivier

2017-01-01

Introduction: When treating periprosthetic joint infections with a two-stage procedure, antibiotic-impregnated spacers are used in the interval between removal of prosthesis and reimplantation. According to our experience, cultures of sonicated spacers are most often negative. The objective of our study was to investigate whether PCR analysis would improve the detection of bacteria in the spacer sonication fluid. Methods: A prospective monocentric study was performed from September 2014 to January 2016. Inclusion criteria were two-stage procedure for prosthetic infection and agreement of the patient to participate in the study. Beside tissues samples and sonication, broad range bacterial PCRs, specific S. aureus PCRs and Unyvero-multiplex PCRs were performed on the sonicated spacer fluid. Results: 30 patients were identified (15 hip, 14 knee and 1 ankle replacements). At reimplantation, cultures of tissue samples and spacer sonication fluid were all negative. Broad range PCRs were all negative. Specific S. aureus PCRs were positive in 5 cases. We had two persistent infections and four cases of infection recurrence were observed, with bacteria different than for the initial infection in three cases. Conclusion: The three different types of PCRs did not detect any bacteria in spacer sonication fluid that was culture-negative. In our study, PCR did not improve the bacterial detection and did not help to predict whether the patient will present a persistent or recurrent infection. Prosthetic 2-stage exchange with short interval and antibiotic-impregnated spacer is an efficient treatment to eradicate infection as both culture- and molecular-based methods were unable to detect bacteria in spacer sonication fluid after reimplantation.

Fluid mechanics in fluids at rest.

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Brenner, Howard

2012-07-01

Using readily available experimental thermophoretic particle-velocity data it is shown, contrary to current teachings, that for the case of compressible flows independent dye- and particle-tracer velocity measurements of the local fluid velocity at a point in a flowing fluid do not generally result in the same fluid velocity measure. Rather, tracer-velocity equality holds only for incompressible flows. For compressible fluids, each type of tracer is shown to monitor a fundamentally different fluid velocity, with (i) a dye (or any other such molecular-tagging scheme) measuring the fluid's mass velocity v appearing in the continuity equation and (ii) a small, physicochemically and thermally inert, macroscopic (i.e., non-Brownian), solid particle measuring the fluid's volume velocity v(v). The term "compressibility" as used here includes not only pressure effects on density, but also temperature effects thereon. (For example, owing to a liquid's generally nonzero isobaric coefficient of thermal expansion, nonisothermal liquid flows are to be regarded as compressible despite the general perception of liquids as being incompressible.) Recognition of the fact that two independent fluid velocities, mass- and volume-based, are formally required to model continuum fluid behavior impacts on the foundations of contemporary (monovelocity) fluid mechanics. Included therein are the Navier-Stokes-Fourier equations, which are now seen to apply only to incompressible fluids (a fact well-known, empirically, to experimental gas kineticists). The findings of a difference in tracer velocities heralds the introduction into fluid mechanics of a general bipartite theory of fluid mechanics, bivelocity hydrodynamics [Brenner, Int. J. Eng. Sci. 54, 67 (2012)], differing from conventional hydrodynamics in situations entailing compressible flows and reducing to conventional hydrodynamics when the flow is incompressible, while being applicable to both liquids and gases.

Calcium carbonate mediates higher lignin peroxidase activity in the culture supernatant of Streptomyces Viridosporus T7A

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J. M. B. MACEDO

1999-06-01

Full Text Available Lignin peroxidase (LiP production has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by S. viridosporus T7A which have been studied include the improvement of yield and enzyme stabilization. In experiments performed in agitated flasks containing culture media composed of yeast extract as the source of nitrogen, mineral salts and different carbon sources, the use of glucose resulted in the highest values for LiP activity (350 U/L, specific LiP activity (450 U/g and productivity (7 U/L/h. As the profile obtained with glucose-containing medium suggested enzyme instability, the effect of calcium carbonate was evaluated. The addition of CaCO3 in two different concentrations, 0.5% and 5.0%, resulted in higher values of maximum LiP activity, 600 and 900 U/L, respectively. The presence of this salt also anticipated enzyme activity peaks and allowed the detection of higher enzyme activities in the extracellular medium for longer periods of time. These results indicate a positive effect of calcium carbonate on LiP production, which is extremely relevant for industrial processes.

Cultivation characteristics and gene expression profiles of Aspergillus oryzae by membrane-surface liquid culture, shaking-flask culture, and agar-plate culture.

Science.gov (United States)

Imanaka, Hiroyuki; Tanaka, Soukichi; Feng, Bin; Imamura, Koreyoshi; Nakanishi, Kazuhiro

2010-03-01

We cultivated a filamentous fungus, Aspergillus oryzae IAM 2706 by three different cultivation methods, i.e., shaking-flask culture (SFC), agar-plate culture (APC), and membrane-surface liquid culture (MSLC), to elucidate the differences of its behaviors by different cultivation methods under the same media, by measuring the growth, secretion of proteases and alpha-amylase, secreted protein level, and gene transcriptional profile by the DNA microarray analysis. The protease activities detected by MSLC and APC were much higher than that by SFC, using both modified Czapek-Dox (mCD) and dextrin-peptone-yeast extract (DPY) media. The alpha-amylase activity was detected in MSLC and APC in a much larger extent than that in SFC when DPY medium was used. On the basis of SDS-PAGE analyses and N-terminal amino acid sequences, 6 proteins were identified in the supernatants of the culture broths using DPY medium, among which oryzin (alkaline protease) and alpha-amylase were detected at a much higher extent for APC and MSLC than those for SFC while only oryzin was detected in mCD medium, in accordance with the activity measurements. A microarray analysis for the fungi cultivated by SFC, APC, and MSLC using mCD medium was carried out to elucidate the differences in the gene transcriptional profile by the cultivation methods. The gene transcriptional profile obtained for the MSLC sample showed a similar tendency to the APC sample while it was quite different from that for the SFC sample. Most of the genes specifically transcribed in the MSLC sample versus those in the SFC sample with a 10-fold up-regulation or higher were unknown or predicted proteins. However, transcription of oryzin gene was only slightly up-regulated in the MSLC sample and that of alpha-amylase gene, slightly down-regulated. Copyright 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Development of a novel method for amniotic fluid stem cell storage.

Science.gov (United States)

Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; De Pol, Anto; La Sala, Giovanni B; Maraldi, Tullia

2017-08-01

Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

DEFF Research Database (Denmark)

Gräs, S; Ovesen, P; Andersen, A N

1994-01-01

Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicular....../l, respectively. VIP at a concentration of 10 nmol/l caused a significant increase in [3H]thymidine incorporation, and at 1000 nmol/l a significant increase in oestradiol secretion was observed. VIP had no effect on progesterone secretion. PHM at the concentrations tested did not influence any of the activities...

[Lessening effect of hypoxia-preconditioned rat cerebrospinal fluid on oxygen-glucose deprivation-induced injury of cultured hippocampal neurons in neonate rats and possible mechanism].

Science.gov (United States)

Niu, Jing-Zhong; Zhang, Yan-Bo; Li, Mei-Yi; Liu, Li-Li

2011-12-25

The present study was to investigate the effect of cerebrospinal fluid (CSF) from the rats with hypoxic preconditioning (HPC) on apoptosis of cultured hippocampal neurons in neonate rats under oxygen glucose deprivation (OGD). Adult Wistar rats were exposed to 3 h of hypoxia for HPC, and then their CSF was taken out. Cultured hippocampal neurons from the neonate rats were randomly divided into four groups (n = 6): normal control group, OGD group, normal CSF group and HPC CSF group. OGD group received 1.5 h of incubation in glucose-free Earle's solution containing 1 mmol/L Na2S2O4, and normal and HPC CSF groups were subjected to 1 d of corresponding CSF treatments followed by 1.5 h OGD. The apoptosis of neurons was analyzed by confocal laser scanning microscope and flow cytometry using Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in normal control group, whereas the number of apoptotic cells was greatly increased in OGD group. Both normal and HPC CSF could decrease the apoptosis of cultured hippocampal neurons injured by OGD (P neurons by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

Immunopotentiation by a new antitumor polysaccharide, DMG, a degraded D-manno-D-glucan from Microellobosporia grisea culture fluid.

Science.gov (United States)

Nakajima, H; Kita, Y; Takashi, T; Akasaki, M; Yamaguchi, F; Ozawa, S; Tsukada, W; Abe, S; Mizuno, D

1984-03-01

The immunopharmacological behavior of DMG, an antitumor polysaccharide, was studied in mice. DMG administered ip or sc stimulated peritoneal macrophages to produce high levels of interleukin-1 activity, which can amplify successive immune responses. DMG dose-dependently and schedule-dependently increased the cellular immune response against allogeneic tumor cells and the humoral immune response to sheep erythrocytes. DMG also enhanced nonspecific antitumor effector functions, such as natural killer activity of spleen and peritoneal cells, and the cytostatic activity of peritoneal macrophages. Peritoneal macrophages activated by ip or sc injection of DMG exhibited high cytostatic activity, especially after exposure in vitro to lymphokine supernatants containing macrophage activation factor. Moreover, granulocyte/macrophage colony-stimulating activity in the serum increased 2-10 hr after DMG administration. Thus, DMG potentiated antigen-specific immunological functions and nonspecific functions of host defense systems against cancer both qualitatively and quantitatively.

Isolation and risk assessment of Geotrichum spp. in the white shrimp (Litopenaeus vannamei Boone, 1931 from culture ponds

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José Luis Ochoa

2015-09-01

Full Text Available The present study was done in order to identify the fungus invading some of the supralittoral ponds used for shrimp aquaculture in the CIBNOR facilities in La Paz, Baja California Sur (BCS, México during the summer season. From the walls and bottoms of the ponds, two strains of Geotrichum spp. were isolated and morphologically identified. Fungal adhesion towards hemocytes and primary cultures of various white shrimp (Litopeneaus vannamei tissues (gill, tegument, and gut was analyzed to determine infectivity. Extracellular protease, lipase, and amylase activity were evaluated as virulence factors. Survival of shrimp post-larvae (PL8 exposed to fungal culture supernatant or to their filaments was also investigated. The results showed that shrimp tegument cells and hemocytes were very susceptible to Geotrichum spp. invasion, and that this fungus provokes great mortality of post-larvae. Hence, Geotrichum spp. could be considered an opportunistic pathogen that might represent a serious health risk to shrimp in culture.

Effect of phenobarbital pretreatment on benzene biotransformation in the rat. Pt. 2. 9. 000 g supernatant and isolated perfused liver versus living rat

Energy Technology Data Exchange (ETDEWEB)

Gut, I.; Hatle, K.; Zizkova, L.

1981-03-01

Factors responsible for different quantitative effect of phenobarbital (PB) pretreatment on benzene metabolism to phenol in vivo and in vitro were studied in male Wistar rats. A more than 4-fold increase of benzene metabolism was observed with 9,000 g supernatant of liver homogenate, 2.8- to 4-fold increase with isolated perfused liver; phenol formation in vivo after oral benzene was increased by PB 2-fold, but only shortly following benzene administration and the enhancement rapidly diminished to 1.15-fold increase in the total excreted phenol. Benzene concentrations in 9,000 g supernatant incubations were 2 mM, those with isolated perfused livers were up to 4 mM, but those in blood in vivo were below 0.3 mM; the effect of PB induction in vivo disappeared along with decreasing benzene and increasing phenol blood concentrations which surpassed benzene 2-3 h after oral benzene administration. The effect of benzene concentration on the manifestation of PB induction is also supported by almost a 2-fold increased phenol formation in PB rats over controls in vivo after repeated administration of benzene. The elimination of radioactive metabolites of orally administered benzene-/sup 14/C, in urine was markedly inhibited by intraperitoneal administration of phenol, but not by pyrocatechol, resorcinol or hydroquinol suggesting that phenol might inhibit benzene metabolism in vivo especially when its concentration exceeds that of benzene.

Antibody-based enzyme-linked lectin assay (ABELLA) for the sialylated recombinant human erythropoietin present in culture supernatant.

Science.gov (United States)

Kim, Hyoung Jin; Lee, Seung Jae; Kim, Hong-Jin

2008-11-04

The terminal sialic acid of human erythropoietin (hEPO) is essential for in vivo activity. The current resorcinol and HPLC methods for analyzing alpha2,3-linked sialic acid require more than a microgram of purified rhEPO, and purification takes a great deal of time and labor. In this study, we assessed the use of an antibody-based enzyme-linked lectin assay (ABELLA) for analyzing non-purified recombinant hEPO (rhEPO). The major problem of this method was the high background due to terminal sialylation of components of the assay (antibody and bovine serum albumin) other than rhEPO. To solve this problem, we used a monoclonal antibody (Mab 287) to capture the rhEPO, and oxidized the bovine serum albumin used for blocking with meta-periodate. The sialic acid content of non-purified rhEPO measured by ABELLA was similar to that obtained by the resorcinol method on purified rhEPO. ABELLA has advantages such as adaptability and need for minimal amounts of rhEPO (40 ng/ml). Our observations suggest that ABELLA should reduce the time and labor needed to improve culture conditions so as to increase protein sialylation, and also facilitate the study of sialylation mechanisms.

Novel culturing platform for brain slices and neuronal cells

DEFF Research Database (Denmark)

Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

2015-01-01

In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions....

TR146 cells grown on filters as a model of human buccal epithelium

DEFF Research Database (Denmark)

Mørck Nielsen, H; Rømer Rassing, M; Nielsen, Hanne Mørck

2000-01-01

cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity...... of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70+/-2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase...

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital

OpenAIRE

Tadros, Manal; Petrich, Astrid

2013-01-01

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit – the Sepsityper Kit (Bruker Daltonik, Germany) – and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in com...

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa.

Science.gov (United States)

Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

2015-04-01

The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the

Experimental Clostridium perfringens type D enterotoxemia in goats.

Science.gov (United States)

Uzal, F A; Kelly, W R

1998-03-01

The effects of intraduodenal administration of Clostridium perfringens cultures and culture products in goats were evaluated to develop a reliable experimental model of enterotoxemia in this species. Five conventionally reared, 11-16-week-old Angora goat kids were dosed intraduodenally with whole cultures of C. perfringens type D; five similar animals were dosed with C. perfringens type D filtered culture supernatant; and a third group of five kids was dosed with C. perfringens type D washed cells. Two kids were used as controls and received sterile, nontoxic culture medium intraduodenally. All animals received starch solution into the abomasum. All five kids inoculated with whole culture and three of five dosed with culture supernatant and with washed cells developed central nervous system signs. Diarrhea was observed in two of five kids inoculated with whole culture, in all five of those dosed with culture supernatant, and in three of five of those that received washed cells. The most striking postmortem findings consisted of lung edema, necrotizing pseudomembranous colitis, and cerebral vasogenic edema. The protocol thus provided a reasonable model of naturally occurring enterotoxemia in goats, producing a range of clinical signs and postmortem changes similar to those observed in the natural disease.

Synthesis of silver nanoparticles by Bacillus subtilis T-1 growing on agro-industrial wastes and producing biosurfactant.

Science.gov (United States)

Płaza, Grażyna Anna; Chojniak, Joanna; Mendrek, Barbara; Trzebicka, Barbara; Kvitek, Libor; Panacek, Ales; Prucek, Robert; Zboril, Radek; Paraszkiewicz, Katarzyna; Bernat, Przemysław

2016-04-01

In this study, culture supernatnats of Bacillus subtilis T-1 growing on brewery effluents and molasses was used for silver nanoparticles (Ag-NPs) synthesis. The biosurfactant production of B. subtilis T-1 was confirmed by the detection of genes in the genome and by the identification of the product in the supernatants. The genes for synthesis of surfactin (sfp, srfAA) and iturin (ituC) were noted by PCR reactions. Also, in examined culture supernatants the presence of C13, C14 and C15 surfactin homologues with the sodiated molecules [M + Na](+) at m/z 1030, 1044 and 1058 was confirmed using LC/MS/MS analysis. The formation of NPs in the culture supernatants was confirmed by UV-vis spectroscopy. The dynamic light scattering measurements and transmission electron microscopy images showed the nanometric sizes of the biosynthesised Ag-NPs which ranged from several nm to several tens of nm depending on the used culture supernatant. Biological properties of Ag-NPs were evaluated by binding of Ag-NPs with DNA isolated from the Escherichia coli ATCC 25922 and B. subtilis ATCC 6633. Biogenic Ag-NPs were actively bound to DNA in increased concentration which could be the one important mode of antibacterial action of the Ag-NPs.

Fluids in crustal deformation: Fluid flow, fluid-rock interactions, rheology, melting and resources

Science.gov (United States)

Lacombe, Olivier; Rolland, Yann

2016-11-01

Fluids exert a first-order control on the structural, petrological and rheological evolution of the continental crust. Fluids interact with rocks from the earliest stages of sedimentation and diagenesis in basins until these rocks are deformed and/or buried and metamorphosed in orogens, then possibly exhumed. Fluid-rock interactions lead to the evolution of rock physical properties and rock strength. Fractures and faults are preferred pathways for fluids, and in turn physical and chemical interactions between fluid flow and tectonic structures, such as fault zones, strongly influence the mechanical behaviour of the crust at different space and time scales. Fluid (over)pressure is associated with a variety of geological phenomena, such as seismic cycle in various P-T conditions, hydrofracturing (including formation of sub-horizontal, bedding-parallel veins), fault (re)activation or gravitational sliding of rocks, among others. Fluid (over)pressure is a governing factor for the evolution of permeability and porosity of rocks and controls the generation, maturation and migration of economic fluids like hydrocarbons or ore forming hydrothermal fluids, and is therefore a key parameter in reservoir studies and basin modeling. Fluids may also help the crust partially melt, and in turn the resulting melt may dramatically change the rheology of the crust.

Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model.

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Aurelia Syngkon

Full Text Available BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP and V. cholerae protease (PrtV. The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL. METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3, CHA6.8 (FA ratio 1.08+/-0.2 n = 3, CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3 and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3 induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3 and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3 induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

Poly-N-acetylglucosamine matrix polysaccharide impedes fluid convection and transport of the cationic surfactant cetylpyridinium chloride through bacterial biofilms.

Science.gov (United States)

Ganeshnarayan, Krishnaraj; Shah, Suhagi M; Libera, Matthew R; Santostefano, Anthony; Kaplan, Jeffrey B

2009-03-01

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-beta(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.

DNA methylation patterns of imprinting centers for H19, SNRPN, and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell

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Hsiu-Huei Peng

2012-09-01

Conclusion: In conclusion, human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature during in vitro cell culture. H19 and KCNQ1OT1 possessed a substantial degree of hypermethylation status, and variable DNA methylation patterns of SNRPN was observed during in vitro cell culture of human amniotic fluid mesenchymal stem cells. Our results urge further understanding of epigenetic status of human amniotic fluid mesenchymal stem cells before it is applied in cell replacement therapy.

The experimental study of VEGF antisense oligodeoxynucleotides with lipiodol in arterial embolization of liver cancer in rats

International Nuclear Information System (INIS)

Wu Hanping; Feng Gansheng; Li Xin; Liang Huimin; Zheng Chuansheng

2003-01-01

Objective: To study the inhibitory effects of VEGF antisense oligodeoxynucleotides (asODN) on cultured Walker-256 cells' VEGF expression, and to observe the anti-tumor effects of intraarterial infusion of asODN mixed with lipiodol on rat liver cancer. Methods: VEGF asODN and sense ODN were added to the media of non-serum cultured Walker-256 cells, and the VEGF concentrations of the supernatants were detected by using ELISA 48 hours later. Cells of endothelial cell line ECV-304 were cultured in the supernatants. The growth of ECV-304 cells was observed by MTT method. 30 rats with Walker-256 carcinoma cells implanted into left liver lobe were randomly divided into 3 groups. 0.2 ml ultra-fluid lipiodol (UFLP group, n=10), 3OD asODN mixed with 0.2 ml ultra-fluid lipiodol (UFLP + asODN group, n=10), and 0.2 ml normal saline (control group, n=10) were infused into the hepatic artery. The volumes of tumors were measured by using MRI before and 7 days after the treatment. VEGF mRNA in cancerous and peri-cancerous tissues was detected by RT-PCR. The microvessel density (MVD) and VEGF expression were observed by immunohistochemistry. Results: asODN could inhibit Walker-256 cells' VEGF expression. The tumor growth rate was lower in UFLP + asODN group than that in UFLP and control groups [(140.1±33.8)%, (177.9±64.9)%, and (403.9± 69.4)%, respectively, F=60.02, P 0.05). The MVD in UFLP + asODN group (53.1±18.4) was significantly less than that of control group (73.2±20.4) and UFLP group (80.3±18.5) (F=5.44, P<0.05). Conclusion: VEGF asODN could inhibit VEGF expression of Walker-256 cells. It may be an antiangiogenesis therapy drug in malignant tumor. VEGF asODN mixed with UFLP in embolizing liver cancer could decrease liver cancer growth, VEGF expression, and microvessel density better than UFLP alone

Solid-phase radioimmunoassay for Epstein-Barr virus-associated membrane antigen prepared from B95-8 cell culture supernatants

International Nuclear Information System (INIS)

Doelken, G.; Klein, G.

1977-01-01

Epstein-Barr virus (EBV)-associated membrane antigen (MA) was concentrated from B95-8 cell culture media by precipitation with polyethylene glycol followed by chromatography on Bio-Gel A-50m. In a RAJI cell-binding assay, MA-positive material could only be found in the void volume of the column. After ultracentrifugation all antigenic activity appeared in the pellet, which suggested that MA was present in aggregates, presumably fragments of cellular membranes and/or virus envelopes. The MA-containing preparation was photopolymerized in polyacrylamide gel. The homogenized gel was used in a solid-phase radioimmunoassay with 125 I-labeled IgG from an anti-MA positive reference serum and an anti-MA negative control serum. The specificity of the reaction was confirmed in blocking tests with anti-EBV positive and negative sera. A good correlation was found between the results obtained in the radioimmunoassay and the results obtained in direct immunofluorescence tests for the detection of MA. The existence of at least two subspecificities of the MA complex could be confirmed by this radioimmunoassay

Specific proliferative response of human lymphocytes to purified soluble antigens from Plasmodium falciparum in vitro cultures and to antigens from malaria patients' sera

DEFF Research Database (Denmark)

Bygbjerg, I C; Jepsen, S; Theander, T G

1985-01-01

Antigens of Plasmodium falciparum, in supernatants of in vitro cultures of the parasite were affinity purified on columns prepared with the IgG fraction of the serum of an immune individual. The purified antigens induced proliferation of lymphocytes from persons who had recently had malaria....... The responses were strongest with lymphocytes from individuals infected with falciparum and ovale malaria; vivax malaria infections induced a lower level of response and lymphocytes of unsensitized individuals were little affected. Lymphocytes from unsensitized individuals did not respond to the affinity...

Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17β and progesterone concentrations in preovulatory follicular fluid

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M. Matsuo

2017-10-01

Full Text Available The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM culture system imitating estradiol-17β (E2 and progesterone (P4 concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs were collected from follicles (2 to 8 mm in diameter of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1 culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group; (2 culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group; or (3 culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group. The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group. After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

International Nuclear Information System (INIS)

Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

1985-01-01

It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [ 14 C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [ 14 C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen

Role of interleukin-1 in antigen presentation by normal articular chondrocytes

International Nuclear Information System (INIS)

Tiku, M.L.; Liu, S.; Tiku, K.

1986-01-01

Recently the authors have described that normal articular chondrocytes of rabbits present antigen to immune T cells. In the present study the authors investigated the role of interleukin-1 (IL-1) on antigen presentation by chondrocytes. For these experiments the antigen pulsed chondroyctes were either untreated or fixed with paraformaldehyde and then co-cultured with immune T cells. T cell proliferation was measured by 3 H-thymidine incorporation. Pulsed non-fixed chondrocytes presented antigen, as expected, but pulsed and fixed cells failed to present antigen to T cells. The 3 H-TdR incorporation was partially restored by addition of purified human IL-1. Next, IL-1 activity was measured in primary chondrocyte culture supernatants stimulated with or without lipopolysaccharide (LPS) in comitogen thymocyte assay. No activity was detected in chondrocyte supernatants. Propagated chondrocyte culture supernatants also lacked IL-1 activity when stimulated with LPS in the presence of increasing concentration of indomethacin. On the other hand the authors observed that chondrocyte culture supernatants in a dose dependent manner inhibited human IL-1 induced 3 H-TdR incorporation of murine thymocytes. This suggested that these cells may produce an inhibitor of IL-1 and IL-1 production by chondrocytes may be essential for T cell proliferation by these cells. Inability to detect IL-1 in chondrocyte supernatants may be due to the presence of an inhibitor to IL-1. These findings may help in elucidating the immunological mechanisms in situations where chondrocytes and T cell interact, such as in arthritis

Immunoregulation of antitumor response; differential secretion of arachidonic acid metabolites by macrophages during stimulation ''in vitro'' with BCG and ''Corynebacterium parvum''

International Nuclear Information System (INIS)

Tomecki, Jaroslaw; Sukiennik, Jadwiga; Kordowiak, Anna

1993-01-01

The level of arachidonic acid (AA) metabolites in the supernatants of cultures peritoneal exudate cells (PEC) were studied under various conditions using BCG and ''Corynebacterium parvum'' as stimulators. The metabolite levels were analyzed by thin layer chromatography (TLC). The degree of macrophage cytotoxic/cytostatic activity was dependent on the dose and character of stimulators used and the source of macrophages. The application of micro cytotoxicity assay for the evaluation of tumor cell lysis (lung sarcoma SaL-1) ''in vitro'' revealed that peritoneal macrophages from healthy and tumor bearing BALB/c mice may affect the degree of antitumor response. In the supernatants of cultured PEC from tumor bearing mice AA level increased (by 10-fold) in comparison with PEC from healthy mice. Stimulation with BCG induced over a double level of AA in PEC isolated from tumor bearing mice non-stimulated or stimulated with ''C.parvum''. A lower level of prostaglandins (PGs) was found in the supernatants of cultured PEC isolated from healthy mice (stimulated and non-stimulated), but the highest level of PGs was observed in the supernatants of cultured PEC isolated from tumor bearing mice stimulated with BCG. The unique metabolite of AA was found only in the supernatants form non-stimulated PEC from tumor bearing mice. PEC from tumor bearing mice produced metabolites of AA which were not detected in control group. These results suggest that macrophages also play a regulatory role by secretion of AA. This process can be modified by bacterial antigens. (author). 21 refs, 7 figs

Effect of Triptolide on Functions of Monocytes/ Macrophages in ...

African Journals Online (AJOL)

The number of monocytes/macrophages under the varying conditions was subsequently determined by methyl thiazolyl tetrazolium (MTT) assay. The supernatants were collected after 24-h culture, and the content of VEGF and VEGF-C in each supernatant measured by enzyme-linked immunosorbent assay (ELISA).

A thin-layer liquid culture technique for the growth of Helicobacter pylori.

Science.gov (United States)

Joo, Jung-Soo; Park, Kyung-Chul; Song, Jae-Young; Kim, Dong-Hyun; Lee, Kyung-Ja; Kwon, Young-Cheol; Kim, Jung-Min; Kim, Kyung-Mi; Youn, Hee-Shang; Kang, Hyung-Lyun; Baik, Seung-Chul; Lee, Woo-Kon; Cho, Myung-Je; Rhee, Kwang-Ho

2010-08-01

Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.

Kadar Prostaglandin F2? pada Cairan Vesikula Seminalis dan Produk Sel Monolayer Vesikula Seminalis Sapi Bali (CONCENTRATIONS OF PROSTAGLANDIN F2? IN SEMINAL VESICLE FLUID AND PRODUCT OF SEMINAL VESICLE MONOLAYER CELLS OF BALI CATTLE

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Tjok Gde Oka Pemayun

2007-12-01

Full Text Available In this study, the concentration of prostaglandin F2 ? (PGF2? in seminal vesicle fluid and seminal vesicle monolayer cell cultures of Bali cattle was determined. The seminal vesicle fluid was aspirated and the epithelial cells of the seminal vesicles were cultured in tissue culture medium (TCM 199 growth medium containing 10% fetal calf serum (FCS and 10% oestrus mares serum (EMS with a density of 1.9 x 106 cells / ml medium. Following an incubation at 38.50 C in 5% CO2 atmosphere for 6 days and the level of PGF2 ? in the original seminal vesicle fluid and in the cell culture medium were determined by radioimmunoassay techniques (RIA. The results showed that the level of PGF2 ? in the non-extracted monolayer culture of seminal vesicle (1287,50 ± 3,39 pg/ml was significantly higher than that of detected in non-extracted seminal vesicle fluid (1,23 ± 0,79 pg/ml. In contrast, after extraction the level of PGF2 ? in seminal vesicle monolayer cell cultures (218,33 ± 2,87 pg/ml significantly decreased as compared to seminal vesicle fluid (1750,83 ± 2,71 pg/ml. In conclusion the highest level of PGF2 ? was found in the extract of seminal vesicle fluid.

Revision washout decreases implant capsule tissue culture positivity: a multicenter study.

Science.gov (United States)

Henry, Gerard D; Carson, Culley C; Wilson, Steven K; Wiygul, Jeremy; Tornehl, Chris; Cleves, Mario A; Simmons, Caroline J; Donatucci, Craig F

2008-01-01

Positive cultures, visible biofilm and confocal micrography confirm bacterial presence on clinically uninfected inflatable penile prostheses at revision surgery. Salvage irrigation has been proved to rescue patients with clinically infected inflatable penile prostheses. Similar washout at revision for noninfectious reasons significantly lowers subsequent infection rates. We investigated a larger series of patients for positive culture rates and evaluated implant capsule tissue culture rates before and after revision washout. At 4 institutions a total of 148 patients with inflatable penile prostheses underwent revision surgery for noninfectious reasons between June 2001 and September 2005. Swab cultures of the fluid around the pump and visible biofilm were obtained. Also, in 65 patients a wedge of tissue from the capsule that forms around the pump was cultured. After implant removal revision washout of the implant spaces was performed and a second wedge of tissue was cultured. Of the 148 patients 97 (66%) had positive bacterial swab cultures of the fluid around the pump or biofilm. A total of 124 isolates were cultured. Of the 65 implant capsule tissue cultures obtained before washout 28 (43%) were positive for bacteria, while 16 (25%) obtained after revision washout were positive. Positive cultures and visible bacterial biofilm are present on clinically uninfected inflatable penile prostheses at revision surgery in most patients. Revision washout appears to decrease the bacterial load on implant capsule tissue at revision surgery of inflatable penile prostheses for noninfectious reasons.

Antisera production to detect indoleacetic acid in cultures of plant-growth promoting bacteria

International Nuclear Information System (INIS)

Rojas, Marcia M; Hernandez, Annia; Rives, Narovis; Tejera, Berto; Acebo, Yanelis; Heydrich, Mayra

2012-01-01

Rabbit polyclonal antisera against indoleacetic acid (IAA) bound to nitrocellulose membrane were obtained, which exhibited a high titer and specificity. The dot immunobinding technique with colloidal gold was used to detect auxin production by several strains belonging to Gluconacetobacter, Herbaspirillum, Azospirillum, Pseudomonas, Burkholderia and Bacillus genera, using culture supernatants as antigens. Moreover, auxin production was quantified by the Salkowski's method to corroborate the previous results. It was found that that all the studied microorganisms produce IAA and the feasibility of using these antisera to detect the metabolite was confirmed. Taking into account the potentialities of plant growth promoting bacteria as biofertilizers, the use of these antisera for a rapid and easy detection of IAA in bacteria associated with important crops is thus recommended.

Connection Between Thermodynamics and Dynamics of Simple Fluids in Pores: Impact of Fluid-Fluid Interaction Range and Fluid-Solid Interaction Strength.

Science.gov (United States)

Krekelberg, William P; Siderius, Daniel W; Shen, Vincent K; Truskett, Thomas M; Errington, Jeffrey R

2017-08-03

Using molecular simulations, we investigate how the range of fluid-fluid (adsorbate-adsorbate) interactions and the strength of fluid-solid (adsorbate-adsorbent) interactions impact the strong connection between distinct adsorptive regimes and distinct self-diffusivity regimes reported in [Krekelberg, W. P.; Siderius, D. W.; Shen, V. K.; Truskett, T. M.; Errington, J. R. Langmuir 2013 , 29 , 14527-14535]. Although increasing the fluid-fluid interaction range changes both the thermodynamics and the dynamic properties of adsorbed fluids, the previously reported connection between adsorptive filling regimes and self-diffusivity regimes remains. Increasing the fluid-fluid interaction range leads to enhanced layering and decreased self-diffusivity in the multilayer-formation regime but has little effect on the properties within film-formation and pore-filling regimes. We also find that weakly attractive adsorbents, which do not display distinct multilayer formation, are hard-sphere-like at super- and subcritical temperatures. In this case, the self-diffusivity of the confined and bulk fluid has a nearly identical scaling-relationship with effective density.

Differential expression of genes of Xylella fastidiosa in xylem fluid of citrus and grapevine.

Science.gov (United States)

Shi, Xiangyang; Bi, Jianlong; Morse, Joseph G; Toscano, Nick C; Cooksey, Donald A

2010-03-01

Xylella fastidiosa causes a serious Pierce's disease (PD) in grapevine. Xylella fastidiosa cells from a PD strain were grown in a pure xylem fluid of a susceptible grapevine cultivar vs. xylem fluid from citrus, which is not a host for this strain of X. fastidiosa. When grown in grapevine xylem fluid, cells of the PD strain formed clumps and biofilm formed to a greater extent than in citrus xylem fluid, although the PD strain did grow in xylem fluid of three citrus varieties. The differential expression of selected genes of a PD X. fastidiosa strain cultured in the two xylem fluids was analyzed using a DNA macroarray. Compared with citrus xylem fluid, grapevine xylem fluid stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as gacA, algU, xrvA, and hsq, and also genes involved in the biogenesis of pili and twitching motility, such as fimT, pilI, pilU, and pilY1. Increased gene expression likely contributes to PD expression in grapevine, whereas citrus xylem fluid did not support or possibly suppressed the expression of these virulence genes.

Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

Science.gov (United States)

Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

2011-01-01

We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

Effect of fluid motion on colony formation in Microcystis aeruginosa

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Lin Li

2013-01-01

Full Text Available Microcystis aeruginosa, generally occurring in large colonies under natural conditions, mainly exists as single cells in laboratory cultures. The mechanisms involved in colony formation in Microcystis aeruginosa and their roles in algal blooms remain unknown. In this study, based on previous research findings that fluid motion may stimulate the colony formation in green algae, culture experiments were conducted under axenic conditions in a circular water chamber where the flow rate, temperature, light, and nutrients were controlled. The number of cells of Microcystis aeruginosa, the number of cells per colony, and the colonial characteristics in various growth phases were observed and measured. The results indicated that the colony formation in Microcystis aeruginosa, which was not observed under stagnant conditions, was evident when there was fluid motion, with the number of cells per largest colony reaching 120 and the proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colony reaching their peak values at a flow rate of 35 cm/s. Based on the analysis of colony formation process, fluid motion stimulates the colony formation in Microcystis aeruginosa in the lag growth phase, while flushes and disaggregates the colonies in the exponential growth phase. The stimulation effect in the lag growth phase may be attributable to the involvement of fluid motion in a series of physiological processes, including the uptake of trace elements and the synthesis and secretion of polysaccharides. In addition, the experimental groups exhibiting typical colonial characteristics in the lag growth phase were found to have higher cell biomass in the later phase.

Decreased levels of sRAGE in follicular fluid from patients with PCOS.

Science.gov (United States)

Wang, BiJun; Li, Jing; Yang, QingLing; Zhang, FuLi; Hao, MengMeng; Guo, YiHong

2017-03-01

This study aimed to explore the association between soluble receptor for advanced glycation end products (sRAGE) levels in follicular fluid and the number of oocytes retrieved and to evaluate the effect of sRAGE on vascular endothelial growth factor (VEGF) in granulosa cells in patients with polycystic ovarian syndrome (PCOS). Two sets of experiments were performed in this study. In part one, sRAGE and VEGF protein levels in follicular fluid samples from 39 patients with PCOS and 35 non-PCOS patients were measured by ELISA. In part two, ovarian granulosa cells were isolated from an additional 10 patients with PCOS and cultured. VEGF and SP1 mRNA and protein levels, as well as pAKT levels, were detected by real-time PCR and Western blotting after cultured cells were treated with different concentrations of sRAGE. Compared with the non-PCOS patients, patients with PCOS had lower sRAGE levels in follicular fluid. Multi-adjusted regression analysis showed that high sRAGE levels in follicular fluid predicted a lower Gn dose, more oocytes retrieved, and a better IVF outcome in the non-PCOS group. Logistic regression analysis showed that higher sRAGE levels predicted favorably IVF outcomes in the non-PCOS group. Multi-adjusted regression analysis also showed that high sRAGE levels in follicular fluid predicted a lower Gn dose in the PCOS group. Treating granulosa cells isolated from patients with PCOS with recombinant sRAGE decreased VEGF and SP1 mRNA and protein expression and pAKT levels in a dose-dependent manner. © 2017 Society for Reproduction and Fertility.

Aneuploidy assessed by DNA index influences the effect of iron status on plasma and/or supernatant cytokine levels and progression of cells through the cell cycle in a mouse model.

Science.gov (United States)

Kuvibidila, Solo; Porretta, Connie; Baliga, Surendra

2014-02-01

Aneuploidy, a condition associated with altered chromosome number, hence DNA index, is frequently seen in many diseases including cancers and affects immunity. Iron, an essential nutrient for humans, modulates the immune function and the proliferation of normal and cancer cells. To determine whether impaired immunity seen in iron-deficient subjects may be related to aneuploidy, we measured spleen cell DNA index, percent of cells in different phases of the cell cycle, plasma and/or supernatant IL-2, IL-10, IL-12, and interferon-gamma in control, pair-fed, iron-deficient, and iron-replete mice (N=20-22/group). The test and control diets differed only in iron content (0.09mmol/kg versus 0.9mmol/kg) and were fed for 68days. Mean levels of hemoglobin and liver iron stores of iron-deficient and iron-replete mice were 40-60% lower than those of control and pair-fed mice (P<0.05). Mean plasma levels of IL-10, interferon-gamma and percent of cells in S+G2/M phases were lower in mice with than in those without aneuploidy (P<0.05). Lowest plasma IL-12 and interferon-gamma concentrations were observed in iron-deficient mice with aneuploidy. Mean percents of cultures with aneuploidy and DNA indexes were higher in iron-deficient and iron-replete than in control and pair-fed mice likely due to delayed cell division (P<0.05). Aneuploidy decreased the concentration of IL-2 and interferon-gamma in baseline cultures while it increased that of interferon-gamma in anti-CD3 treated cultures. Aneuploidic indexes negatively correlated with cytokine levels, percents of cells in S+G2/M phases and indicators of iron status (P<0.05). Although chromosome cytogenetics was not performed, for the first time, we report that increased aneuploidy rate may modulate the immune function during iron-deficiency. Copyright © 2014. Published by Elsevier Ltd.

Development program for magnetically assisted chemical separation: Evaluation of cesium removal from Hanford tank supernatant

International Nuclear Information System (INIS)

Nunez, L.; Buchholz, B.A.; Ziemer, M.; Dyrkacz, G.; Kaminski, M.; Vandegrift, G.F.; Atkins, K.J.; Bos, F.M.; Elder, G.R.; Swift, C.A.

1994-12-01

Magnetic particles (MAG*SEP SM ) coated with various absorbents were evaluated for the separation and recovery of low concentrations of cesium from nuclear waste solutions. The MAG*SEP SM particles were coated with (1) clinoptilolite, (2) transylvanian volcanic tuff, (3) resorcinol formaldehyde, and (4) crystalline silico-titanate, and then were contacted with a Hanford supernatant simulant. Particles coated with the crystalline silico-titanate were identified by Bradtec as having the highest capacity for cesium removal under the conditions tested (variation of pH, ionic strength, cesium concentration, and absorbent/solution ratio). The MAG*SEP SM particles coated with resorcinol formaldehyde had high distribution ratios values and could also be used to remove cesium from Hanford supernant simulant. Gamma irradiation studies were performed on the MAG*SEP SM particles with a gamma dose equivalent to 100 cycles of use. This irradiation decreased the loading capacity and distribution ratios for the particles by greater than 75%. The particles demonstrated high sensitivity to radiolytic damage due to the degradation of the polymeric regions. These results were supported by optical microscopy measurements. Overall, use of magnetic particles for cesium separation under nuclear waste conditions was found to be marginally effective

DEVELOPMENT OF PRIMARY CELL CULTURE FROM TAIL EPIDERMAL TISSUE OF KOI CARP (Cyprinus carpio koi

Directory of Open Access Journals (Sweden)

Lila Gardenia

2014-06-01

Full Text Available Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin. Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Primary cells infected with koi herpes virus produced typical cytopathic effects characterized by severe vacuolation and deformation of nuclei, which is consistent with those of previous reports. Artificial injection experiment by using supernatant koi herpes virus SKBM-1 isolate revealed that it could cause 90% mortality in infected fish within two weeks. PCR test with Sph I-5 specific primers carried out with DNA template from supernatant virus, pellet cell, and gills of infected fish showed positive results in all samples (molecular weight of DNA target 290 bp. The cells were found to be susceptible to koi herpes virus and can be used for virus propagation.

Closed-loop feedback control for microfluidic systems through automated capacitive fluid height sensing.

Science.gov (United States)

Soenksen, L R; Kassis, T; Noh, M; Griffith, L G; Trumper, D L

2018-03-13

Precise fluid height sensing in open-channel microfluidics has long been a desirable feature for a wide range of applications. However, performing accurate measurements of the fluid level in small-scale reservoirs (sensor contact needs to be avoided. In particular, gravity-driven systems used in several microfluidic applications to establish pressure gradients and impose flow remain open-loop and largely unmonitored due to these sensing limitations. Here we present an optimized self-shielded coplanar capacitive sensor design and automated control system to provide submillimeter fluid-height resolution (∼250 μm) and control of small-scale open reservoirs without the need for direct fluid contact. Results from testing and validation of our optimized sensor and system also suggest that accurate fluid height information can be used to robustly characterize, calibrate and dynamically control a range of microfluidic systems with complex pumping mechanisms, even in cell culture conditions. Capacitive sensing technology provides a scalable and cost-effective way to enable continuous monitoring and closed-loop feedback control of fluid volumes in small-scale gravity-dominated wells in a variety of microfluidic applications.

Two-phase cooling fluids; Les fluides frigoporteurs diphasiques

Energy Technology Data Exchange (ETDEWEB)

Lallemand, A. [Institut National des Sciences Appliquees (INSA), 69 - Lyon (France)

1997-12-31

In the framework of the diminution of heat transfer fluid consumption, the concept of indirect refrigerating circuits, using cooling intermediate fluids, is reviewed and the fluids that are currently used in these systems are described. Two-phase cooling fluids advantages over single-phase fluids are presented with their thermophysical characteristics: solid fraction, two-phase mixture enthalpy, thermal and rheological properties, determination of heat and mass transfer characteristics, and cold storage through ice slurry

Effects of 17β-estradiol on the release of monocyte chemotactic protein-1 and MAPK activity in monocytes stimulated with peritoneal fluid from endometriosis patients.

Science.gov (United States)

Lee, Dong-Hyung; Kim, Seung-Chul; Joo, Jong-Kil; Kim, Hwi-Gon; Na, Young-Jin; Kwak, Jong-Young; Lee, Kyu-Sup

2012-03-01

Hormones and inflammation have been implicated in the pathological process of endometriosis; therefore, we investigated the combined effects of 17β-estradiol (E2) and peritoneal fluid obtained from patients with endometriosis (ePF) or a control peritoneal fluid (cPF) obtained from patients without endometriosis on the release of monocyte chemotactic protein-1 (MCP-1) by monocytes and the role of signaling pathways. Monocytes were cultured with ePF and cPF in the presence of E2; the MCP-1 levels in the supernatants were then measured by ELISA. In addition, mitogen activated protein kinase (MAPK) activation was measured by Western blotting of phosphorylated proteins. E2 down-regulated MCP-1 release by lipopolysaccharide- or cPF-treated monocytes, but failed to suppress its release by ePF-treated monocytes. The release of MCP-1 by ePF- and cPF-treated monocytes was efficiently abrogated by p38 mitogen activated protein kinase (MAPK) inhibitors; however, the MCP-1 release by cPF-treated monocytes, but not by ePF-treated monocytes, was blocked by a MAPK kinase inhibitor. In addition, ePF and cPF induced the phosphorylation of extracellular stress regulated kinase (ERK)1/2, p38 MAPK and c-Jun N-terminal kinase (JNK). E2 decreased the phosphorylation of p38 MAPK, but not ERK1/2 in ePF-treated monocytes; however, E2 decreased the phosphorylation of p38 MAPK, ERK1/2 and JNK in cPF-treated monocytes. The ability of E2 to modulate MCP-1 production is impaired in ePF-treated monocytes, which may be related to regulation of MAPK activity. These findings suggest that the failure of E2 to suppress ePF-treated production of MCP-1 may be involved in the pathogenesis of endometriosis. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Use of radioactive double labelling technique in the chemical analysis of the mediators of cellular immunity

International Nuclear Information System (INIS)

Sorg, C.

1978-01-01

Radioactive double labelling was adapted for the analysis of mediators of cellular immunity. Two identical lymphocyte cultures were simultaneously labelled with [ 3 H]- or [ 14 C] leucine. Each of the cultures was stimulated with antigen or mitogen. The combined supernatants were then subjected to various fractionation procedures. By determining the isotope ratio in each fraction it is possible to identify those products of activated lymphocytes that have been produced either de novo or in increased amounts. The method proved sensitive enough to detect lymphocyte activation products in supernatants of activated lymphocyte cultures from guinea pig, mouse, and man

Viscous Flow with Large Fluid-Fluid Interface Displacement

DEFF Research Database (Denmark)

Rasmussen, Henrik Koblitz; Hassager, Ole; Saasen, Arild

1998-01-01

The arbitrary Lagrange-Euler (ALE) kinematic description has been implemented in a 3D transient finite element program to simulate multiple fluid flows with fluid-fluid interface or surface displacements. The description of fluid interfaces includes variable interfacial tension, and the formulation...... is useful in the simulation of low and intermediate Reynolds number viscous flow. The displacement of two immiscible Newtonian fluids in a vertical (concentric and eccentric) annulus and a (vertical and inclined)tube is simulated....

Fluid transport due to nonlinear fluid-structure interaction

DEFF Research Database (Denmark)

Jensen, Jakob Søndergaard

1997-01-01

This work considers nonlinear fluid-structure interaction for a vibrating pipe containing fluid. Transverse pipe vibrations will force the fluid to move relative to the pipe creating unidirectional fluid flow towards the pipe end. The fluid flow induced affects the damping and the stiffness...... of the pipe. The behavior of the system in response to lateral resonant base excitation is analysed numerically and by the use of a perturbation method (multiple scales). Exciting the pipe in the fundamental mode of vibration seems to be most effective for transferring energy from the shaker to the fluid......, whereas higher modes of vibration can be used to transport fluid with pipe vibrations of smaller amplitude. The effect of the nonlinear geometrical terms is analysed and these terms are shown to affect the response for higher modes of vibration. Experimental investigations show good agreement...

BK virus has tropism for human salivary gland cells in vitro: Implications for transmission

International Nuclear Information System (INIS)

Jeffers, Liesl K.; Madden, Vicki; Webster-Cyriaque, Jennifer

2009-01-01

Background: In this study, it was determined that BKV is shed in saliva and an in vitro model system was developed whereby BKV can productively infect both submandibular (HSG) and parotid (HSY) salivary gland cell lines. Results: BKV was detected in oral fluids using quantitative real-time PCR (QRTPCR). BKV infection was determined using quantitative RT-PCR, immunofluorescence and immunoblotting assays. The infectivity of BKV was inhibited by pre-incubation of the virus with gangliosides that saturated the major capsid protein, VP1, halting receptor mediated BKV entry into salivary gland cells. Examination of infected cultures by transmission electron microscopy revealed 45-50 nm BK virions clearly visible within the cells. Subsequent to infection, encapsidated BK virus was detected in the supernatant. Conclusion: We thus demonstrated that BKV was detected in oral fluids and that BK infection and replication occur in vitro in salivary gland cells. These data collectively suggest the potential for BKV oral route of transmission and oral pathogenesis.

Finite element study of scaffold architecture design and culture conditions for tissue engineering.

Science.gov (United States)

Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

2009-10-01

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

Identifying yeast isolated from spoiled peach puree and assessment of its batch culture for invertase production

Directory of Open Access Journals (Sweden)

Marcela Vega FERREIRA

Full Text Available Abstract The identification of yeasts isolated from spoiled Jubileu peach puree using the API 20C AUX method and a commercial yeast as witness were studied. Subsequently, the yeast’s growth potential using two batch culture treatments were performed to evaluate number of colonies (N, reducing sugar concentration (RS, free-invertase (FI, and culture-invertase activity (CI. Stock cultures were maintained on potato dextrose agar (PDA slants at 4 °C and pH 5 for later use for batch-culture (150 rpm at 30°C for 24 h, then they were stored at 4 °C for subsequent invertase extraction. The FI extract was obtained using NaHCO3 as autolysis agent, and CI activity was determined on the supernatant after batch-cultured centrifugation. The activity was followed by an increase in absorbance at 490 nm using the acid 3,5-DNS method with glucose standard. Of the four yeasts identified, Saccharomyces cerevisiae was chosen for legal reasons. It showed logarithmic growth up to 18 h of fermentation with positive correlation CI activity and inverse with RS. FI showed greater activity by the end of the log phase and an inverse correlation with CI activity. Finally, it was concluded that treatment “A” is more effective than “B” to produce invertase (EC 3.2.1.26.

Improved neurological outcome by intramuscular injection of human amniotic fluid derived stem cells in a muscle denervation model.

Directory of Open Access Journals (Sweden)

Chun-Jung Chen

Full Text Available The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model.Seventy two Sprague-Dawley rats weighing 200-250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups.NT-3 (Neurotrophin 3, BDNF (Brain derived neurotrophic factor, CNTF (Ciliary neurotrophic factor, and GDNF (Glia cell line derived neurotrophic factor were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology.Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve

Allergen-stimulated T lymphocytes from allergic patients induce vascular cell adhesion molecule-1 (VCAM-1) expression and IL-6 production by endothelial cells.

Science.gov (United States)

Delneste, Y; Jeannin, P; Gosset, P; Lassalle, P; Cardot, E; Tillie-Leblond, I; Joseph, M; Pestel, J; Tonnel, A B

1995-01-01

Adhesion of inflammatory cells to endothelium is a critical step for their transvascular migration to inflammatory sites. To evaluate the relationship between T lymphocytes (TL) and vascular endothelium, supernatants from allergen-stimulated TL obtained from patients sensitive to Dermatophagoides pteronyssinus (Dpt) versus healthy subjects were added to endothelial cell (EC) cultures. TL were stimulated by autologous-activated antigen-presenting cells (APC) previously fixed in paraformaldehyde to prevent monokine secretion. Two parameters were measured: the expression of adhesion molecule and the production of IL-6. Related allergen-stimulated TL supernatants from allergic patients induced an increase of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) expression when supernatants of the control groups (TL exposed to an unrelated allergen or not stimulated or TL obtained from healthy subjects) did not. E-selectin expression was not modulated whatever the supernatant added to EC culture. IL-6 production by EC was significantly enhanced after activation with related allergen-stimulated TL supernatants from allergics compared with control supernatants. Induction of VCAM-1 expression was inhibited by adding neutralizing antibodies against IL-4, whereas IL-6 production and ICAM-1 expression were inhibited by anti-interferon-gamma (IFN-gamma) antibodies. Enhanced production of IL-4 and IFN-gamma was detected in related allergen-stimulated TL supernatants from allergic subjects compared with the different supernatants. These data suggest that allergen-specific TL present in the peripheral blood of allergic patients are of Th1 and Th2 subtypes. Their stimulation in allergic patients may lead to the activation of endothelial cells and thereby participate in leucocyte recruitment towards the inflammatory site. PMID:7542574

Fluid-fluid level on MR image: significance in Musculoskeletal diseases

Energy Technology Data Exchange (ETDEWEB)

Chung, Hye Won; Lee, Kyung Won [Seoul Naitonal University, Seoul (Korea, Republic of). Coll. of Medicine; Song, Chi Sung [Seoul City Boramae Hospital, Seoul (Korea, Republic of); Han, Sang Wook; Kang, Heung Sik [Seoul Naitonal University, Seoul (Korea, Republic of). Coll. of Medicine

1998-01-01

To evaluate the frequency, number and signal intensity of fluid-fluid levels of musculoskeletal diseases on MR images, and to determine the usefulness of this information for the differentiation of musculoskeletal diseases. MR images revealed fluid-fluid levels in the following diseases : giant cell tumor(6), telangiectatic osteosarcoma(4), aneurysmal bone cyst(3), synovial sarcoma(3), chondroblastoma(2), soft tissue tuberculous abscess(2), hematoma(2), hemangioma (1), neurilemmoma(1), metastasis(1), malignant fibrous histiocytoma(1), bursitis(1), pyogenic abscess(1), and epidermoid inclusion cyst(1). Fourteen benign tumors and ten malignant, three abscesses, and the epidermoid inclusion cyst showed only one fluid-fluid level in a unilocular cyst. On T1-weighted images, the signal intensities of fluid varied, but on T2-weighted images, superior layers were in most cases more hyperintense than inferior layers. Because fluid-fluid layers are a nonspecific finding, it is difficult to specifically diagnose each disease according to the number of fluid-fluid levels or signal intensity of fluid. In spite of the nonspecificity of fluid-fluid levels, they were frequently seen in cases of giant cell tumor, telangiectatic osteosarcoma, aneurysmal bone cycle, and synovial sarcoma. Nontumorous diseases such abscesses and hematomas also demonstrated this finding. (author). 11 refs., 1 tab., 4 figs.

Fluid-fluid level on MR image: significance in Musculoskeletal diseases

International Nuclear Information System (INIS)

Chung, Hye Won; Lee, Kyung Won; Han, Sang Wook; Kang, Heung Sik

1998-01-01

To evaluate the frequency, number and signal intensity of fluid-fluid levels of musculoskeletal diseases on MR images, and to determine the usefulness of this information for the differentiation of musculoskeletal diseases. MR images revealed fluid-fluid levels in the following diseases : giant cell tumor(6), telangiectatic osteosarcoma(4), aneurysmal bone cyst(3), synovial sarcoma(3), chondroblastoma(2), soft tissue tuberculous abscess(2), hematoma(2), hemangioma (1), neurilemmoma(1), metastasis(1), malignant fibrous histiocytoma(1), bursitis(1), pyogenic abscess(1), and epidermoid inclusion cyst(1). Fourteen benign tumors and ten malignant, three abscesses, and the epidermoid inclusion cyst showed only one fluid-fluid level in a unilocular cyst. On T1-weighted images, the signal intensities of fluid varied, but on T2-weighted images, superior layers were in most cases more hyperintense than inferior layers. Because fluid-fluid layers are a nonspecific finding, it is difficult to specifically diagnose each disease according to the number of fluid-fluid levels or signal intensity of fluid. In spite of the nonspecificity of fluid-fluid levels, they were frequently seen in cases of giant cell tumor, telangiectatic osteosarcoma, aneurysmal bone cycle, and synovial sarcoma. Nontumorous diseases such abscesses and hematomas also demonstrated this finding. (author). 11 refs., 1 tab., 4 figs

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles.

Science.gov (United States)

Gotoh, Aina; Nara, Misaki; Sugiyama, Yuta; Sakanaka, Mikiyasu; Yachi, Hiroyuki; Kitakata, Aya; Nakagawa, Akira; Minami, Hiromichi; Okuda, Shujiro; Katoh, Toshihiko; Katayama, Takane; Kurihara, Shin

2017-10-01

Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.

Blood culture bottles are superior to lysis-centrifugation tubes for bacteriological diagnosis of spontaneous bacterial peritonitis.

OpenAIRE

Siersema, P D; de Marie, S; van Zeijl, J H; Bac, D J; Wilson, J H

1992-01-01

The conventional method of ascitic fluid culturing was compared with the bedside inoculation of ascites into blood culture bottles and into lysis-centrifugation tubes. The conventional culture method was compared with the blood culture bottle method in 31 episodes of spontaneous bacterial peritonitis (SBP). Cultures were positive with the conventional culture method in 11 (35%) episodes and with the blood culture bottle method in 26 (84%) episodes (P less than 0.001). The lysis-centrifugation...

G protein-coupled receptor 120 (GPR120) transcription in intestinal epithelial cells is significantly affected by bacteria belonging to the Bacteroides, Proteobacteria, and Firmicutes phyla

DEFF Research Database (Denmark)

Fredborg, Marlene; Theil, Peter Kappel; Jensen, Bent Borg

2012-01-01

RNA abundance. Supernatants of the 12 bacteria were added to differentiated Caco-2 intestinal epithelial cells cultured on filter inserts in concentrations corresponding to a cell:bacteria ratio of 1:200. After 4 h of incubation, changes in cellular mRNA of GLP-1 and GPR120 by bacterial supernatant were...

Outbreak of Serratia marcescens postsurgical bloodstream infection due to contaminated intravenous pain control fluids.

Science.gov (United States)

Chiang, Ping-Cherng; Wu, Tsu-Lan; Kuo, An-Jing; Huang, Yhu-Chering; Chung, Ting-Ying; Lin, Chun-Sui; Leu, Hsieh-Shong; Su, Lin-Hui

2013-09-01

Serratia marcescens is an important nosocomial pathogen causing significant outbreaks. Here we report an outbreak of bloodstream infection caused by S. marcescens at a 3500-bed hospital in Taiwan. The effective cooperative efforts of both laboratory personnel and infection control practitioners (ICPs) jointly contributed to the total control of the outbreak. A sudden increase in the isolation of S. marcescens from blood cultures was noted in the Clinical Microbiology Laboratory. The information was passed to the ICPs and an investigation was initiated. Pulsed-field gel electrophoresis was used to study the relationships among the isolates. Pulsotype A was identified in 43 (82.7%) of the 52 blood isolates studied. They were isolated from 52 patients distributed across 22 wards that were surveyed by seven ICPs. All patients had undergone surgery before the infection, and fentanyl-containing intravenous fluids were used for pain control in 43 of them. Isolates from 42 belonged to pulsotype A. Three S. marcescens isolates, all from fentanyl-containing fluids and demonstrating pulsotype A, were identified from 251 environmental cultures. All fentanyl-containing fluids that were in use were withdrawn and the outbreak was stopped. The outbreak of S. marcescens bloodstream infection apparently occurred through the use of fentanyl-containing fluids contaminated by a pulsotype A S. marcescens. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes.

Science.gov (United States)

Wang, Yuxi; McAllister, Tim A

2010-08-30

The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH(2)O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 x g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26-32% remaining in supernatant from incubation with a cell-associated enzymes fraction. Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content. Copyright (c) 2008 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.

Prophylactic cefazolin in amnioinfusions administered for meconium-stained amniotic fluid.

Science.gov (United States)

Edwards, R K; Duff, P

1999-01-01

OBJECTIVE: To determine if amnioinfusion with an antibiotic solution decreased the rate of clinical chorioamnionitis and puerperal endometritis in patients with meconium-stained amniotic fluid. METHODS: Patients in labor at 36 weeks of gestation or greater with singleton pregnancies and meconium-stained amniotic fluid were randomized to receive either cefazolin, 1 g/1,000 mL, of normal saline (n = 90) or normal saline (n = 93) amnioinfusion. Rates of clinically diagnosed chorioamnionitis and endometritis and of suspected and culture-proven neonatal infection were determined. RESULTS: Between the study and control groups, the incidences of clinical chorioamnionitis (7.8% vs. 8.6%), endometritis (2.4% vs. 3.5%), aggregate intrauterine infection (10.0% vs. 11.8%), suspected neonatal infection (17.8% vs. 21.5%), and proven neonatal infection (0.0% vs. 2.2%) were not significantly different. CONCLUSIONS: Prophylactic use of cefazolin in amnioinfusions did not significantly reduce rates of maternal or neonatal infection in patients with meconium-stained amniotic fluid. PMID:10371474

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Science.gov (United States)

Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Culture-Negative Neonatal Meningitis and Endocarditis Caused by Streptococcus agalactiae

OpenAIRE

Nègre, Valérie Lefranc; Colin-Gorski, Anne-Marie; Magnier, Suzel; Maisonneuve, Lydia; Aujard, Yannick; Bingen, Edouard; Bonacorsi, Stéphane

2004-01-01

We describe a case of culture-negative meningitis and endocarditis caused by Streptococcus agalactiae in a 27-day-old boy. S. agalactiae was detected in cerebrospinal fluid and serum by broad-spectrum PCR amplification.

Increased hydrazine during partial nitritation process in upflow air-lift reactor fed with supernatant of anaerobic digester effluent

Energy Technology Data Exchange (ETDEWEB)

Choi, Jeongdong [University of Alberta, Alberta (Canada); Jung, Sokhee [Samsung SDS, Seoul (Korea, Republic of); Ahn, Young-Ho [Yeungnam University, Gyungsan (Korea, Republic of)

2013-06-15

The optimal balance of ammonium and nitrite is essential for successful operation of the subsequent anammox process. We conducted a partial nitritation experiment using an upflow air-lift reactor to provide operational parameters for achieving the optimal ratio of ammonium to nitrite, by feeding supernatant of anaerobic digester effluent, high-nitrogen containing rejection water. Semi-continuous operation results show that HRT should be set between 15 and 17 hours to achieve the optimum ration of 1.3 of NO{sub 2}-N/NH{sub 4}-N. In the UAR, nitritation was the dominant reaction due to high concentration of ammonia and low biodegradable organics. The influent contained low concentrations of hydroxylamine and hydrazine. However, hydrazine increased during partial nitritation by ⁓60-130% although there was no potential anammox activity in the reactor. The partial nitritation process successfully provided the ratio of nitrogen species for the anammox reaction, and relived the nitrite restraint on the anammox activity by increasing hydrazine concentration.

Auxillary Fluid Flowmeter

DEFF Research Database (Denmark)

RezaNejad Gatabi, Javad; Forouzbakhsh, Farshid; Ebrahimi Darkhaneh, Hadi

2010-01-01

The Auxiliary Fluid Flow meter is proposed to measure the fluid flow of any kind in both pipes and open channels. In this kind of flow measurement, the flow of an auxiliary fluid is measured Instead of direct measurement of the main fluid flow. The auxiliary fluid is injected into the main fluid ...

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

Science.gov (United States)

Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

2015-11-10

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

Science.gov (United States)

Dutta, Amit K.; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A.; Zhang, Ada W.; Tustian, Andrew D.; Zydney, Andrew L.; Shinkazh, Oleg

2015-01-01

Recent studies using simple model systems have demonstrated that Continuous Countercurrent Tangential Chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an “after binder” to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (~0.67 g/L) and one with high titer (~6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to that obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. PMID:25747172

Fluids engineering

International Nuclear Information System (INIS)

Anon.

1991-01-01

Fluids engineering has played an important role in many applications, from ancient flood control to the design of high-speed compact turbomachinery. New applications of fluids engineering, such as in high-technology materials processing, biotechnology, and advanced combustion systems, have kept up unwaining interest in the subject. More accurate and sophisticated computational and measurement techniques are also constantly being developed and refined. On a more fundamental level, nonlinear dynamics and chaotic behavior of fluid flow are no longer an intellectual curiosity and fluid engineers are increasingly interested in finding practical applications for these emerging sciences. Applications of fluid technology to new areas, as well as the need to improve the design and to enhance the flexibility and reliability of flow-related machines and devices will continue to spur interest in fluids engineering. The objectives of the present seminar were: to exchange current information on arts, science, and technology of fluids engineering; to promote scientific cooperation between the fluids engineering communities of both nations, and to provide an opportunity for the participants and their colleagues to explore possible joint research programs in topics of high priority and mutual interest to both countries. The Seminar provided an excellent forum for reviewing the current state and future needs of fluids engineering for the two nations. With the Seminar ear-marking the first formal scientific exchange between Korea and the United States in the area of fluids engineering, the scope was deliberately left broad and general

Personalised fluid resuscitation in the ICU: still a fluid concept?

Science.gov (United States)

van Haren, Frank

2017-12-28

The administration of intravenous fluid to critically ill patients is one of the most common, but also one of the most fiercely debated, interventions in intensive care medicine. Even though many thousands of patients have been enrolled in large trials of alternative fluid strategies, consensus remains elusive and practice is widely variable. Critically ill patients are significantly heterogeneous, making a one size fits all approach unlikely to be successful.New data from basic, animal, and clinical research suggest that fluid resuscitation could be associated with significant harm. There are several important limitations and concerns regarding fluid bolus therapy as it is currently being used in clinical practice. These include, but are not limited to: the lack of an agreed definition; limited and short-lived physiological effects; no evidence of an effect on relevant patient outcomes; and the potential to contribute to fluid overload, specifically when fluid responsiveness is not assessed and when targets and safety limits are not used.Fluid administration in critically ill patients requires clinicians to integrate abnormal physiological parameters into a clinical decision-making model that also incorporates the likely diagnosis and the likely risk or benefit in the specific patient's context. Personalised fluid resuscitation requires careful attention to the mnemonic CIT TAIT: context, indication, targets, timing, amount of fluid, infusion strategy, and type of fluid.The research agenda should focus on experimental and clinical studies to: improve our understanding of the physiological effects of fluid infusion, e.g. on the glycocalyx; evaluate new types of fluids; evaluate novel fluid minimisation protocols; study the effects of a no-fluid strategy for selected patients and scenarios; and compare fluid therapy with other interventions. The adaptive platform trial design may provide us with the tools to evaluate these types of interventions in the intrinsically

CodY-mediated regulation of Streptococcus pyogenes exoproteins

Directory of Open Access Journals (Sweden)

McDowell Emily J

2012-06-01

Full Text Available Abstract Background The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. Results We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB, and a putative adhesion factor (Spy49_0549 were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA, CAMP factor (Cfa, a prophage encoded nuclease (Spd-3, and an uncharacterized extracellular protein (Spy49_0015 were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY and chemically defined media (CDM. No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. Conclusions Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate

MicroRNA Expression in Salivary Supernatant of Patients with Pancreatic Cancer and Its Relationship with ZHENG

Directory of Open Access Journals (Sweden)

Song Gao

2014-01-01

Full Text Available In traditional Chinese medicine (TCM, diagnosis and prescriptions are based on the signs and symptoms which are recognized as ZHENG. The cornerstone of TCM is to differentially treat one ZHENG from others, which is also known as syndrome differentiation, and this relies on the gathering of clinical information through inspection, auscultation and olfaction, inquiry, and palpation. However, the biomolecular basis of the ZHENG remains unclear. In this study, the expressions of 384 cancer-related miRNAs in salivary supernatant of patients with pancreatic cancer were assessed by miRNA polymerase chain reaction (PCR array, and the different expression patterns of miRNA in three different groups of ZHENG were studied with use of real-time quantitative PCR (qRT-PCR. Some miRNAs were found to be specifically expressed in some ZHENGs, for instance, miR-17, miR-21, and miR-181b in Shi-Re ZHENG and miR-196a in Pi-Xu ZHENG. This indicates that these miRNAs may play important roles in different ZHENG condition. Therefore, this study to some extent revealed the molecular basis of TCM ZHENG in pancreatic cancer.

Bench-scale cross flow filtration of Tank S-107 sludge slurries and Tank C-107 supernatant

International Nuclear Information System (INIS)

Geeting, J.G.H.; Reynolds, B.A.

1996-10-01

Hanford tank waste filtration experiments were conducted using a bench-scale cross flow filter on 8 wt%, 1.5 wt%, and 0.05 wt% Tank S- 107 sludge slurries and on Tank C-107 supernatant. For comparison, two simulants each with solids loadings of 8 wt% and 0.05 wt% were also tested. The purpose of the tests was to determine the efficacy of cross flow filtration on slurries of various solids loadings. -In addition, filtrate flux dependency on axial velocity and transmembrane pressure was sought so that conditions for future experiments might be better selected. The data gathered are compared to the simulants and three cross flow filtration models. A two- parameter central composite design which tested. transmembrane pressure from 5 to 40 psig and axial Velocity from 3 to 9 ft/s was used for all feeds. The cross flow filter effectively removed solids from the liquid, as 19 of 20 filtrate samples had particle concentrations below the resolution limit of the photon correlation spectrometer used in the Hanford Radiocolloid Laboratory. Radiochemical analysis indicate that all filtrate samples were below Class A waste classification standards for 9OSr and transuranics

Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

Directory of Open Access Journals (Sweden)

Jiali Xing

Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

Response to Dengue virus infections altered by cytokine-like substances from mosquito cell cultures

Directory of Open Access Journals (Sweden)

Laosutthipong Chaowanee

2010-11-01

Full Text Available Abstract Background With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells. Results Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2 and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects. Conclusions This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.

Modern fluid dynamics

CERN Document Server

Kleinstreuer, Clement

2018-01-01

Modern Fluid Dynamics, Second Edition provides up-to-date coverage of intermediate and advanced fluids topics. The text emphasizes fundamentals and applications, supported by worked examples and case studies. Scale analysis, non-Newtonian fluid flow, surface coating, convection heat transfer, lubrication, fluid-particle dynamics, microfluidics, entropy generation, and fluid-structure interactions are among the topics covered. Part A presents fluids principles, and prepares readers for the applications of fluid dynamics covered in Part B, which includes computer simulations and project writing. A review of the engineering math needed for fluid dynamics is included in an appendix.

Evaluation of phytate-degrading Lactobacillus culture administration to broiler chickens.

Science.gov (United States)

Askelson, Tyler E; Campasino, Ashley; Lee, Jason T; Duong, Tri

2014-02-01

Probiotics have been demonstrated to promote growth, stimulate immune responses, and improve food safety of poultry. While widely used, their effectiveness is mixed, and the mechanisms through which they contribute to poultry production are not well understood. Microbial phytases are increasingly supplemented in feed to improve digestibility and reduce antinutritive effects of phytate. The microbial origin of these exogenous enzymes suggests a potentially important mechanism of probiotic functionality. We investigated phytate degradation as a novel probiotic mechanism using recombinant Lactobacillus cultures expressing Bacillus subtilis phytase. B. subtilis phyA was codon optimized for expression in Lactobacillus and cloned into the expression vector pTRK882. The resulting plasmid, pTD003, was transformed into Lactobacillus acidophilus, Lactobacillus gallinarum, and Lactobacillus gasseri. SDS-PAGE revealed a protein in the culture supernatants of Lactobacillus pTD003 transformants with a molecular weight similar to that of the B. subtilis phytase. Expression of B. subtilis phytase increased phytate degradation of L. acidophilus, L. gasseri, and L. gallinarum approximately 4-, 10-, and 18-fold over the background activity of empty-vector transformants, respectively. Phytase-expressing L. gallinarum and L. gasseri were administered to broiler chicks fed a phosphorus-deficient diet. Phytase-expressing L. gasseri improved weight gain of broiler chickens to a level comparable to that for chickens fed a control diet adequate in phosphorus, demonstrating proof of principle that administration of phytate-degrading probiotic cultures can improve performance of livestock animals. This will inform future studies investigating whether probiotic cultures are able to provide both the performance benefits of feed enzymes and the animal health and food safety benefits traditionally associated with probiotics.

Skin-to-skin contact: multicultural perspectives on birth fluids and birth 'dirt'.

Science.gov (United States)

Finigan, V; Long, T

2014-06-01

To explore the experiences of women from three population groups of immediate skin-to-skin contact (SSC) with their newborn babies. A mixed methods approach was adopted in a phenomenological study to elicit the experiences of English, Pakistani and Bangladeshi women. Audiotaped diaries, semi-structured interviews, photographs and video recordings were employed. Concept mapping was central to data analysis. This paper reports novel findings that women contextualized and accepted secretions and bodily fluids from birth. This contradicts the beliefs of midwives that Asian women find bodily secretions abhorrent and culturally unacceptable. All participants reported positive experiences of SSC despite varying degrees of soiling from birth fluids. The study was conducted in a single setting, and participants may not have been representative of others in their cultural groups. Third-party translation may have added an unsought layer of interpretation. The imposition of cultural expectations by peers in the recruitment process excluded some potential participants. Stereotypical assumptions about cultural background often characterize professional responses. When this stereotyping was put aside, women of all three cultures, whether breastfeeding or bottle-feeding, were able to enjoy SSC with their babies. The findings suggest that changes will be needed in professional practice to be more open to women's expressed preferences, in local policy to ensure that choices are made clear and are available, and in national strategic direction to ensure widespread adoption of positive practices for opportunities to increase breastfeeding, promote parent-child bonding and support patient choice to be realized. © 2014 The Authors. International Nursing Review published by John Wiley & Sons Ltd on behalf of International Council of Nurses.

Skin-to-skin contact: multicultural perspectives on birth fluids and birth ‘dirt’

Science.gov (United States)

Finigan, V; Long, T

2014-01-01

Aim To explore the experiences of women from three population groups of immediate skin-to-skin contact (SSC) with their newborn babies. Method A mixed methods approach was adopted in a phenomenological study to elicit the experiences of English, Pakistani and Bangladeshi women. Audiotaped diaries, semi-structured interviews, photographs and video recordings were employed. Concept mapping was central to data analysis. Results This paper reports novel findings that women contextualized and accepted secretions and bodily fluids from birth. This contradicts the beliefs of midwives that Asian women find bodily secretions abhorrent and culturally unacceptable. All participants reported positive experiences of SSC despite varying degrees of soiling from birth fluids. Limitations The study was conducted in a single setting, and participants may not have been representative of others in their cultural groups. Third-party translation may have added an unsought layer of interpretation. The imposition of cultural expectations by peers in the recruitment process excluded some potential participants. Conclusion Stereotypical assumptions about cultural background often characterize professional responses. When this stereotyping was put aside, women of all three cultures, whether breastfeeding or bottle-feeding, were able to enjoy SSC with their babies. Implications for Nursing and Health Policy The findings suggest that changes will be needed in professional practice to be more open to women's expressed preferences, in local policy to ensure that choices are made clear and are available, and in national strategic direction to ensure widespread adoption of positive practices for opportunities to increase breastfeeding, promote parent–child bonding and support patient choice to be realized. PMID:24712443

The protective effect of follicular fluid against the emerging mycotoxins alternariol and beauvericin

NARCIS (Netherlands)

Santos, R. R.; Schoevers, E. J.; Wu, X.; Roelen, B. A. J.; Fink-Gremmels, J.

2015-01-01

Porcine granulosa cells were cultured in the absence or presence of 10% porcine follicular fluid (FF) at different concentrations (0-20 mu M) of the mycotoxins alternariol (AOH) and beauvericin (BEA). The analyses were performed after exposure to these mycotoxins in a medium supplemented or not with

Boildown Study on Supernatant Liquid Retrieved from AP-107 in May 2010

Energy Technology Data Exchange (ETDEWEB)

Callaway, W. S. [Washington River Protection Solutions, LLC, Richland, WA (United States); Page, J. S. [Washington River Protection Solutions, LLC, Richland, WA (United States)

2013-02-12

A boildown study was completed on a composite prepared from supernatant liquid grab samples retrieved from tank 241-AP-107 in May of 2010. The composite was a clear, yellow liquid containing no visible solids at hot cell ambient temperatures (25-27 °C). The density of the test composite was 1.216 g/mL at 26.8 °C. The boiling temperature curves generated at three reduced pressures—40-, 60-, and 80 Torr—displayed steadily increasing boiling temperatures with increasing volume reduction with no significant discontinuities. Only minimal foaming was observed after the volume reduction proceeded beyond 50 %WVR (percent waste volume reduction). The bulk densities (D{sub Bulk}{sup 18 °C}) and quantities of settled and centrifuged solids present were measured on samples of the boildown concentrates that were kept at 18 °C for 7-8 days. Estimated values of the bulk densities of the concentrates at 60-Torr boiling temperatures (D{sub Bulk}{sup 60 Torr}) were also calculated. Solids were observed in all boildown concentrates at process temperatures, at hot cell ambient temperatures (25-27 °C), and at 18 °C. The quantity of solids found in the cooled concentrates increased slowly through 50.2 %WVR. The quantity of solids found in concentrates after 54.0 %WVR was noticeably greater. Beyond 54.0 %WVR, the quantity of solids found in cooled concentrates increased dramatically. Analysis of boildown test samples indicated that sodium oxalate and sodium carbonate solids form in cooled concentrates after volume reduction of 8.4 %WVR or less. The major contributors to the large increase in the quantity of solids found in concentrates after 54 %WVR were sodium nitrate and sodium carbonate.

The work of cultural transition

DEFF Research Database (Denmark)

Ryba, T. V.; Stambulova, N. B.; Ronkainen, Noora J.

2016-01-01

In today's uncertain, fluid job market, transnational mobility has intensified. Though the concept of cultural transition is increasingly used in sport and career research, insight into the processes of how individuals produce their own development through work and relationships in shifting...... provide professionals working in the fields of career counseling and migrant support with a content framework for enhancing migrant workers' adaptabilities and psychological wellbeing. © 2016 Ryba, Stambulova and Ronkainen....

[Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein].

Science.gov (United States)

Huang, Shigao; Yin, Yuting; Xiong, Chunhui; Wang, Caihong; Lü, Jianxin; Gao, Jimin

2013-01-01

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

The Omega-3 Fatty Acid Docosahexaenoic Acid Modulates Inflammatory Mediator Release in Human Alveolar Cells Exposed to Bronchoalveolar Lavage Fluid of ARDS Patients

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Paolo Cotogni

2015-01-01

Full Text Available Background. This study investigated whether the 1 : 2 ω-3/ω-6 ratio may reduce proinflammatory response in human alveolar cells (A549 exposed to an ex vivo inflammatory stimulus (bronchoalveolar lavage fluid (BALF of acute respiratory distress syndrome (ARDS patients. Methods. We exposed A549 cells to the BALF collected from 12 ARDS patients. After 18 hours, fatty acids (FA were added as docosahexaenoic acid (DHA, ω-3 and arachidonic acid (AA, ω-6 in two ratios (1 : 2 or 1 : 7. 24 hours later, in culture supernatants were evaluated cytokines (TNF-α, IL-6, IL-8, and IL-10 and prostaglandins (PGE2 and PGE3 release. The FA percentage content in A549 membrane phospholipids, content of COX-2, level of PPARγ, and NF-κB binding activity were determined. Results. The 1 : 2 DHA/AA ratio reversed the baseline predominance of ω-6 over ω-3 in the cell membranes (P < 0.001. The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001 but was increased by the 1 : 7 ratio (P < 0.01. The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001 as well as NF-κB translocation into the nucleus (P < 0.01, while it increased activation of PPARγ and IL-10 release (P < 0.001. Conclusion. This study demonstrated that shifting the FA supply from ω-6 to ω-3 decreased proinflammatory mediator release in human alveolar cells exposed to BALF of ARDS patients.

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital.

Science.gov (United States)

Tadros, Manal; Petrich, Astrid

2013-01-01

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

The allogeneic umbilical cord mesenchymal stem cells regulate the function of T helper 17 cells from patients with rheumatoid arthritis in an in vitro co-culture system

Directory of Open Access Journals (Sweden)

Wang Qin

2012-12-01

Full Text Available Abstract Background Previous in vivo studies have shown that mesenchymal stem cell (MSC transplantation significantly improves the condition of a number of autoimmune diseases including autoimmune cerebrospinal meningitis, multiple sclerosis, glomerulonephritis and systemic lupus erythematosus. Methods To investigate the immunoregulatory effect of stem cell transplantation, human umbilical cord MSCs were co-cultured with peripheral blood mononuclear cells (PBMCs from patients with rheumatoid arthritis (RA. Orphan nuclear receptor gamma (ROR-γ mRNA and protein expression was detected with real-time PCR and Western blotting. Interleukin (IL-17, IL-6 and tumor necrosis factor (TNF-α in the cell culture supernatant were measured using a flow cytometric bead capture method. Results After 72 hours of co-culture, the mRNA and protein expression levels of ROR-γ in co-cultured PBMCs were decreased compared with that in PBMC of RA patients cultured alone (p  Conclusions In vitro co-culture with MSCs down-regulated the inflammatory response of PBMCs from RA patients with severe disease activity, but had no significant effect on PBMCs from healthy controls or patients with mild disease activity, suggesting that the immunoregulatory role of MSCs may associate with the occurrence of inflammatory mediators.

Effects of waste drilling fluid on bacterial isolates from a mangrove swamp oilfield location in the Niger delta of Nigeria

Energy Technology Data Exchange (ETDEWEB)

Benka-Coker, M.O.; Olumagin, A. [Benin Univ. (Nigeria). Dept. of Microbiology

1996-03-01

Four bacteria strains isolated from a mangrove swamp oilfield location in the Niger Delta of Nigeria were cultured aerobically in the presence of 1.0% waste drilling fluid, to determine the effect of the waste on their growth. A 2-h lag phase of growth was produced by the waste in cultures of Micrococcus and Pseudomonas species, while the waste increased the lag phases of Alcaligenes and Staphylococcus species to 4 h. The exponential phase of growth of Pseudomonas sp. was depressed by the waste drilling fluid but fluid stimulated the exponential phases of Micrococcus and Alcaligenes spp. There was enhancement of the growth rate of Alcaligenes and Micrococcus spp. while those of Staphylococcus and Pseudonomas spp. were decreased. The depressed growth rates of Staphylococcus and Pseudonomas spp. in the presence of the waste drilling fluid might lead to a decrease in their contribution to the removal of the waste from the environment during spillage or disposal and, therefore, may result in an accumulation of the waste in the environment. (author)

Effects of waste drilling fluid on bacterial isolates from a mangrove swamp oilfield location in the Niger delta of Nigeria

International Nuclear Information System (INIS)

Benka-Coker, M.O.; Olumagin, A.

1996-01-01

Four bacteria strains isolated from a mangrove swamp oilfield location in the Niger Delta of Nigeria were cultured aerobically in the presence of 1.0% waste drilling fluid, to determine the effect of the waste on their growth. A 2-h lag phase of growth was produced by the waste in cultures of Micrococcus and Pseudomonas species, while the waste increased the lag phases of Alcaligenes and Staphylococcus species to 4 h. The exponential phase of growth of Pseudomonas sp. was depressed by the waste drilling fluid but fluid stimulated the exponential phases of Micrococcus and Alcaligenes spp. There was enhancement of the growth rate of Alcaligenes and Micrococcus spp. while those of Staphylococcus and Pseudonomas spp. were decreased. The depressed growth rates of Staphylococcus and Pseudonomas spp. in the presence of the waste drilling fluid might lead to a decrease in their contribution to the removal of the waste from the environment during spillage or disposal and, therefore, may result in an accumulation of the waste in the environment. (author)

Community-acquired Pseudomonas aeruginosa-pneumonia in a previously healthy man occupationally exposed to metalworking fluids

Directory of Open Access Journals (Sweden)

Fernando Peixoto Ferraz de Campos

2014-09-01

Full Text Available Although the Pseudomonas aeruginosa infection is well known and frequently found in hospitals and nursing care facilities, many cases are also reported outside these boundaries. In general, this pathogen infects debilitated patients either by comorbidities or by any form of immunodeficiency. In cases of respiratory infection, tobacco abuse seems to play an important role as a risk factor. In previously healthy patients, community-acquired pneumonia (CAP with P. aeruginosa as the etiological agent is extremely rare, and unlike the cases involving immunocompromised or hospitalized patients, the outcome is severe, and is fatal in up to 61.1% of cases. Aerosolized contaminated water or solutions are closely linked to the development of respiratory tract infection. In this setting, metalworking fluids used in factories may be implicated in CAP involving previously healthy people. The authors report the case of a middle-aged man who worked in a metalworking factory and presented a right upper lobar pneumonia with a rapid fatal outcome. P. aeruginosa was cultured from blood and tracheal aspirates. The autopsy findings confirmed a hemorrhagic necrotizing pneumonia with bacteria-invading vasculitis and thrombosis. A culture of the metalworking fluid of the factory was also positive for P. aeruginosa. The pulsed-field gel electrophoresis showed that both strains (blood culture and metalworking fluid were genetically indistinguishable. The authors highlight the occupational risk for the development of this P. aeruginosa-infection in healthy people.

Variation in FCN1 affects biosynthesis of ficolin-1 and is associated with outcome of systemic inflammation

DEFF Research Database (Denmark)

Munthe-fog, L; Hummelshoj, T; Honoré, C

2012-01-01

), -271 (rs28909976), -144 (rs10117466) and +7918 (rs1071583) were determined in 100 healthy individuals. FCN1 expression by isolated monocytes and granulocytes and ficolin-1 levels in monocyte culture supernatants were assessed in 21 FCN1-genotyped individuals. FCN1 polymorphisms were determined...... in a cohort of 251 patients with systemic inflammation. High ficolin-1 plasma levels were significantly associated with the minor alleles in position -542 and -144. These alleles were also significantly associated with high FCN1 mRNA expression. The level of ficolin-1 in culture supernatants was significantly...

Doppler optical coherence tomography imaging of local fluid flow and shear stress within microporous scaffolds

Science.gov (United States)

Jia, Yali; Bagnaninchi, Pierre O.; Yang, Ying; Haj, Alicia El; Hinds, Monica T.; Kirkpatrick, Sean J.; Wang, Ruikang K.

2009-05-01

Establishing a relationship between perfusion rate and fluid shear stress in a 3D cell culture environment is an ongoing and challenging task faced by tissue engineers. We explore Doppler optical coherence tomography (DOCT) as a potential imaging tool for in situ monitoring of local fluid flow profiles inside porous chitosan scaffolds. From the measured fluid flow profiles, the fluid shear stresses are evaluated. We examine the localized fluid flow and shear stress within low- and high-porosity chitosan scaffolds, which are subjected to a constant input flow rate of 0.5 ml.min-1. The DOCT results show that the behavior of the fluid flow and shear stress in micropores is strongly dependent on the micropore interconnectivity, porosity, and size of pores within the scaffold. For low-porosity and high-porosity chitosan scaffolds examined, the measured local fluid flow and shear stress varied from micropore to micropore, with a mean shear stress of 0.49+/-0.3 dyn.cm-2 and 0.38+/-0.2 dyn.cm-2, respectively. In addition, we show that the scaffold's porosity and interconnectivity can be quantified by combining analyses of the 3D structural and flow images obtained from DOCT.

Lactic acid bacteria as functional probiotic isolates for inhibiting the growth of Aspergillus flavus, A. parasiticus, A. niger and Penicillium chrysogenum.

Science.gov (United States)

Abbaszadeh, S; Tavakoli, R; Sharifzadeh, A; Shokri, H

2015-12-01

The aim of this study was to assess the potential of lactic acid bacteria (LAB) such as Lactobacillus acidophilus, L. rhamnosus, L. casei, L. paracasei and Bifidobacterium bifidum to inhibit the outgrowth of some common food-spoiling fungi including Aspergillus niger, A. flavus, A. parasiticus and Penicillium chrysogenum. Bacterial isolates were cultured on Mann Rogosa Sharpe (MRS) broth and liquid cultures and supernatants were prepared. The antifungal activity was tested using the agar well diffusion method. Both liquid culture and supernatant of L. casei isolate exhibited high antifungal activity, followed by L. acidophilus and L. paracasei isolates. The least activity was recorded for the isolates B. bifidum, while the isolate L. rhamnosus was moderately active against tested fungi. The antifungal activity of the supernatants obtained from all probiotic isolates against fungi was significantly less than that of liquid cultures (Pniger and A. parasiticus. These results suggest that probiotic bacteria strains have the ability to prevent the growth of pathogenic and mycotoxigenic fungi as antifungal agents for various biomedical applications. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Fixed, Fluid, and Transient: Negotiating Layers of Art Classroom Material Culture

Science.gov (United States)

Woywod, Christine

2015-01-01

Objects of material culture have meaning. American flags, worktables, bulletin boards, interactive whiteboards, and large white-faced clocks signify "classroom" while color wheels, cupboards, cabinets, sinks, drawing supplies, and that particular scent that lingers after years of exposure to painting materials even more specifically…

A Comparison of Aspergillus and Mucorales PCR Testing of Different Bronchoalveolar Lavage Fluid Fractions from Patients with Suspected Invasive Pulmonary Fungal Disease.

Science.gov (United States)

Springer, Jan; White, P Lewis; Kessel, Johanna; Wieters, Imke; Teschner, Daniel; Korczynski, Daniel; Liebregts, Tobias; Cornely, Oliver A; Schwartz, Stefan; Elgeti, Thomas; Meintker, Lisa; Krause, Stefan W; Posso, Raquel B; Heinz, Werner J; Fuhrmann, Sandra; Vehreschild, Jörg Janne; Einsele, Hermann; Rickerts, Volker; Loeffler, Juergen

2018-02-01

In patients with hematological malignancies, bronchoalveolar lavage fluid (BALF) specimens are commonly used for the diagnosis of mold infections. However, it is not clear whether the cell pellet (P) or the supernatant fraction (S) of the BALF specimen is optimal for molecular diagnostic testing. Thus, 99 BALF specimens were collected from 96 hematology patients with or without allogeneic hematopoietic stem cell transplant. The cell pellets and supernatants were processed alone and in combination (S/P) for testing by two fungus-specific real-time PCR assays compliant with international recommendations. The results achieved with S/P were revealed to be superior in comparison to those achieved with S and P alone, with the use of each single fraction showing a reduced sensitivity for the detection of Aspergillus DNA (82% and 43% for S and P, respectively). In 57% of the samples, testing of the combination of S and P generated a lower quantification cycle value than testing of S or P alone. Molds would have been missed in 5 and 16 out of 28 samples if only S or P, respectively, was analyzed. No sample was positive by testing of S or P only. Similar results were obtained for the detection of Mucorales DNA in BALF specimens (reduced sensitivity of 67% and 50% for S and P, respectively). Study patients were categorized according to the current European Organization for the Research and Treatment of Cancer/Mycoses Study Group classification for invasive fungal disease (IFD), revealing that 35 patients had proven/probable IFD (36%), 47 patients had possible IFD (49%), and 14 patients had undetermined IFD (15%). Copyright © 2018 American Society for Microbiology.

Evaluation of MALDI-TOF Mass Spectrometry and Sepsityper Kit™ for the Direct Identification of Organisms from Sterile Body Fluids in a Canadian Pediatric Hospital

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Manal Tadros

2013-01-01

Full Text Available Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit – the Sepsityper Kit (Bruker Daltonik, Germany – and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2% were blood culture specimens and seven (8.7% were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8% and 39 of 50 (78% blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%.

Changes in surfactant in bronchoalveolar lavage fluid after hemithorax irradiation in patients with mesothelioma

International Nuclear Information System (INIS)

Hallman, M.; Maasilta, P.; Kivisaari, L.; Mattson, K.

1990-01-01

Experimental studies have shown that the surfactant system of the lung is affected shortly after irradiation. It is unclear, however, whether surfactant plays a role in the pathogenesis of radiation pneumonitis. In the present study surfactant components (saturated phosphatidylcholine, surfactant protein A, phosphatidylglycerol, and phosphatidylinositol) and other phospholipids of bronchoalveolar lavage fluid (BAL) were studied in four patients with pleural mesothelioma before and during hemithorax irradiation (70 Gy) as well as zero, 1, 2, 3, and 4 months following irradiation. The concentrations of these same components and of soluble proteins were also estimated in the epithelial lining fluid (ELF) using urea as a marker of dilution. After radiotherapy, the concentrations of the surfactant components in ELF decreased to 12 to 55% of the control values before radiation, whereas the concentration of sphingomyelin in ELF increased ninefold. There were small changes in the other phospholipids. The concentration of soluble protein in ELF increased sevenfold. The minimum surface activity of crude BAL increased from 12 +/- 4 to 32 +/- 6 mN/m, and that of the sediment fraction of BAL increased from 7 +/- 4 to 22 +/- 6 mN/m, p less than 0.001. The protein-rich supernatant fraction of BAL from irradiated lung had a inhibitory effect on normal surfactant. There were significant correlations between the increasing severity of the radiologic changes on the one hand and, on the other, the saturated phosphatidylcholine/sphingomyelin ratio (p less than 0.001), the concentrations of soluble protein (p less than 0.001), and the concentrations of the surfactant components (p less than 0.02-0.001) in ELF

Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and pharmaceutical research

International Nuclear Information System (INIS)

Minuth, Will W; Strehl, Raimund

2007-01-01

Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container. (topical review)

Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and pharmaceutical research

Energy Technology Data Exchange (ETDEWEB)

Minuth, Will W [Department of Molecular and Cellular Anatomy, University of Regensburg, D-93053 Regensburg, University Street 31 (Germany); Strehl, Raimund [Cellartis AB, S-41346 Goeteborg, Arvid Wallgrens Backe 20 (Sweden)

2007-06-01

Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container. (topical review)

Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture.

Science.gov (United States)

Miranda, Melissa; Pino, Ana María; Fuenzalida, Karen; Rosen, Clifford J; Seitz, Germán; Rodríguez, J Pablo

2016-10-01

Bone marrow adipose tissue (BMAT) is associated with low bone mass, although the functional consequences for skeletal maintenance of increased BMAT are currently unclear. BMAT might have a role in systemic energy metabolism, and could be an energy source as well as an endocrine organ for neighboring bone cells, releasing cytokines, adipokines and free fatty acids into the bone marrow microenvironment. The aim of the present report was to compare the fatty acid composition in the bone marrow supernatant fluid (BMSF) and blood plasma of postmenopausal women women (65-80 years old). BMSF was obtained after spinning the aspirated bone marrow samples; donors were classified as control, osteopenic or osteoporotic after dual-energy X-ray absorptiometry. Total lipids from human bone marrow fluid and plasma were extracted, converted to the corresponding methyl esters, and finally analyzed by a gas chromatographer coupled with a mass spectrometer. Results showed that fatty acid composition in BMSF was dynamic and distinct from blood plasma, implying significance in the locally produced lipids. The fatty acid composition in the BMSF was enriched in saturated fatty acid and decreased in unsaturated fatty acids as compared to blood plasma, but this relationship switched in women who suffered a hip fracture. On the other hand, there was no relationship between BMSF and bone mineral density. In conclusion, lipid composition of BMSF is distinct from the circulatory compartment, most likely reflecting the energy needs of the marrow compartment. J. Cell. Biochem. 117: 2370-2376, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

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Drevets, Peter; Chucair-Elliott, Ana; Shrestha, Priyadarsini; Jinkins, Jeremy; Karamichos, Dimitrios; Carr, Daniel J J

2015-10-01

To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

Science.gov (United States)

Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

2015-01-01

Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

Biocontrol Potential of Streptomyces hydrogenans strain DH16 Towards Alternaria brassicicola to Control Damping Off and Black Leaf Spot of Raphanus sativus

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Rajesh Kumari Manhas

2016-12-01

Full Text Available Biocontrol agents and their bioactive metabolites provide one of the best alternatives to decrease the use of chemical pesticides. In light of this, the present investigation reports the biocontrol potential of Streptomyces hydrogenans DH16 and its metabolites towards Alternaria brassicicola, causal agent of black leaf spot and damping off of seedlings of crucifers. In vitro antibiosis of strain against pathogen revealed complete suppression of mycelial growth of pathogen, grown in potato dextrose broth supplemented with culture supernatant (20% v/v of Streptomyces hydrogenans DH16. Microscopic examination of the fungal growth showed severe morphological abnormalities in the mycelium caused by antifungal metabolites. In vivo studies showed the efficacy of streptomycete cells and culture supernatant as seed dressings to control damping off of Raphanus sativus seedlings. Treatment of pathogen infested seeds with culture supernatant (10% and streptomycete cells significantly improved seed germination (75-80% and vigour index (1167-1538. Furthermore, potential of cells and culture supernatant as foliar treatment to control black leaf spot was also evaluated. Clearly visible symptoms of disease were observed in the control plants with 66.81% disease incidence and retarded growth of root system. However, disease incidence reduced to 6.78 and 1.47% in plants treated with antagonist and its metabolites, respectively. Additionally, treatment of seeds and plants with streptomycete stimulated various growth traits of plants over uninoculated control plants in the absence of pathogen challenge. These results indicate that S. hydrogenans and its culture metabolites can be developed as biofungicides as seed dressings to control seed borne pathogens, and as sprays to control black leaf spot of crucifers.

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Directory of Open Access Journals (Sweden)

Irma Gonzalez-Curiel

Full Text Available Diabetic foot ulcers (DFU are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH2 D3 and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Near infrared spectroscopy combined with multivariate analysis for monitoring the ethanol precipitation process of fraction I + II + III supernatant in human albumin separation

Science.gov (United States)

Li, Can; Wang, Fei; Zang, Lixuan; Zang, Hengchang; Alcalà, Manel; Nie, Lei; Wang, Mingyu; Li, Lian

2017-03-01

Nowadays, as a powerful process analytical tool, near infrared spectroscopy (NIRS) has been widely applied in process monitoring. In present work, NIRS combined with multivariate analysis was used to monitor the ethanol precipitation process of fraction I + II + III (FI + II + III) supernatant in human albumin (HA) separation to achieve qualitative and quantitative monitoring at the same time and assure the product's quality. First, a qualitative model was established by using principal component analysis (PCA) with 6 of 8 normal batches samples, and evaluated by the remaining 2 normal batches and 3 abnormal batches. The results showed that the first principal component (PC1) score chart could be successfully used for fault detection and diagnosis. Then, two quantitative models were built with 6 of 8 normal batches to determine the content of the total protein (TP) and HA separately by using partial least squares regression (PLS-R) strategy, and the models were validated by 2 remaining normal batches. The determination coefficient of validation (Rp2), root mean square error of cross validation (RMSECV), root mean square error of prediction (RMSEP) and ratio of performance deviation (RPD) were 0.975, 0.501 g/L, 0.465 g/L and 5.57 for TP, and 0.969, 0.530 g/L, 0.341 g/L and 5.47 for HA, respectively. The results showed that the established models could give a rapid and accurate measurement of the content of TP and HA. The results of this study indicated that NIRS is an effective tool and could be successfully used for qualitative and quantitative monitoring the ethanol precipitation process of FI + II + III supernatant simultaneously. This research has significant reference value for assuring the quality and improving the recovery ratio of HA in industrialization scale by using NIRS.

Near infrared spectroscopy combined with multivariate analysis for monitoring the ethanol precipitation process of fraction I+II+III supernatant in human albumin separation.

Science.gov (United States)

Li, Can; Wang, Fei; Zang, Lixuan; Zang, Hengchang; Alcalà, Manel; Nie, Lei; Wang, Mingyu; Li, Lian

2017-03-15

Nowadays, as a powerful process analytical tool, near infrared spectroscopy (NIRS) has been widely applied in process monitoring. In present work, NIRS combined with multivariate analysis was used to monitor the ethanol precipitation process of fraction I+II+III (FI+II+III) supernatant in human albumin (HA) separation to achieve qualitative and quantitative monitoring at the same time and assure the product's quality. First, a qualitative model was established by using principal component analysis (PCA) with 6 of 8 normal batches samples, and evaluated by the remaining 2 normal batches and 3 abnormal batches. The results showed that the first principal component (PC1) score chart could be successfully used for fault detection and diagnosis. Then, two quantitative models were built with 6 of 8 normal batches to determine the content of the total protein (TP) and HA separately by using partial least squares regression (PLS-R) strategy, and the models were validated by 2 remaining normal batches. The determination coefficient of validation (R p 2 ), root mean square error of cross validation (RMSECV), root mean square error of prediction (RMSEP) and ratio of performance deviation (RPD) were 0.975, 0.501g/L, 0.465g/L and 5.57 for TP, and 0.969, 0.530g/L, 0.341g/L and 5.47 for HA, respectively. The results showed that the established models could give a rapid and accurate measurement of the content of TP and HA. The results of this study indicated that NIRS is an effective tool and could be successfully used for qualitative and quantitative monitoring the ethanol precipitation process of FI+II+III supernatant simultaneously. This research has significant reference value for assuring the quality and improving the recovery ratio of HA in industrialization scale by using NIRS. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis.

Science.gov (United States)

Tan, Susanna K; Burgener, Elizabeth B; Waggoner, Jesse J; Gajurel, Kiran; Gonzalez, Sarah; Chen, Sharon F; Pinsky, Benjamin A

2016-01-01

Background.  Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods.  Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results.  Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions.  Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.

Prenatal diagnosis of congenital toxoplasmosis: comparative value of fetal blood and amniotic fluid using serological techniques and cultures.

Science.gov (United States)

Fricker-Hidalgo, H; Pelloux, H; Muet, F; Racinet, C; Bost, M; Goullier-Fleuret, A; Ambroise-Thomas, P

1997-09-01

The prenatal diagnosis of congenital toxoplasmosis is mainly based on biological tests performed on fetal blood and amniotic fluid. We studied the performance of neonatal diagnosis procedures and the results of fetal blood and amniotic fluid analysis. Of 127 women who contracted toxoplasmosis and underwent prenatal diagnosis, the postnatal serological follow-up was long enough to definitively diagnose congenital toxoplasmosis in 19 cases and to exclude it in 27 cases. Prenatal diagnosis allowed the detection of 94.7 per cent (18/19) of the infected fetuses. The sensitivities of tests in amniotic fluid and fetal blood were equivalent, 88.2 per cent (15/17) and 87.5 per cent (14/16), respectively. In fetal blood, biological techniques were positive in 12/16 cases and in 2/16 cases, serological tests were the only positive sign. The specificities of tests in amniotic fluid and fetal blood were respectively 100 per cent (23/23) and 86.3 per cent (19/22) (three false-positive serological results). These results, added to the lower morbidity of amniocentesis compared with cordocentesis, might lead to cordocentesis being abandoned in the prenatal diagnosis of congenital toxoplasmosis.

Cerebrospinal fluid cytotoxicity does not affect survival in amyotrophic lateral sclerosis.

Science.gov (United States)

Galán, L; Matías-Guiu, J; Matias-Guiu, J A; Yáñez, M; Pytel, V; Guerrero-Sola, A; Vela-Souto, A; Arranz-Tagarro, J A; Gómez-Pinedo, U; García, A G

2017-09-01

Cerebrospinal fluid (CSF) from some patients with amyotrophic lateral sclerosis (ALS) has been demonstrated to significantly reduce the neuronal viability of primary cell cultures of motor neurons. We aimed to study the potential clinical consequences associated with the cytotoxicity of CSF in a cohort of patients with ALS. We collected CSF from thirty-one patients with ALS. We analysed cytotoxicity by incubating it into the primary cultures of motor cortex neurons. Neural viability was quantified after 24 hours using the colorimetric MTT reduction assay. All patients were followed up from the moment of diagnosis to death, and a complete evaluation during disease progression and survival was performed, including gastrostomy and respiratory assistance. Twenty-one patients (67.7%) presented a cytotoxic CSF. There were no significant differences between patients with and without cytotoxicity regarding mean time from symptom onset to the diagnosis, from the diagnosis to death, from the diagnosis to respiratory assistance with BIPAP, from diagnosis to gastrostomy and from the onset of symptoms to death. In Cox regression analysis, bulbar onset, but not cytotoxicity, gender or age at onset, was associated with a lower risk of survival. Cerebrospinal fluid cytotoxicity was not associated with differential survival rates. This suggests that the presence of cytotoxicity in CSF, measured through neuronal viability in primary cultures of motor cortex neurons, could reflect different mechanisms of the disease, but it does not predict disease outcome. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor

International Nuclear Information System (INIS)

Aizaki, Hideki; Nagamori, Seishi; Matsuda, Mami; Kawakami, Hayato; Hashimoto, Osamu; Ishiko, Hiroaki; Kawada, Masaaki; Matsuura, Tomokazu; Hasumura, Satoshi; Matsuura, Yoshiharu; Suzuki, Tetsuro; Miyamura, Tatsuo

2003-01-01

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy

Fluid Mechanics.

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Drazin, Philip

1987-01-01

Outlines the contents of Volume II of "Principia" by Sir Isaac Newton. Reviews the contributions of subsequent scientists to the physics of fluid dynamics. Discusses the treatment of fluid mechanics in physics curricula. Highlights a few of the problems of modern research in fluid dynamics. Shows that problems still remain. (CW)

Electric fluid pump

Science.gov (United States)

Van Dam, Jeremy Daniel; Turnquist, Norman Arnold; Raminosoa, Tsarafidy; Shah, Manoj Ramprasad; Shen, Xiaochun

2015-09-29

An electric machine is presented. The electric machine includes a hollow rotor; and a stator disposed within the hollow rotor, the stator defining a flow channel. The hollow rotor includes a first end portion defining a fluid inlet, a second end portion defining a fluid outlet; the fluid inlet, the fluid outlet, and the flow channel of the stator being configured to allow passage of a fluid from the fluid inlet to the fluid outlet via the flow channel; and wherein the hollow rotor is characterized by a largest cross-sectional area of hollow rotor, and wherein the flow channel is characterized by a smallest cross-sectional area of the flow channel, wherein the smallest cross-sectional area of the flow channel is at least about 25% of the largest cross-sectional area of the hollow rotor. An electric fluid pump and a power generation system are also presented.

Principles of fluid mechanics

International Nuclear Information System (INIS)

Kreider, J.F.

1985-01-01

This book is an introduction on fluid mechanics incorporating computer applications. Topics covered are as follows: brief history; what is a fluid; two classes of fluids: liquids and gases; the continuum model of a fluid; methods of analyzing fluid flows; important characteristics of fluids; fundamentals and equations of motion; fluid statics; dimensional analysis and the similarity principle; laminar internal flows; ideal flow; external laminar and channel flows; turbulent flow; compressible flow; fluid flow measurements

Rapid detection of cytomegalovirus in bronchoalveolar lavage fluid and serum samples by polymerase chain reaction: correlation of virus isolation and clinical outcome for patients with human immunodeficiency virus infection

DEFF Research Database (Denmark)

Hansen, K K; Vestbo, Jørgen; Benfield, T

1997-01-01

Bronchoalveolar lavage (BAL) fluids and serum samples from 153 patients with pulmonary symptoms who were infected with human immunodeficiency virus (HIV) and underwent BAL were examined for the presence of cytomegalovirus (CMV) by conventional culture and by polymerase chain reaction (PCR...... technique than conventional culture. Detection of CMV DNA in BAL fluid or serum predicted subsequent development of extrapulmonary CMV disease but not death for HIV-infected patients with pulmonary symptoms....

DNA hydrogel-based supercapacitors operating in physiological fluids

Science.gov (United States)

Hur, Jaehyun; Im, Kyuhyun; Hwang, Sekyu; Choi, ByoungLyong; Kim, Sungjee; Hwang, Sungwoo; Park, Nokyoung; Kim, Kinam

2013-01-01

DNA nanostructures have been attractive due to their structural properties resulting in many important breakthroughs especially in controlled assemblies and many biological applications. Here, we report a unique energy storage device which is a supercapacitor that uses nanostructured DNA hydrogel (Dgel) as a template and layer-by-layer (LBL)-deposited polyelectrolyte multilayers (PEMs) as conductors. Our device, named as PEM-Dgel supercapacitor, showed excellent performance in direct contact with physiological fluids such as artificial urine and phosphate buffered saline without any need of additional electrolytes, and exhibited almost no cytotoxicity during cycling tests in cell culture medium. Moreover, we demonstrated that the PEM-Dgel supercapacitor has greater charge-discharge cycling stability in physiological fluids than highly concentrated acid electrolyte solution which is normally used for supercapacitor operation. These conceptually new supercapacitors have the potential to be a platform technology for the creation of implantable energy storage devices for packageless applications directly utilizing biofluids. PMID:23412432

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly.

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Yue Liu

Full Text Available The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS and inulin both stimulated bifidobacteria compared to other treatments (p<0.05. Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05. IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05. To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters.

A study of the localized humoral immune response to implicated microorganisms in juvenile periodontitis

International Nuclear Information System (INIS)

Hall, E.R.

1988-01-01

A study was undertaken using an in vitro explant culture system to determine the presence of immunoglobulins (IgG, IgA, and IgM) in the supernatant fluids (SF) of disease gingival tissue explant cultures. Studies were also undertaken to determine if the de novo biosynthesis of 14 C-immunoglobulins could be observed in the explant cultures of diseased tissues from juvenile periodontitis (JP) patients. Radiolabeled proteins were detected in the SF and immunodiffusion studies using goat antihuman gamma, alpha or mu chain serum revealed the presence of IgG and IgA but no IgM present in the SF of the JP gingival tissue explant cultures. Immunodiffusion studies using goat anti-human gamma chain serum with Staph protein A isolated IgG fractions of the SF, followed by autoradiography of the IgG precipitation lines demonstrated the biosynthesis of IgG by the JP gingival tissue explant cultures. The serological studies suggested that local immune response in JP was to a polymicrobic infection. The SF of JP showed significantly higher levels of antibody reactivity to B. intermedius, C. ochracea, E. nodatum and P. micros as compared to healthy tissues. The local antibody response to the microorganisms tested differed from that observed in the sera of the patients

A study of the localized humoral immune response to implicated microorganisms in juvenile periodontitis

Energy Technology Data Exchange (ETDEWEB)

Hall, E.R.

1988-01-01

A study was undertaken using an in vitro explant culture system to determine the presence of immunoglobulins (IgG, IgA, and IgM) in the supernatant fluids (SF) of disease gingival tissue explant cultures. Studies were also undertaken to determine if the de novo biosynthesis of {sup 14}C-immunoglobulins could be observed in the explant cultures of diseased tissues from juvenile periodontitis (JP) patients. Radiolabeled proteins were detected in the SF and immunodiffusion studies using goat antihuman gamma, alpha or mu chain serum revealed the presence of IgG and IgA but no IgM present in the SF of the JP gingival tissue explant cultures. Immunodiffusion studies using goat anti-human gamma chain serum with Staph protein A isolated IgG fractions of the SF, followed by autoradiography of the IgG precipitation lines demonstrated the biosynthesis of IgG by the JP gingival tissue explant cultures. The serological studies suggested that local immune response in JP was to a polymicrobic infection. The SF of JP showed significantly higher levels of antibody reactivity to B. intermedius, C. ochracea, E. nodatum and P. micros as compared to healthy tissues. The local antibody response to the microorganisms tested differed from that observed in the sera of the patients.

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

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Suassuna José HR

2011-08-01

Full Text Available Abstract Background ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1. Methods Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection. Results In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively. Conclusion ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in

Soft sensor for monitoring biomass subpopulations in mammalian cell culture processes.

Science.gov (United States)

Kroll, Paul; Stelzer, Ines V; Herwig, Christoph

2017-11-01

Biomass subpopulations in mammalian cell culture processes cause impurities and influence productivity, which requires this critical process parameter to be monitored in real-time. For this reason, a novel soft sensor concept for estimating viable, dead and lysed cell concentration was developed, based on the robust and cheap in situ measurements of permittivity and turbidity in combination with a simple model. It could be shown that the turbidity measurements contain information about all investigated biomass subpopulations. The novelty of the developed soft sensor is the real-time estimation of lysed cell concentration, which is directly correlated to process-related impurities such as DNA and host cell protein in the supernatant. Based on data generated by two fed-batch processes the developed soft sensor is described and discussed. The presented soft sensor concept provides a tool for viable, dead and lysed cell concentration estimation in real-time with adequate accuracy and enables further applications with respect to process optimization and control.

Numerical Modeling of Fluid-Structure Interaction with Rheologically Complex Fluids

OpenAIRE

Chen, Xingyuan

2014-01-01

In the present work the interaction between rheologically complex fluids and elastic solids is studied by means of numerical modeling. The investigated complex fluids are non-Newtonian viscoelastic fluids. The fluid-structure interaction (FSI) of this kind is frequently encountered in injection molding, food processing, pharmaceutical engineering and biomedicine. The investigation via experiments is costly, difficult or in some cases, even impossible. Therefore, research is increasingly aided...

Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients.

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Salunya Tancharoen

Full Text Available Lysine-specific gingipain (Kgp is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis, a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F. We investigated the release of K6F and its induction of cytokine secretion.K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359-378, in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

Science.gov (United States)

Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

2015-01-01

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and

Maintenance fluid therapy and fluid creep impose more significant fluid, sodium, and chloride burdens than resuscitation fluids in critically ill patients: a retrospective study in a tertiary mixed ICU population.

Science.gov (United States)

Van Regenmortel, Niels; Verbrugghe, Walter; Roelant, Ella; Van den Wyngaert, Tim; Jorens, Philippe G

2018-04-01

Research on intravenous fluid therapy and its side effects, volume, sodium, and chloride overload, has focused almost exclusively on the resuscitation setting. We aimed to quantify all fluid sources in the ICU and assess fluid creep, the hidden and unintentional volume administered as a vehicle for medication or electrolytes. We precisely recorded the volume, sodium, and chloride burdens imposed by every fluid source administered to 14,654 patients during the cumulative 103,098 days they resided in our 45-bed tertiary ICU and simulated the impact of important strategic fluid choices on patients' chloride burdens. In septic patients, we assessed the impact of the different fluid sources on cumulative fluid balance, an established marker of morbidity. Maintenance and replacement fluids accounted for 24.7% of the mean daily total fluid volume, thereby far exceeding resuscitation fluids (6.5%) and were the most important sources of sodium and chloride. Fluid creep represented a striking 32.6% of the mean daily total fluid volume [median 645 mL (IQR 308-1039 mL)]. Chloride levels can be more effectively reduced by adopting a hypotonic maintenance strategy [a daily difference in chloride burden of 30.8 mmol (95% CI 30.5-31.1)] than a balanced resuscitation strategy [daily difference 3.0 mmol (95% CI 2.9-3.1)]. In septic patients, non-resuscitation fluids had a larger absolute impact on cumulative fluid balance than did resuscitation fluids. Inadvertent daily volume, sodium, and chloride loading should be avoided when prescribing maintenance fluids in view of the vast amounts of fluid creep. This is especially important when adopting an isotonic maintenance strategy.

Accuracy of the cytopathology, bacterioscopy, and vaginal flora culture.

Science.gov (United States)

Almeida, P Q; Pereira, M A P; Palomo, F S; Okazaki, C; Schimidt, M A; Speck, N M G; Ribalta, J C L

2013-01-01

An over-population of vaginal microorganisms causing inflammatory processes renders it difficult to properly assess the cytopathological exam that aims to screen precedent cervical lesions. On the contrary, the occurrence of the microbial flora saprophyte does not influence correct cythodiagnosis. To assess the composition of vaginal tract aerobic microorganisms of asymptomatic women in menacme and post-menopause, and to analyze the accuracy of cytopathologic, bacterioscopic exams, and culturing of the flora. The women were first submitted to a focused anamnestic interrogatory and then submitted to gynecological exam. A sample of the vaginal fluid was collected with a culture swab and a smear was made on two glass slides for stained bacterioscopic exam (GRAM). The collection of material was then compiled in a cytopathologic smear analysis. All women signed the free and informed consent letter and the project was approved by the Ethics Research Board of Hospital São Paulo - UNIFESP. Bacterioscopy and culture proved to be better than the cytopathologic exam in featuring the bacilli and cocci. The bacterioscopy provided a better detection of the presence of bacilli (p < 0.001); no statistical difference was seen between both exams with respect to the detected cocci. The beta-hemolytic Streptococcus group was of significance in post-menopausal women (p < 0.05). In this study, the bacterioscopic and culture exams of the vaginal fluid were more effective in assessing the vaginal flora and in the detection of bacilli, compared to the cytopathological exam.

Effect of oxidative stress induced by Brevibacterium sp. BS01 on a HAB causing species--Alexandrium tamarense.

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Huajun Zhang

Full Text Available Harmful algal blooms occur all over the world, destroying aquatic ecosystems and threatening other organisms. The culture supernatant of the marine algicidal actinomycete BS01 was able to lysis dinoflagellate Alexandrium tamarense ATGD98-006. Physiological and biochemical responses to oxidative stress in A. tamarense were investigated to elucidate the mechanism involved in BS01 inhibition of algal growth. Transmission electron microscope analysis revealed that there were some chloroplast abnormalities in response to BS01 supernatant. The decrease in cellular-soluble protein content suggested that cell growth was greatly inhibited at high concentration of BS01 supernatant. The increase in the levels of reactive oxygen species (ROS and malondialdehyde contents following exposure to BS01 supernatant indicated that algal cells suffered from oxidative damage. The content of pigment was significantly decreased after 12 h treatment, which indicated that the accumulation of ROS destroyed pigment synthesis. Moreover, the decrease of Fv/Fm ratio suggested that in the photosynthetic system, the dominant sites producing ROS were destroyed by the supernatant of the BS01 culture. The activities of the antioxidant enzymes including superoxide dismutase and peroxidase increased in a short time and decreased slightly with increasing exposure time. A real-time PCR assay showed changes in the transcript abundances of two photosynthetic genes, psbA and psbD. The results showed that BS01 supernatant reduced the expression of the psbA gene after 2 h exposure, but the expression of the psbD gene was increased at concentrations of 1.0 and 1.5%. Our results demonstrated that the expression of the psbA gene was inhibited by the BS01 supernatant, which might block the electron transport chain, significantly enhancing ROS level and excess activity of the antioxidant system. The accumulation of ROS destoryed pigment synthesis and membrane integrity, and inhibited or

Green Algae as Model Organisms for Biological Fluid Dynamics

Science.gov (United States)

Goldstein, Raymond E.

2015-01-01

In the past decade, the volvocine green algae, spanning from the unicellular Chlamydomonas to multicellular Volvox, have emerged as model organisms for a number of problems in biological fluid dynamics. These include flagellar propulsion, nutrient uptake by swimming organisms, hydrodynamic interactions mediated by walls, collective dynamics and transport within suspensions of microswimmers, the mechanism of phototaxis, and the stochastic dynamics of flagellar synchronization. Green algae are well suited to the study of such problems because of their range of sizes (from 10 μm to several millimeters), their geometric regularity, the ease with which they can be cultured, and the availability of many mutants that allow for connections between molecular details and organism-level behavior. This review summarizes these recent developments and highlights promising future directions in the study of biological fluid dynamics, especially in the context of evolutionary biology, that can take advantage of these remarkable organisms.

Fermentation supernatants of Lactobacillus delbrueckii inhibit growth of human colon cancer cells and induce apoptosis through a caspase 3-dependent pathway.

Science.gov (United States)

Wan, Ying; Xin, Yi; Zhang, Cuili; Wu, Dachang; Ding, Dapeng; Tang, Li; Owusu, Lawrence; Bai, Jing; Li, Weiling

2014-05-01

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Therefore, the present study explored the effect of the supernatants obtained from Lactobacillus delbrueckii fermentation (LBF) on colon cancer. The results indicated that the proliferation of LBF solution-treated colon cancer SW620 cells was arrested and accumulated in the G1 phase in a concentration-dependent manner. The LBF solution efficiently induced apoptosis through the intrinsic caspase 3-depedent pathway, with a corresponding decreased expression of Bcl-2. The activity of matrix metalloproteinase 9, which is associated with the invasion of colon cancer cells, was also decreased in the LBF-treated cells. In conclusion, the results demonstrate the antitumor effect of LBF in vitro and may contribute to the development of novel therapies for the treatment of colon cancer.

Flow field measurements in the cell culture unit

Science.gov (United States)

Walker, Stephen; Wilder, Mike; Dimanlig, Arsenio; Jagger, Justin; Searby, Nancy

2002-01-01

The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the

Self lubricating fluid bearings

International Nuclear Information System (INIS)

Kapich, D.D.

1980-01-01

The invention concerns self lubricating fluid bearings, which are used in a shaft sealed system extending two regions. These regions contain fluids, which have to be isolated. A first seal is fluid tight for the first region between the carter shaft and the shaft. The second seal is fluid tight between the carter and the shaft, it communicates with the second region. The first fluid region is the environment surrounding the shaft carter. The second fluid region is a part of a nuclear reactor which contains the cooling fluid. The shaft is conceived to drive a reactor circulating and cooling fluid [fr

REMEDIAL APPLICATIONS OF EXOSOMES IN CANCER, INFECTIONS AND DIABETES.

Science.gov (United States)

Wang, Qianhuai; Ding, Xiaoqing; Zhen, Fei; Ma, Jianwei; Meng, Fanxing

2017-03-01

Different cell types under normal and diseased states constantly secrete numerous membrane vesicles including exosomes into extracellular space wkich can be isolated from various biological fluids and cell culture supernatants. Exosomal diameter ranges between 40-100 nm. In current research, exosomes are being exploited as biomarkers for pathological diagnosis and potential remedy against various disease conditions such as infections and autoimmune disorders. In cancer immunotherapy, exosomes have promisingly been employed due to the identical immunogenic antigens of exosomes produced by neoplastic cells and the originating tissues. Antigen-specific T-cell activation and immunomodulatory activity is observed to be performed by exosomes isolated from tumor and dendritic cells (DCs). However, more research is still required to uncover the realistic uses of exosomes, particularly as drug delivery tool in autoimmune diseases, cancers and diabetes.

The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

Science.gov (United States)

Tian, Shan; Zhu, Fengping; Hu, Ruiping; Tian, Song; Chen, Xingxing; Lou, Dan; Cao, Bing; Chen, Qiulei; Li, Bai; Li, Fang; Bai, Yulong; Wu, Yi; Zhu, Yulian

2018-01-01

Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface. Copyright © 2017 Elsevier B.V. All rights reserved.

Synovial fluid white cell count and histopathological examination of periprosthetic tissue samples (frozen and permanent sections in the diagnosis of prosthetic knee infection

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Obada B.

2017-02-01

Full Text Available The aim of the study was to determine prospectively the importance of synovial fluid white cell count and intraoperative frozen and permanent sections analysis (number of polymorphonuclear leukocytes per high-power field in the diagnosis of septic total knee arthroplasty. There were studied prospectively 72 patients who needed a revision total knee arthroplasty between 2013-2015. 30 patients were diagnosed with prosthetic joint infection due to high rates of ESR (93% and CRP (90% and preoperative positive culture from aspirated synovial fluid and 42 patients were considered to have aseptic failure according to negative preoperative culture from joint aspirate. For all the patients was analysed synovial fluid white cell count and histopathological aspect of intraoperative frozen and permanent sections of periprosthetic tissue. The results showed a median value of 13800 of sinovial white cells count for infected knee and 92 for noninfected knee. 90% of the patients with joint infection had more than 5 polymorphonuclear leukocytes per high power field on intraoperative frozen sections and 83% on permanent sections. None of the patients from aseptic group had more than 5 polymorphonuclear leukocytes per field on permanent sections. The erythrocyte sedimentation rate and C-reactive protein level can be supplemented with cultures of aspirated joint fluid and fluid white cell count to confirm the diagnosis of periprosthetic infection. When the preoperative diagnosis remain unclear, the histological examination of frozen or permanent sections of periprosthetic tissue with at least 5 polymorphonuclear leukocytes per high power field, is predictive for the presence of infection.

[Effects of non-saccharomyces albicans metabolic products on the proliferation of human umbilical vein endothelial cell ECV304].

Science.gov (United States)

Chen, Bin; Che, Tuanjie; Bai, Decheng; He, Xiangyi

2013-04-01

To evaluate the effects of non-Saccharomyces albicans metabolic products on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro. The parallel dilution supernatant of Saccharomyces tropicalis, Saccharomyces krusei and Saccharomyces glabrata were prepared, and 1, 4, 16-fold(s) diluted concentration and control group were set up. The line of human umbilical vein endothelial cell ECV304 was cultured in vitro and treated by non-Saccharomyces albicans supernatant. The proliferous effect of ECV304 induced by non-Saccharomyces albicans supernatant after 24, 48, 72 h was detected by the methods of MTT, and the changes of cell density and cycle after 48 h were investigated by inverted microscope and flow cytometry. At the 24th hour, all of the higher concentration (1-fold) of non-Saccharomyces albicans supernatant and the 4-folds diluted Saccharomyces krusei could promote ECV304 proliferation(P Saccharomyces albicans supernatant at 48h and 72th hour, Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant significantly increased proliferation rate of ECV304, while Saccharomyces tropicalis supernatant group showed no significant change no matter which concentration was tested. At 48th hour after adding the non-Saccharomyces albicans supernatant, the ECV304 cells density treated by Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant were significantly higher under the inverted microscope. The G0/G1 population of ECV304 cells decreased while cell proliferation index (PI) increased after incubated with Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant for 48 hours (P Saccharomyces tropicalis group showed no significant change (P > 0.05). The metabolic products of Sacharoymces krusei and Saccharomyces glabrata could induce proliferation of ECV304 cell, which suggests non-Saccharomyces albicans should be undergone more attention clinically in detection and treatment.

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

Science.gov (United States)

Entcheva, P; Liebl, W; Johann, A; Hartsch, T; Streit, W R

2001-01-01

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

Directory of Open Access Journals (Sweden)

Sergio E Palma-Vera

Full Text Available Ovarian steroid hormones (mainly E2 and P4 regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.A cell line derived from the porcine oviductal epithelium (CCLV-RIE270 was characterized (lineage markers, proliferation characteristics and transformation status. Primary porcine oviduct epithelial cells (POEC were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml. Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR and inflammation was quantified.CCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8. Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.CCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

One-step immunochromatographic visual assay for anti-transglutaminase detection in organ culture system: An easy and prompt method to simplify the in vitro diagnosis of celiac disease.

Science.gov (United States)

Di Tola, Marco; Marino, Mariacatia; Casale, Rossella; Borghini, Raffaele; Tiberti, Antonio; Donato, Giuseppe; Occhiuzzi, Umberto; Picarelli, Antonio

2018-01-01

Anti-tissue transglutaminase (anti-tTG) and endomysium antibodies (EMA) are detectable in duodenal culture media of celiac disease (CD) patients. To improve the management of this organ culture system, we evaluated the anti-tTG occurrence by immunochromatographic assay (ICA). A total of 103 CD patients and 41 disease controls underwent duodenal biopsy for the organ culture. In culture supernatants, IgA anti-tTG were tested by both enzyme-linked immunosorbent assay (ELISA) and ICA, IgA EMA were searched by indirect immunofluorescence analysis (iIFA). Endomysium antibodies and anti-tTG measured by ELISA were positive in culture media of all CD patients, while anti-tTG detected by ICA were positive in culture media of 87/103 CD patients. Anti-tTG ICA scores significantly correlated with anti-tTG ELISA values (r=.71, Pculture media of most CD patients and the intensity of indicative lines depends on the anti-tTG concentration. Sensitivity and diagnostic accuracy achieved with ICA are lower than those obtained with ELISA but, given that the first is a more easy and prompt method, data suggest the possibility of utilizing it in the in vitro diagnosis of CD. © 2017 Wiley Periodicals, Inc.

Long-term storage of clinical samples in CyMol® medium for PNA- FISH® and culturing from the eSwab™ system

DEFF Research Database (Denmark)

Larsen, Lone Heimann; Xu, Yijuan; Pedersen, Malene Schibler

Objectives: A steadily growing diversity of bacteria is reported in foreign body infections, and culture-independent methods have been shown to supplement established culture methods. Therefore, sampling and preservation of specimens have become an important issue. We report here experience from...... analytical methods. Methods: Sampling for both culture-dependent and -independent analyses were done over a period of two years. Specimens were transferred directly to the lab, and cultures of tissue biopsies, joint fluid, sonication fluid from the prosthesis components, and eSwab™ (Copan, Italy) were...... performed within 24 h after sampling. The corresponding specimens for culture-independent methods were stored at -80°C until analyzed in batchs. Specimens for fluorescence in situ hybridization (FISH) analysis were stored for app. one year at -80°C in CyMol® (Copan, Italy), an alcohol based media, before...

Standardization of Thermo-Fluid Modeling in Modelica.Fluid

Energy Technology Data Exchange (ETDEWEB)

Franke, Rudiger; Casella, Francesco; Sielemann, Michael; Proelss, Katrin; Otter, Martin; Wetter, Michael

2009-09-01

This article discusses the Modelica.Fluid library that has been included in the Modelica Standard Library 3.1. Modelica.Fluid provides interfaces and basic components for the device-oriented modeling of onedimensional thermo-fluid flow in networks containing vessels, pipes, fluid machines, valves and fittings. A unique feature of Modelica.Fluid is that the component equations and the media models as well as pressure loss and heat transfer correlations are decoupled from each other. All components are implemented such that they can be used for media from the Modelica.Media library. This means that an incompressible or compressible medium, a single or a multiple substance medium with one or more phases might be used with one and the same model as long as the modeling assumptions made hold. Furthermore, trace substances are supported. Modeling assumptions can be configured globally in an outer System object. This covers in particular the initialization, uni- or bi-directional flow, and dynamic or steady-state formulation of mass, energy, and momentum balance. All assumptions can be locally refined for every component. While Modelica.Fluid contains a reasonable set of component models, the goal of the library is not to provide a comprehensive set of models, but rather to provide interfaces and best practices for the treatment of issues such as connector design and implementation of energy, mass and momentum balances. Applications from various domains are presented.

Differential contributions of specimen types, culturing, and 16S rRNA sequencing in diagnosis of prosthetic joint infections

DEFF Research Database (Denmark)

Larsen, Lone Heimann; Khalid, Vesal; Xu, Yijuan

2018-01-01

Prosthetic joint failure is mainly caused by infection, aseptic failure (AF), and mechanical problems. Infection detection has been improved with modified culture methods and molecular diagnostics. However, comparisons between modified and conventional microbiology methods are difficult due...... to variations in specimen sampling. In this prospective, multidisciplinary study of hip or knee prosthetic failures, we assessed the contributions of different specimen types, extended culture incubations, and 16S rRNA sequencing for diagnosing prosthetic joint infections (PJI). Project specimens included joint...... fluid (JF), bone biopsy specimens (BB), soft-tissue biopsy specimens (STB), and swabs (SW) from the prosthesis, collected in situ, and sonication fluid collected from prosthetic components (PC). Specimens were cultured for 6 (conventional) or 14 days, and 16S rRNA sequencing was performed at study...

Thermophysical properties of supercritical fluids and fluid mixtures

International Nuclear Information System (INIS)

Sengers, J.V.

1991-07-01

This research is concerned with the development of a quantitative scientific description of the thermodynamic and transport properties of supercritical and subcritical fluids and fluid mixtures. It is known that the thermophysical properties of fluids and fluid mixtures asymptotically close to the critical point satisfy scaling laws with universal critical exponents and universal scaling functions. However, the range of validity of these asymptotic scaling laws is quite small. As a consequence, the impact of the modern theory of critical phenomena on chemical engineering has been limited. On the other hand, an a priori estimate of the range of temperatures and densities, where critical fluctuations become significant, can be made on the basis of the so-called Ginzburg criterion. A recent analysis of this criterion suggests that this range is actually quite large and for a fluid like carbon dioxide can easily extend to 100 degrees or so above the critical temperature. Hence, the use of traditional engineering equations like cubic equations is not scientifically justified in a very wide range of temperatures and densities around the critical point. We have therefore embarked on a scientific approach to deal with the global effects of critical fluctuations on the thermophysical properties of fluids and fluid mixtures. For this purpose it is not sufficient to consider the asymptotic critical fluctuations but we need to deal also with the nonasymptotic critical fluctuations. The goal is to develop scientifically based questions that account for the crossover of the thermophysical properties from their asymptotic singular behavior in the near vicinity of the critical point to their regular behavior very far away from the critical point

Effect of L-arginine on the growth of Plasmodium falciparum and immune modulation of host cells.

Science.gov (United States)

Awasthi, Vikky; Chauhan, Rubika; Chattopadhyay, Debprasad; Das, Jyoti

2017-01-01

Malaria is a life-threatening disease caused by Plasmodium parasites. The life-cycle of Plasmodium species involves several stages both in mosquito and the vertebrate host. In the erythrocytic stage, Plasmodium resides inside the red blood cells (RBCs), where it meets most of its nutritional requirement by degrad- ing host's haemoglobin. L-arginine is required for growth and division of cells. The present study was aimed to demonstrate the effect of supplementation of different concentrations of L-arginine and L-citrulline on the growth of parasite, and effect of the culture supernatant on the host's peripheral blood mononuclear cells (PBMCs). To examine the effect of supplementation of L-arginine and L-citrulline, Plasmodium falciparum (3D7 strain) was cultured in RPMI 1640, L-arginine deficient RPMI 1640, and in different concentrations of L-arginine, and L-citrulline supplemented in arginine deficient RPMI 1640 medium. To have a holistic view of in vivo cell activation, the PBMCs isolated from healthy human host were cultured in the supernatant collected from P. falciparum culture. Growth of the parasite was greatly enhanced in L-arginine supplemented media and was found to be concentration dependent. However, parasite growth was compromised in L-citrulline supplemented and L-arginine deficient media. The supernatant collected from L-arginine supplemented parasite media (sArg) showed increased FOXP3 and interleukin-10 (IL-10) expression as compared to the supernatant collected from L-citrulline supple- mented parasite media (sCit). The in vitro culture results showed, decreased parasite growth, and decreased expression of programmed cell death-1 (PD-1) (a coinhibitory molecule) and IL-10 in the L-citrulline supplemented media as compared to L-arginine supplemented media. Hence, it was concluded that L-citrulline supplementation would be a better alternative than L-arginine to inhibit the parasite growth.

N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

Directory of Open Access Journals (Sweden)

Costa Nicola

2007-12-01

Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

The changes of production of lymphokines from gamma irradiated human tonsillar lymphocytes: Pt. 2

International Nuclear Information System (INIS)

Miao Jingcheng; Zhang Lansheng

1989-02-01

The human tonsillar lymphocytes were exposed to gamma rays in various doses (0 ∼ 40 Gy) and stimulated by PHA, then cultured for 24 to 96 hours. The activities of NKCF in the supernatants were assayed by MTT colorimetric method. The results showed: (1) The activity of NKCF was slightly inhibited by irradiation of 2.5 ∼ 40 Gy; (2) The activity of NKCF in the supernatants cultured for 48 to 96 hours is obviously higher than that for 24 hours. Both the irradiatiion doses and cultural periods had no interactiion on the changes of the production of NKCF. The radiation resistance of NK cells in the experiment is similar to other results. The tonsillar Nk cells activated in the state of chronic inflamation has higher radioresistance

CT findings of a unicameral calcaneal bone cyst containing a fluid-fluid level.

Science.gov (United States)

Gallagher, Thomas A; Lim-Dunham, Jennifer E; Vade, Aruna

2007-03-01

Calcaneal unicameral bone cysts often contain fluid, but rarely contain fluid-fluid levels. We present a case focusing on the CT findings of a large calcaneal bone cyst with a fluid-fluid level and a review of the literature.

Atomistic Modeling of the Fluid-Solid Interface in Simple Fluids

Science.gov (United States)

Hadjiconstantinou, Nicolas; Wang, Gerald

2017-11-01

Fluids can exhibit pronounced structuring effects near a solid boundary, typically manifested in a layered structure that has been extensively shown to directly affect transport across the interface. We present and discuss several results from molecular-mechanical modeling and molecular-dynamics (MD) simulations aimed at characterizing the structure of the first fluid layer directly adjacent to the solid. We identify a new dimensionless group - termed the Wall number - which characterizes the degree of fluid layering, by comparing the competing effects of wall-fluid interaction and thermal energy. We find that in the layering regime, several key features of the first layer layer - including its distance from the solid, its width, and its areal density - can be described using mean-field-energy arguments, as well as asymptotic analysis of the Nernst-Planck equation. For dense fluids, the areal density and the width of the first layer can be related to the bulk fluid density using a simple scaling relation. MD simulations show that these results are broadly applicable and robust to the presence of a second confining solid boundary, different choices of wall structure and thermalization, strengths of fluid-solid interaction, and wall geometries.

Rupatadine inhibits inflammatory mediator release from human laboratory of allergic diseases 2 cultured mast cells stimulated by platelet-activating factor.

Science.gov (United States)

Alevizos, Michail; Karagkouni, Anna; Vasiadi, Magdalini; Sismanopoulos, Nikolaos; Makris, Michael; Kalogeromitros, Dimitrios; Theoharides, Theoharis C

2013-12-01

Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 μmol/L) and substance P (1 μmol/L) with or without pretreatment with RUP (2.5 and 25 μmol/L), which was added 10 minutes before stimulation. Release of β-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001-0.1 μmol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 μmol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 μmol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Radiographical diagnosis of posttraumatic knee joints hemarthrosis and lipohemarthrosis

International Nuclear Information System (INIS)

Li Shuling; Liu Xulin; Ma Heng; Zhang Guanghui; Zhou Chengtao; Tang Xiaofeng; Li Wenle; Liu Zhongguang; Zhang Baozheng; Sun Qingju

2008-01-01

Objective: To explore the imaging findings and diagnostic values of X-ray, CT, MR, and ultrasonography in traumatic knee joints hemarthrosis and lipohemarthrosis. Methods: Traumatic knee joints hemarthrosis (12 knees) and lipohemarthrosis (18 knees) proved by operation (27 knees) or puncturation (3 knees) were included in the study. Horizontal-beam plain radiographs (16 knees), CT (30 knees), MRI (30 knees) and ultrasonography (24 knees) in supine position were investigated. Results (1)supine position horizontal-beam plain radiographs: Fat-liquid layer was found in 8 cases of lipohemarthrosis. Dense supragenual bursa was found in 1 case of lipohemarthrosis and 7 cases of hemarthrosis. Fracture (13 knees) was diagnosed correctly. (2) CT findings: double fluid-fluid layer was found in 11 of all 18 cases, and single fluid-fluid layer was found in 7 of 11 cases of lipohemarthrosis. Single fluid-fluid layer was found in 3 of 12 cases of hemarthrosis. Isodensity was detected in 9 cases, and high- density blood clot was found in 4 cases. Fracture (30 knees) was diagnosed correctly. (3) MRI findings: in 12 of 18 cases of lipohemarthrosis, double fluid-fluid layer was shown including supernatant layer as short T 1 , long T 2 signal and low signal after fat-suppression, middle layer as long T 1 , long T 2 signal and high signal after fat-suppression, and dependent layer as iso-T 1 , iso-T 2 and slight high signal after fat- suppression. Single fluid-fluid layer was seen in 6 cases, only had aforementioned upper and under layer. Only aforementioned supernatant layer and dependent layer were seen in 12 cases of hemarthrosis. 4 cases showed entire blood clot in fluid, T 1 WI showed middle signal or center iso-signal accompanied with peripheral high signal ring, and fat-suppression imaging showed high signal. T 2 WI and fat-suppression imaging showed middling or high signal accompanied with peripheral low signal ring. Fracture (30 knees) was diagnosed correctly. (4) Ultrasound

Synovial fluid analysis

Science.gov (United States)

Joint fluid analysis; Joint fluid aspiration ... El-Gabalawy HS. Synovial fluid analysis, synovial biopsy, and synovial pathology. In: Firestein GS, Budd RC, Gabriel SE, McInnes IB, O'Dell JR, eds. Kelly's Textbook of ...

Assessment of the pro-inflammatory activity of water sampled from ...

African Journals Online (AJOL)

2014-03-20

Mar 20, 2014 ... nursing sister and obtaining informed consent, MNL were pre- pared from heparinised (5 ... and discarding of the supernatant fluid, the cells were re-sus- pended in sterile tissue ... The balance is likely to consist of natural ...

Quantitative cultures of bronchoscopically obtained specimens should be performed for optimal management of ventilator-associated pneumonia.

Science.gov (United States)

Baselski, Vickie; Klutts, J Stacey; Baselski, Vickie; Klutts, J Stacey

2013-03-01

Ventilator-associated pneumonia (VAP) is a leading cause of health care-associated infection. It has a high rate of attributed mortality, and this mortality is increased in patients who do not receive appropriate empirical antimicrobial therapy. As a result of the overuse of broad-spectrum antimicrobials such as the carbapenems, strains of Acinetobacter, Enterobacteriaceae, and Pseudomonas aeruginosa susceptible only to polymyxins and tigecycline have emerged as important causes of VAP. The need to accurately diagnose VAP so that appropriate discontinuation or de-escalation of antimicrobial therapy can be initiated to reduce this antimicrobial pressure is essential. Practice guidelines for the diagnosis of VAP advocate the use of bronchoalveolar lavage (BAL) fluid obtained either bronchoscopically or by the use of a catheter passed through the endotracheal tube. The CDC recommends that quantitative cultures be performed on these specimens, using ≥ 10(4) CFU/ml to designate a positive culture (http://www.cdc.gov/nhsn/TOC_PSCManual.html, accessed 30 October 2012). However, there is no consensus in the clinical microbiology community as to whether these specimens should be cultured quantitatively, using the aforementioned designated bacterial cell count to designate infection, or by a semiquantitative approach. We have asked Vickie Baselski, University of Tennessee Health Science Center, who was the lead author on one of the seminal papers on quantitative BAL fluid culture, to explain why she believes that quantitative BAL fluid cultures are the optimal strategy for VAP diagnosis. We have Stacey Klutts, University of Iowa, to advocate the semiquantitative approach.

Editorial Special Issue on Fluid Mechanics and Fluid Power (FMFP ...

Indian Academy of Sciences (India)

This special issue of Sadhana contains selected papers from two conferences related to fluid mechanics held in India recently, Fluid Mechanics and Fluid Power conference at NIT, Hamirpur, and an International Union of Theoretical ... A simple, well thought out, flow visualization experiment or a computation can sometimes ...

FOREWORD Fluid Mechanics and Fluid Power (FMFP)

Indian Academy of Sciences (India)

This section of the Special Issue carries selected articles from the Fluid Mechanics and Fluid. Power Conference held during 12–14 December 2013 at the National Institute of Technology,. Hamirpur (HP). The section includes three review articles and nine original research articles. These were selected on the basis of their ...

Mycobacterium tuberculosis bacteremia detected by the Isolator lysis-centrifugation blood culture system.

OpenAIRE

Kiehn, T E; Gold, J W; Brannon, P; Timberger, R J; Armstrong, D

1985-01-01

Mycobacterium tuberculosis was detected by the Isolator lysis-centrifugation blood culture system from the blood of a patient with tuberculosis of the breast. The organism also grew on conventional laboratory media inoculated with pleural fluid from the patient.

statistical fluid theory for associating fluids containing alternating ...

Indian Academy of Sciences (India)

Statistical associating fluid theory of homonuclear dimerized chain fluids and homonuclear ... The proposed models account for the appropriate .... where gHNM(1,1) is the expression for the contact value of the correlation func- tion of two ...

Mixed species biofilms of Fusobacterium necrophorum and Porphyromonas levii impair the oxidative response of bovine neutrophils in vitro.

Science.gov (United States)

Lockhart, Joey S; Buret, Andre G; Ceri, Howard; Storey, Douglas G; Anderson, Stefanie J; Morck, Douglas W

2017-10-01

Biofilms composed of anaerobic bacteria can result in persistent infections and chronic inflammation. Host immune cells have difficulties clearing biofilm-related infections and this can result in tissue damage. Neutrophils are a vital component of the innate immune system and help clear biofilms. The comparative neutrophilic response to biofilms versus planktonic bacteria remains incompletely understood, particularly in the context of mixed infections. The objective of this study was to generate mixed species anaerobic bacterial biofilms composed of two opportunistic pathogens, Fusobacterium necrophorum and Porphyromonas levii, and evaluate neutrophil responses to extracellular fractions from both biofilms and planktonic cell co-cultures of the same bacteria. Purified bovine neutrophils exposed to culture supernatants from mixed species planktonic bacteria showed elevated oxidative activity compared to neutrophils exposed to biofilms composed of the same bacteria. Bacterial lipopolysaccharide plays a significant role in the stimulation of neutrophils; biofilms produced substantially more lipopolysaccharide than planktonic bacteria under these experimental conditions. Removal of lipopolysaccharide significantly reduced neutrophil oxidative response to culture supernatants of planktonic bacteria. Oxidative responses to LPS-removed biofilm supernatants and LPS-removed planktonic cell supernatants were similar. The limited neutrophil response to biofilm bacteria observed in this study supports the reduced ability of the innate immune system to eradicate biofilm-associated infections. Lipopolysaccharide is likely important in neutrophil response; however, the presence of other extracellular, immune modifying molecules in the bacterial media also appears to be important in altering neutrophil function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diagnostic significance of pleural fluid adenosine deaminase activity in tuberculous pleurisy

Directory of Open Access Journals (Sweden)

Sharmeen Ahmed

2011-01-01

Full Text Available Diagnosis of tuberculous pleural effusion (TPE is difficult because of its non-specific clinical presentation and insufficient efficiency of conventional diagnostic methods. The study was carried out to evaluate the utility of adenosine deaminase (ADA activity in pleural fluid for the diagnosis of TPE. ADA activity was measured in pleural fluid of 103 pleural effusion patients by colorimetric method using a commercial ADA assay kit. The diagnosis of TPE was made from pleural fluid examinations (including cytology, biochemistry, and bacteriology and pleural biopsy. Patient with negative result of this methods were diagnosed by response of empirical treatment. Out of 130 cases, 62 (61.1% had TPE and the remaining 41 (39.8% had pleural effusion due to non tuberculous diseases. There was statistically significant difference (p < 0.001 between the mean of pleural fluid ADA levels (70.82±22.54 U/L in TPE group and (30.07±22.93 U/L in non-TPE group. Of 62 TPE cases, microscopy for AFB and culture for M.tuberculosis in pleural fluid revealed positivity in 9.6% and 22.5% cases respectively, and biopsy of pleura showed typical epithelioid granuloma in only 43.5% cases. The cut-off value of ADA for diagnosing TPE was 40 U/L using a ROC curve, with a sensitivity of 94% and specificity of 88%. Positive and negative predictive value of ADA assay were 92% and 90% respectively. The overall test accuracy was 90%. Pleural fluid ADA assay is therefore a simple, rapid, highly sensitive and specific adjunct test for diagnosis of TPE. Ibrahim Med. Coll. J. 2011; 5(1: 1-5

Fluid inclusion geothermometry

Science.gov (United States)

Cunningham, C.G.

1977-01-01

Fluid inclusions trapped within crystals either during growth or at a later time provide many clues to the histories of rocks and ores. Estimates of fluid-inclusion homogenization temperature and density can be obtained using a petrographic microscope with thin sections, and they can be refined using heating and freezing stages. Fluid inclusion studies, used in conjunction with paragenetic studies, can provide direct data on the time and space variations of parameters such as temperature, pressure, density, and composition of fluids in geologic environments. Changes in these parameters directly affect the fugacity, composition, and pH of fluids, thus directly influencing localization of ore metals. ?? 1977 Ferdinand Enke Verlag Stuttgart.

Regulated gene expression in cultured type II cells of adult human lung

OpenAIRE

Ballard, Philip L.; Lee, Jae W.; Fang, Xiaohui; Chapin, Cheryl; Allen, Lennell; Segal, Mark R.; Fischer, Horst; Illek, Beate; Gonzales, Linda W.; Kolla, Venkatadri; Matthay, Michael A.

2010-01-01

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at ∼95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days...

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

Directory of Open Access Journals (Sweden)

Sunita Nayak

Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro.

Science.gov (United States)

Malandrin, Laurence; Marchand, Anne-Marie; Chauvin, Alain

2004-09-01

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.

Buffer fluid

Energy Technology Data Exchange (ETDEWEB)

Mirzadzhanzade, A Kh; Dedusanko, G Ya; Dinaburg, L S; Markov, Yu M; Rasizade, Ya N; Rozov, V N; Sherstnev, N M

1979-08-30

A drilling fluid is suggested for separating the drilling and plugging fluids which contains as the base increased solution of polyacrylamide and additive. In order to increase the viscoelastic properties of the liquid with simultaneous decrease in the periods of its fabrication, the solution contains as an additive dry bentonite clay. In cases of the use of a buffer fluid under conditions of negative temperatures, it is necessary to add to it table salt or ethylene glycol.

Immunomodulatory effect of non-viable components of probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus on holoxenic mice.

Science.gov (United States)

Ditu, L M; Chifiriuc, M C; Bezirtzoglou, E; Marutescu, L; Bleotu, C; Pelinescu, D; Mihaescu, G; Lazar, V

2014-01-01

Competition of probiotic bacteria with other species from the intestinal microbiota involves different mechanisms that occur regardless of probiotics' viability. The objective of this paper was to assess the cytokine serum levels in holoxenic mice after oral administration of non-viable components (NVC) of Enterococcus faecium probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus in comparison to NVC of unstimulated E. faecium probiotic culture. Probiotic E. faecium CMGb 16 culture, grown in the presence of heat-inactivated cultures of E. coli and B. cereus CMGB 102, was subsequently separated into supernatant (SN) and heat-inactivated cellular sediment (CS) fractions by centrifugation. Each NVC was orally administered to holoxenic mice (balb C mouse strain), in three doses, given at 24 hours. Blood samples were collected from the retinal artery, at 7, 14, and 21 days after the first administration of the NVC. The serum concentrations of IL-12 and tumor necrosis factor-alpha (TNF-α) interleukins were assessed by ELISA method. After the oral administration of SN component obtained from the probiotic culture stimulated with heat-inactivated cultures of B. cereus CMGB 102 and E. coli O28, the serum concentrations of IL-12 were maintained higher in the samples collected at 7 and 14 days post-administration. No specific TNF-α profile could be established, depending on stimulated or non-stimulated probiotic culture, NVC fraction, or harvesting time. The obtained results demonstrate that non-viable fractions of probiotic bacteria, stimulated by other bacterial species, could induce immunostimulatory effects mediated by cytokines and act, therefore, as immunological adjuvants.

Fluid Shifts

Science.gov (United States)

Stenger, M. B.; Hargens, A. R.; Dulchavsky, S. A.; Arbeille, P.; Danielson, R. W.; Ebert, D. J.; Garcia, K. M.; Johnston, S. L.; Laurie, S. S.; Lee, S. M. C.;

Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

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Archana Shrestha

2016-12-01

Full Text Available Clostridium perfringens enterotoxin (CPE binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

Grain dust induces IL-8 production from bronchial epithelial cells: effect on neutrophil recruitment.

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Park, H S; Suh, J H; Kim, S S; Kwon, O J

2000-06-01