Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants.

PubMed

Khalil, Jacques Y B; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2016-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus , we sorted and re-cultured a new, slow-growing virus, which we named "Cedratvirus." The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.

Bioleaching of metals from steel slag by Acidithiobacillus thiooxidans culture supernatant.

PubMed

Hocheng, Hong; Su, Cheer; Jadhav, Umesh U

2014-12-01

The generation of 300–500 kg of slag per ton of the steel produced is a formidable amount of solid waste available for treatment. They usually contain considerable quantities of valuable metals. In this sense, they may become either important secondary resource if processed in eco-friendly manner for secured supply of contained metals or potential pollutants, if not treated properly. It is possible to recover metals from steel slag by applying bioleaching process. Electric arc furnace (EAF) slag sample was used for bioleaching of metals. In the present study, before bioleaching experiment water washing of an EAF slag was carried out. This reduced slag pH from 11.2 to 8.3. Culture supernatants of Acidithiobacillus thiooxidans (At. thiooxidans), Acidithiobacillus ferrooxidans (At. ferrooxidans), and Aspergillus niger (A. niger) were used for metal solubilization. At. thiooxidans culture supernatant containing 0.016 M sulfuric acid was found most effective for bioleaching of metals from an EAF slag. Maximum metal extraction was found for Mg (28%), while it was least for Mo (0.1%) in six days. Repeated bioleaching cycles increased metal recovery from 28% to 75%, from 14% to 60% and from 11% to 27%, for Mg, Zn and Cu respectively.

Relative potency of culture supernatants of Xenorhabdus and Photorhabdus spp. on growth of some fungal phytopathogens

USDA-ARS?s Scientific Manuscript database

We evaluated the potency of 10% v/v cell-free culture supernatants of cultures of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), Fusicladium effusum (pecan scab), Moni...

40 CFR Appendix Vii to Part 86 - Standard Bench Cycle (SBC)

Code of Federal Regulations, 2012 CFR

2012-07-01

.... VII Appendix VII to Part 86—Standard Bench Cycle (SBC) 1. The standard bench aging durability procedures [Ref. § 86.1823-08(d)] consist of aging a catalyst-oxygen-sensor system on an aging bench which follows the standard bench cycle (SBC) described in this appendix. 2. The SBC requires use of an aging...

40 CFR Appendix Vii to Part 86 - Standard Bench Cycle (SBC)

Code of Federal Regulations, 2013 CFR

2013-07-01

.... VII Appendix VII to Part 86—Standard Bench Cycle (SBC) 1. The standard bench aging durability procedures [Ref. § 86.1823-08(d)] consist of aging a catalyst-oxygen-sensor system on an aging bench which follows the standard bench cycle (SBC) described in this appendix. 2. The SBC requires use of an aging...

Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

PubMed

Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

2014-05-01

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

40 CFR Appendix Vii to Part 86 - Standard Bench Cycle (SBC)

Code of Federal Regulations, 2014 CFR

2014-07-01

... VII to Part 86—Standard Bench Cycle (SBC) 1. The standard bench aging durability procedures [Ref. § 86.1823-08(d)] consist of aging a catalyst-oxygen-sensor system on an aging bench which follows the standard bench cycle (SBC) described in this appendix. 2. The SBC requires use of an aging bench with an...

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

NASA Astrophysics Data System (ADS)

Wu, Riqin; Zhang, Peijun; Li, Jun; Xu, Yongli

2005-03-01

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes ( P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Platelet-Rich Gel Supernatants Stimulate the Release of Anti-Inflammatory Proteins on Culture Media of Normal Equine Synovial Membrane Explants

PubMed Central

Ríos, Diana L.; López, Catalina; Carmona, Jorge U.

2015-01-01

The aims were as follows: (1) to evaluate the effects at 48 and 96 h of two concentrations (25 and 50%) of leukocyte and platelet-rich gel (L-PRG) and pure PRG (P-PRG) supernatants on the production/degradation in normal equine synovial membrane explants (SME) of platelet derived growth factor isoform BB, transforming growth factor beta-1, tumor necrosis factor alpha, interleukin (IL-) 4 (IL-4), IL-1 receptor antagonist (IL-1ra), and hyaluronan (HA) synthesis and (2) to correlate these molecules with their respective PRG supernatant treatments. SME from 6 horses were cultured for 96 h with L-PRG and P-PRG supernatants at 25 and 50% concentrations, respectively. SME culture media were changed each 48 h and used for determination by ELISA of the molecules, which were also determined in synovial fluid. 25% L-PRG supernatant produced a sustained release over time of IL-1ra and a gradual release of HA, whereas 50% L-PRG supernatant produced a sustained increase over time of IL-4 and HA. 50% P-PRG supernatant produced an increased and sustained production of IL-1ra and IL-4. The cellular composition and the articular concentration (volume) of a platelet-rich plasma preparation could affect the anti-inflammatory and anabolic joint responses in horses with osteoarthritis. PMID:26090267

[Effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells and ciprofloxacin on Staphylococcus aureus in vitro].

PubMed

Zhou, B; Tu, H L; Ba, T; Wang, L F; Wang, S J; Nie, S Y

2017-06-20

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. Cells in the third passage were used in the experiments after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the experiments. Cells were divided into 0, 10, 100, and 1 000 ng/mL lipopolysaccharide (LPS) groups according to the random number table (the same dividing method below). Cells were cultured with culture medium of mesenchymal stem cells (MSCs) after being treated with medium containing the corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12, and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay. Ninety blood agar plates were divided into ciprofloxacin control group (CC), ciprofloxacin+ supernatant group (CS), and ciprofloxacin+ supernatant+ LL-37 antibody group (CSL), with 30 blood agar plates in each group. Blood agar plates in group CC were coated with 1.5×10(8) colony forming unit (CFU)/mL bacteria solution prepared with normal saline. Blood agar plates in group CS were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline and culture supernatant of hUCMSCs (cultured by culture medium of MSCs, the same below) in double volume of normal saline. Blood agar plates in group CSL were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline, culture supernatant of hUCMSCs in double volume of normal saline, and 2.6 μL LL-37 antibody in the concentration of 2 μg/mL. At PCH 12, 24, and 48, 10 blood agar plates of each group were harvested to observe the distribution of SA colony on blood agar plate and to measure the diameter of

Overcoming bottlenecks of enzymatic biofuel cell cathodes: crude fungal culture supernatant can help to extend lifetime and reduce cost.

PubMed

Sané, Sabine; Jolivalt, Claude; Mittler, Gerhard; Nielsen, Peter J; Rubenwolf, Stefanie; Zengerle, Roland; Kerzenmacher, Sven

2013-07-01

Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

13 CFR 126.202 - Who does SBA consider to control a HUBZone SBC?

Code of Federal Regulations, 2010 CFR

2010-01-01

... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Who does SBA consider to control a HUBZone SBC? 126.202 Section 126.202 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION HUBZONE PROGRAM Requirements to be a Qualified HUBZone SBC § 126.202 Who does SBA consider to control a HUBZone SBC? Control means both the day-to-day...

Method for simple and rapid concentration of Zika virus particles from infected cell-culture supernatants.

PubMed

Richard, Vaea; Aubry, Maite

2018-05-01

Experimental studies on Zika virus (ZIKV) may require improvement of infectious titers in viral stocks obtained by cell culture amplification. The use of centrifugal filter devices to increase infectious titers of ZIKV from cell-culture supernatants is highlighted here. A mean gain of 2.33 ± 0.12 log 10 DICT 50 /mL was easily and rapidly obtained with this process. This efficient method of ultrafiltration may be applied to other viruses and be useful in various experimental studies requiring high viral titers. Copyright © 2018 Elsevier B.V. All rights reserved.

Hepatic microsomal cytochromes P450 in mink fed Saginaw Bay carp (SBC)

USGS Publications Warehouse

Melancon, M.J.; LeCaptain, L.; Rattner, B.A.; Heaton, S.; Aulerich, R.; Tillitt, D.; Stegeman, John J.; Woodin, B.

1992-01-01

Livers from mink fed diets containing 0% (n = 12), 10% (n = 11), 20% (n = 12) and 40% (n = 10) SBC for 6 months contained 0.1, 2.2, 3.6, and 6.3 ug/g total PCBs, respectively. Hepatic microsomes were prepared and assayed for protein, arylhydrocarbon hydroxylase (AHH), benzyloxyresorufin-O-dealkylase (BROD), ethoxy-ROD (ER0D), pentoxy-ROD (PROD), and ethoxycoumarin-OD (ECOD). Mink fed SBC had increased AHH, EROD, and ECOD (group means 2.2-3.4 X control means), decreased BROD and unchanged PROD (the latter 2 assays indicators for phenobarbital-type induction in mammals). Three samples from each group were examined by western blot using a polyclonal anti-P450llB antibody and a monoclonal anti-P450lA antibody (MAb 1-12-3). Mink fed SBC showed induction of a protein recognized by anti-P450lA (8 X control), but had little protein recognized by anti-P450IlB. The monooxygenase activities and western blot data give a consistent picture of MC-type but not PB-type induction in mink fed SBC.

13 CFR 126.613 - How does a price evaluation preference affect the bid of a qualified HUBZone SBC in full and open...

Code of Federal Regulations, 2010 CFR

2010-01-01

... full and open competition, a qualified HUBZone SBC submits an offer of $98, a non-HUBZone SBC submits..., the non-HUBZone SBC's offer at $100 does not displace the large business' offer because a price... and open competition, a qualified HUBZone SBC submits an offer of $98 and a non-HUBZone SBC submits an...

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.

PubMed

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.

PubMed

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.

Regulation of immunophenotype modulation of monocytes-macrophages from M1 into M2 by prostate cancer cell-culture supernatant via transcription factor STAT3.

PubMed

Solís-Martínez, R; Cancino-Marentes, M; Hernández-Flores, G; Ortiz-Lazareno, P; Mandujano-Álvarez, G; Cruz-Gálvez, C; Sierra-Díaz, E; Rodríguez-Padilla, C; Jave-Suárez, L F; Aguilar-Lemarroy, A; Bravo-Cuellar, A

2018-04-01

Transcription factor STAT3 has a prominent innate immunity effect on cancer progression. We determined the regulation of STAT3 in the immunophenotype modulation of macrophages from M1 into M2 induced by the cell-culture supernatant of the Prostate-Cancer line PC3. Monocytes-macrophages from healthy donors were cultured in the supernatant of PC3 cells, membrane proteins, and intracytoplasmic and phosphorylated STAT3 were measured using flow cytometry, while cytokines and growth factors were studied using luminescence. Cytotoxicity and nitric oxide were evaluated via colorimetric assays. The supernatant of PC3 prostate-tumor cells effectively induced macrophages toward an M2 profile, and the expression of phosphorylated STAT3 in the monocytes-macrophages notably increased, and mainly related to IL-10. In the group of monocytes-macrophages treated with a STAT3 inhibitor, the macrophages were induced toward an M1 phenotype. In this study, we showed that the secretion profile of PC3 prostate-cancer cells induces a change in macrophage phenotype from M1 into M2, and that the phenomenon is related to phosphorylation of transcription factor STAT3 and IL-10. Copyright © 2018. Published by Elsevier B.V.

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

PubMed

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.

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

PubMed

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.

PubMed

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.

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

PubMed

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.

PubMed

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.

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

PubMed

Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

2009-07-01

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

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

PubMed

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.

PubMed

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.

Implementation of secondary bacterial culture testing of platelets to mitigate residual risk of septic transfusion reactions.

PubMed

Bloch, Evan M; Marshall, Christi E; Boyd, Joan S; Shifflett, Lisa; Tobian, Aaron A R; Gehrie, Eric A; Ness, Paul M

2018-04-01

Bacterial contamination of platelets remains a major transfusion-associated risk despite long-standing safety measures in the United States. We evaluated an approach using secondary bacterial culture (SBC) to contend with residual risk of bacterial contamination. Phased implementation of SBC was initiated in October 2016 for platelets (all apheresis collected) received at our institution from the blood donor center (Day 3 post collection). Platelet products were sampled aseptically (5 mL inoculated into an aerobic bottle [BacT/ALERT BPA, BioMerieux, Inc.]) by the blood bank staff upon receipt, using a sterile connection device and sampling kit. The platelet sample was inoculated into an aerobic blood culture bottle and incubated at 35°C for 3 days. The cost of SBC was calculated on the basis of consumables and labor costs at time of implementation. In the 13 months following implementation (October 6, 2016, to November 30, 2017), 23,044/24,653 (93.47%) platelet products underwent SBC. A total of eight positive cultures were detected (incidence 1 in 2881 platelet products), seven of which were positive within 24 hours of SBC. Coagulase negative Staphyloccus spp. were identified in four cases. Five of the eight cases were probable true positive (repeat reactive) and interdicted (cost per averted case was US$77,935). The remaining three cases were indeterminate. No septic transfusion reactions were reported during the observation period. We demonstrate the feasibility of SBC of apheresis platelets to mitigate bacterial risk. SBC is lower cost than alternative measures (e.g., pathogen reduction and point-of-release testing) and can be integrated into workflow at hospital transfusion services. © 2018 AABB.

Comparative evaluation of paired blood culture (aerobic/aerobic) and single blood culture, along with clinical importance in catheter versus peripheral line at a tertiary care hospital.

PubMed

Tarai, B; Das, P; Kumar, D; Budhiraja, S

2012-01-01

Paired blood culture (PBC) is uncommon practice in hospitals in India, leading to delayed and inadequate diagnosis. Also contamination remains a critical determinant in hampering the definitive diagnosis. To establish the need of PBC over single blood culture (SBC) along with the degree of contamination, this comparative retrospective study was initiated. We processed 2553 PBC and 4350 SBC in BacT/ALERT 3D (bioMerieux) between October 2010 and June 2011. The positive cultures were identified in VITEK 2 Compact (bioMerieux). True positivity and contaminants were also analyzed in 486 samples received from catheter and peripheral line. Out of 2553 PBC samples, positivity was seen in 350 (13.70%). In 4350 SBC samples, positivity was seen in 200 samples (4.59%). In PBC true pathogens were 267 (10.45%) and contaminants were 83 (3.25%), whereas in SBC 153 (3.51%) were true positives and contaminants were 47 (1.08%). Most of the blood cultures (99.27 %) grew within 72 h and 95.8% were isolated within 48 h. In 486 PBCs received from catheter/periphery (one each), catheter positivity was found in 85 (true positives were 48, false positives 37). In peripheral samples true positives were 50 and false positives were 8. Significantly higher positive rates were seen in PBCs compared with SBCs. Automated blood culture and identification methods significantly reduced the time required for processing of samples and also facilitated yield of diverse/rare organisms. Blood culture from catheter line had higher false positives than peripheral blood culture. Thus every positive result from a catheter must be correlated with clinical findings and requires further confirmation.

40 CFR Appendix Vii to Part 86 - Standard Bench Cycle (SBC)

Code of Federal Regulations, 2011 CFR

2011-07-01

... procedures [Ref. § 86.1823-08(d)] consist of aging a catalyst-oxygen-sensor system on an aging bench which... bench with an engine as the source of feed gas for the catalyst. 3. The SBC is a 60-second cycle which... occurs in the hottest catalyst. Alternatively, the feed gas temperature may be measured and converted to...

40 CFR Appendix Vii to Part 86 - Standard Bench Cycle (SBC)

Code of Federal Regulations, 2010 CFR

2010-07-01

... procedures [Ref. § 86.1823-08(d)] consist of aging a catalyst-oxygen-sensor system on an aging bench which... bench with an engine as the source of feed gas for the catalyst. 3. The SBC is a 60-second cycle which... occurs in the hottest catalyst. Alternatively, the feed gas temperature may be measured and converted to...

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

PubMed

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.

PubMed

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.

Use of supernatant refractive index and supernatant hemoglobin concentration to assess residual glycerol concentration in cryopreserved red blood cells.

PubMed

Wong, Kenneth A; Nsier, Nada; Acker, Jason P

2009-10-01

Red blood cells (RBCs) cryopreserved in glycerol must be deglycerolized prior to transfusion. The adequacy of glycerol removal is commonly assessed by measurement of the refractive index (RI) of the supernatant fluid. However, the presence of free hemoglobin in the supernatant falsely increases the RI and may lead to discard of units that have an acceptable residual glycerol concentration. We performed an analysis of the diagnostic accuracy of 3 methods for residual glycerol measurement - refractometry, osmometry, and a glycerol assay kit. Residual glycerol measurement using these methods was performed on 12 deglycerolized, citrate-phosphate-dextrose (CPD)/saline-adenine-glucose-mannitol (SAGM) leukoreduced RBCs. A calculation that estimates the glycerol concentration based on the refractive index and supernatant hemoglobin concentration was developed and ensures that units with an elevated RI due to the presence of hemoglobin are not discarded if their residual glycerol concentration was <1.0% (w/v). Osmometry was an accurate method for estimating residual glycerol concentration. Refractometry overestimated the residual glycerol concentration due to the interference from hemoglobin. However, when supernatant hemoglobin values were measured and used in the calculation for glycerol concentration, refractometry accurately estimated the residual glycerol concentration. The residual glycerol concentration of cryopreserved, deglycerolized CPD/SAGM RBCs can be accurately estimated using the supernatant refractive index and an equation that accounts for the supernatant hemoglobin concentration.

Assessment of the effect of a Salmonella enterica ser. Typhimurium culture supernatant on the single-cell lag time of foodborne pathogens.

PubMed

Blana, Vasiliki A; Lianou, Alexandra; Nychas, George-John E

2015-12-23

The objective of this study was the in vitro evaluation of the effect of a cell-free microbial supernatant, produced by a luxS-positive Salmonella enterica ser. Typhimurium strain, on the single-cell growth kinetic behavior of two strains of S. enterica (serotypes Enteritidis and Typhimurium) and a methicillin-resistant Staphylococcus aureus strain. The single-cell lag time (λ) of the pathogens was estimated in the absence and presence (20% v/v) of microbial supernatant based on optical density measurements. As demonstrated by the obtained results, the tested microbial supernatant had a strain-specific effect on the single-cell λ and its variability. Although the mean λ values were similar in the absence and presence of microbial supernatant in the case of Salmonella Enteritidis, a significant (P ≤ 0.05) reduction and increase in the mean value of this parameter in the presence of microbial supernatant were observed for Salmonella Typhimurium and St. aureus, respectively. With regard to the effect of the tested microbial supernatant on the single-cell variability of λ, similar λ distributions were obtained in its absence and presence for S. Enteritidis, while considerable differences were noted for the other two tested organisms; the coefficient of variation of λ in the absence and presence of microbial supernatant was 41.6 and 69.8% for S. Typhimurium, respectively, with the corresponding values for St. aureus being 74.0 and 56.9%. As demonstrated by the results of bioassays, the tested microbial supernatant exhibited autoinducer-2 activity, indicating a potential association of such quorum sensing compounds with the observed effects. Although preliminary in nature, the collected data provide a good basis for future research on the role of quorum sensing in the single-cell growth behavior of foodborne pathogens.

Rapid Extracellular Biosynthesis of Silver Nanoparticles by Cunninghamella phaeospora Culture Supernatant

PubMed Central

Ghareib, Mohamed; Tahon, Medhat Abu; Saif, Mona Mostafa; El-Sayed Abdallah, Wafaa

2016-01-01

The development of green approaches for the biosynthesis of silver nanoparticles (AgNPs) is of prime significance in the field of nanotechnology research. A fast and eco-friendly protocol for the biosynthesis of extracellular AgNPs using culture supernatant (CS) from the fungus Cunninghamella phaeospora was studied in this work. This CS was proved as a potential new source for the extracellular biosynthesis of AgNPs. The AgNPs were formed at 100 oC and pH 9 within four min of contact between CS and 1mM silver nitrate (AgNO3) solution. Nitrate reductase (NR) was confirmed to play a pivotal role in the biosynthesis of AgNPs. The enzyme expressed its highest activity at 80 oC and pH 9. At 100 oC the enzyme retained 70% of its original activity for one hour. The half-life (T1/2) of the enzyme activity was calculated to be 1.55 h confirming its thermostability. The produced AgNPs were characterized by UV-Vis spectroscopy, high resolution-transmission electron microscope (HR-TEM) and x-ray diffraction (XRD). These NPs showed an absorption peak at 415 nm in UV-Vis spectrum corresponding to the plasmon resonance of AgNPs. Transmission electron micrographs revealed the production of monodispersed spherical NPs with average particle size 14 nm. XRD spectrum of the NPs confirmed the formation of metallic crystalline silver. It was also suggested that the aromatic amino acids play a role in the biosynthesis process. The current research provided an insight on the green biosynthesis of AgNPs including some mechanistic aspects using a new mycogenic source. PMID:28243290

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

Evaluation of entomopathogenic nematodes and the supernatants of the in vitro culture medium of their mutualistic bacteria for the control of the root-knot nematodes Meloidogyne incognita and M. arenaria.

PubMed

Kepenekci, Ilker; Hazir, Selcuk; Lewis, Edwin E

2016-02-01

The suppressive effects of various formulations of four entomopathogenic nematode (EPN) species and the supernatants of their mutualistic bacteria on the root-knot nematodes (RKNs) Meloidogyne incognita and M. arenaria in tomato roots were evaluated. The EPNs Steinernema carpocapsae, S. feltiae, S. glaseri and Heterorhabditis bacteriophora were applied as either live infective juveniles (IJs) or infected insect cadavers. Spent medium from culturing the bacterial symbionts Xenorhabdus bovienii and Photorhabdus luminescens kayaii with the cells removed was also applied without their nematode partners. The aqueous suspensions of IJs, infected cadaver applications of EPNs and especially treatments of X. bovienii supernatant suppressed the negative impact of RKNs on tomatoes. Specific responses to treatment were reduced RKN egg masses, increased plant height and increased fresh and dry weights compared with the control where only RKNs were applied. Among the treatments tested, the plant-dipping method of X. bovienii into bacterial culture fluid may be the most practical and effective method for M. incognita and M. arenaria control. © 2015 Society of Chemical Industry.

13 CFR 125.30 - What are the requirements for representing SDVO SBC status, and what are the penalties for...

Code of Federal Regulations, 2014 CFR

2014-01-01

... Credit and Assistance SMALL BUSINESS ADMINISTRATION GOVERNMENT CONTRACTING PROGRAMS Penalties and... Management (SAM) database (or any successor thereto), a concern must certify its SDVO SBC status in...

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

PubMed

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.

PubMed

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

PubMed Central

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

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

PubMed Central

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

2017-01-01

Background 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. Methods and findings 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. Conclusions 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. PMID:28828411

13 CFR 126.308 - What happens if SBA inadvertently omits a qualified HUBZone SBC from the List?

Code of Federal Regulations, 2010 CFR

2010-01-01

... writing at U.S. Small Business Administration, 409 Third Street, SW., Washington, DC 20416 or via e-mail... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false What happens if SBA inadvertently omits a qualified HUBZone SBC from the List? 126.308 Section 126.308 Business Credit and Assistance...

13 CFR 125.29 - What are the requirements for representing SDVO SBC status, and what are the penalties for...

Code of Federal Regulations, 2014 CFR

2014-01-01

... include the firm's internal management procedures governing SDVO SBC status representations or certifications, the clarity or ambiguity of the representation or certification requirement, and the efforts made to correct an incorrect or invalid representation or certification in a timely manner. An individual...

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R

PubMed Central

Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

2016-01-01

Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2−) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R. PMID:26927174

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

PubMed

Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

2016-02-25

Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.

Innovation in Vertical Axis Hydrokinetic Turbine – Straight Blade Cascaded (VAHT-SBC) design and testing for low current speed power generation

NASA Astrophysics Data System (ADS)

Hantoro, R.; Utama, I. K. A. P.; Arief, I. S.; Ismail, A.; Manggala, S. W.

2018-05-01

This study examines an innovative turbine with the addition of the number and arrangement of straight blade cascaded (SBC). SBC is a combination of passive variable-pitch and fixed pitch of each turbine arm. This study was conducted in an open channel flow that has a current velocity (V-m/s) of 1.1, 1.2, and 1.3. RPM and torque ware measured for coefficient of performance (Cp) and tip speed ratio (TSR) calculation. Without changing the turbine dimension, the employment of cascaded blade (three blades in each arm) contributes to improve energy extraction significantly. A significant increase in Cp value is seen when 9 blades (3 cascaded blades per arm) are used with a Cp 0.42 value at TSR 2.19. This value has reached 93% of the maximum theoritical Cp value.

13 CFR 125.15 - What requirements must an SDVO SBC meet to submit an offer on a contract?

Code of Federal Regulations, 2010 CFR

2010-01-01

... corresponding to the NAICS code assigned to the contract, provided: (A) For a procurement having a revenue-based... to the contract; or (B) For a procurement having an employee-based size standard, the procurement... specific contract: (1) It is an SDVO SBC; (2) It is small under the NAICS code assigned to the procurement...

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

PubMed

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.

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

PubMed Central

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

2017-01-01

ABSTRACT 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 × 1010/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose. PMID:28326167

Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide

PubMed Central

Ríos, Diana L.; Pérez, Jorge E.

2017-01-01

Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane. PMID:28761774

Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide.

PubMed

Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E

2017-01-01

Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NF κ B), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NF κ B, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NF κ B, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

Lactobacillus paracasei CNCM I-4034 and its culture supernatant modulate Salmonella-induced inflammation in a novel transwell co-culture of human intestinal-like dendritic and Caco-2 cells.

PubMed

Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gómez-Llorente, Carolina; Matencio, Esther; Romero, Fernando; Gil, Angel

2015-04-01

The action of probiotics has been studied in vitro in cells isolated from both mice and humans, particularly enterocytes (IECs), dendritic cells (DCs) and co-cultures of peripheral DCs and IECs. Peripheral DCs and murine DCs differ from human gut DCs, and to date there are no data on the action of any probiotic on co-cultured human IECs and human intestinal DCs. To address this issue, a novel transwell model was used. Human IECs (Caco-2 cells) grown in the upper chamber of transwell filters were co-cultured with intestinal-like human DCs grown in the basolateral compartment of the transwells. The system was apically exposed for 4 h to live probiotic L. paracasei CNCM I-4034 obtained from the faeces of breastfed infants or to its cell-free culture supernatant (CFS) and challenged with Salmonella typhi. The secretion of pro- and anti-inflammatory cytokines in the basolateral compartment was determined by immunoassay, and the DC expression pattern of 20 TLR signaling pathway genes was analysed by PCR array. The presence of the live probiotic alone significantly increased IL-1β, IL-6, IL-8, TGF-β2, RANTES and IP-10 levels and decreased IL-12p40, IL-10, TGF- β1 and MIP-1α levels. This release was correlated with a significant increase in the expression of almost all TLR signaling genes. By contrast, incubation of the co-culture with CFS increased IL-1β, IL-6, TGF-β2 and IP-10 production only when Salmonella was present. This induction was correlated with an overall decrease in the expression of all TLR genes except TLR9, which was strongly up-regulated. The data presented here clearly indicate that L. paracasei CNCM I-4034 significantly increases the release of pro-inflammatory cytokines, enhances TLR signaling pathway activation and stimulates rather than suppresses the innate immune system. Furthermore, our findings provide evidence that the effects of probiotics in the presence of IECs and DCs differ from the effects of probiotics on cultures of each cell type

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women.

PubMed

Aranha, C; Natraj, U; Iyer, K S; Shahani, S

1998-03-01

Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

Kinetics of fluoride bioavailability in supernatant saliva and salivary sediment.

PubMed

Naumova, E A; Sandulescu, T; Bochnig, C; Gaengler, P; Zimmer, S; Arnold, W H

2012-07-01

The assessment of the fluoride kinetics in whole saliva as well as in the different salivary phases (supernatant saliva and sediment) is essential for the understanding of fluoride bioavailability. To assess the fluoride content, provided by sodium fluoride and amine fluoride, in the supernatant saliva and in salivary sediment. Seven trained volunteers were randomly attributed to 2 groups in a cross-over design and brushed their teeth in the morning for 3 min with a product containing either sodium fluoride or amine fluoride. Saliva was collected before, immediately after tooth brushing and 30, 120, and 360 min later and measured. The samples were centrifuged 10 min at 3024 × g. Fluoride content of the supernatant saliva and of the sediment was analysed using a fluoride sensitive electrode. All subjects repeated the study cycles 2 times, and statistical analyses were made using the nonparametric sign test for related samples, the Wilcoxon-Mann-Whitney-test for independent samples. There was a significant increase in fluoride immediately after tooth brushing in both groups in saliva and sediment. The distribution of fluoride between salivary sediment and supernatant saliva (ratio) varied considerably at the different collection times: decreased from 17.87 in baseline samples of saliva to 0.07 immediately and to 0.86 half an hour after tooth brushing in the sodium fluoride group and from 14.33 to 2.85 and to 3.09 in the amine fluoride group. Furthermore after 120 min and after 360 min after tooth brushing the ratio increased from 17.6 to 31.6 in the sodium fluoride group and from 20.5 to 25.76 in the amine fluoride group. No difference was found in the sediment-supernatant saliva ratio between the sodium fluoride and the amine fluoride groups 360 min after tooth brushing. For the assessment of fluoride kinetics in whole saliva it is necessary to pay attention to at least four factors: fluoride formulation, time after fluoride application, fluoride concentration in

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

TANK 26F SUPERNATANT AND 2F EVAPORATOR EDUCTOR PUMP SAMPLE CHARACTERIZATION RESULTS

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, W.; Hay, M.; Coleman, C.

2011-08-23

In an effort to understand the reasons for system plugging problems in the SRS 2F evaporator, supernatant samples were retrieved from the evaporator feed tank (Tank 26F) and solids were collected from the evaporator eductor feed pump for characterization. The variable depth supernatant samples were retrieved from Tank 26F in early December of 2010 and samples were provided to SRNL and the F/H Area laboratories for analysis. Inspection and analysis of the samples at SRNL was initiated in early March of 2011. During the interim period, samples were frequently exposed to temperatures as low as 12 C with daily temperaturemore » fluctuations as high as 10 C. The temperature at the time of sample collection from the waste tank was 51 C. Upon opening the supernatant bottles at SRNL, many brown solids were observed in both of the Tank 26F supernatant samples. In contrast, no solids were observed in the supernatant samples sent to the F/H Area laboratories, where the analysis was completed within a few days after receipt. Based on these results, it is believed that the original Tank 26F supernatant samples did not contain solids, but solids formed during the interim period while samples were stored at ambient temperature in the SRNL shielded cells without direct climate control. Many insoluble solids (>11 wt. % for one sample) were observed in the Tank 26F supernatant samples after three months of storage at SRNL which would not dissolve in the supernatant solution in two days at 51 C. Characterization of these solids along with the eductor pump solids revealed the presence of sodium oxalate and clarkeite (uranyl oxyhydroxide) as major crystalline phases. Sodium nitrate was the dominant crystalline phase present in the unwashed Eductor Pump solids. Crystalline sodium nitrate may have formed during the drying of the solids after filtration or may have been formed in the Tank 26F supernatant during storage since the solution was found to be very concentrated (9-12 M Na

13 CFR 126.618 - How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation...

Code of Federal Regulations, 2014 CFR

2014-01-01

... HUBZone SBC qualifies as small under part 121 of this chapter, SBA will not find affiliation between the... set forth in § 125.6(b) of this chapter. (ii) SBA may find affiliation between a prime HUBZone contractor and its mentor subcontractor where the mentor will perform primary and vital requirements of the...

13 CFR 126.618 - How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation...

Code of Federal Regulations, 2012 CFR

2012-01-01

... HUBZone SBC qualifies as small under part 121 of this chapter, SBA will not find affiliation between the... set forth in § 125.6(b) of this chapter. (ii) SBA may find affiliation between a prime HUBZone contractor and its mentor subcontractor where the mentor will perform primary and vital requirements of the...

13 CFR 126.618 - How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation...

Code of Federal Regulations, 2013 CFR

2013-01-01

... HUBZone SBC qualifies as small under part 121 of this chapter, SBA will not find affiliation between the... set forth in § 125.6(b) of this chapter. (ii) SBA may find affiliation between a prime HUBZone contractor and its mentor subcontractor where the mentor will perform primary and vital requirements of the...

13 CFR 126.618 - How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation...

Code of Federal Regulations, 2011 CFR

2011-01-01

... HUBZone SBC qualifies as small under part 121 of this chapter, SBA will not find affiliation between the... set forth in § 125.6(b) of this chapter. (ii) SBA may find affiliation between a prime HUBZone contractor and its mentor subcontractor where the mentor will perform primary and vital requirements of the...

Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...

13 CFR 126.618 - How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation...

Code of Federal Regulations, 2010 CFR

2010-01-01

... participation in a Mentor-Protégé relationship affect its participation in the HUBZone Program? 126.618... Assistance § 126.618 How does a HUBZone SBC's participation in a Mentor-Protégé relationship affect its participation in the HUBZone Program? (a) Qualified HUBZone SBCs may enter into Mentor-Protégé relationships in...

Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers

PubMed Central

Lau, Susanna KP; Lam, Ching-Wan; Curreem, Shirly OT; Lee, Kim-Chung; Lau, Candy CY; Chow, Wang-Ngai; Ngan, Antonio HY; To, Kelvin KW; Chan, Jasper FW; Hung, Ivan FN; Yam, Wing-Cheong; Yuen, Kwok-Yung; Woo, Patrick CY

2015-01-01

Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography–electrospray ionization–quadruple time of flight–mass spectrometry (UHPLC–ESI–Q–TOF–MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette–Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16∶0/0∶0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic

Analysis of Cervical Supernatant Samples Luminescence Using 355 nm Laser

NASA Astrophysics Data System (ADS)

Vaitkuviene, A.; Gegzna, V.; Kurtinaitiene, R.; Stanikunas, R.; Rimiene, J.; Vaitkus, J.

2010-05-01

The biomarker discovery for accurate detection and diagnosis of cervical carcinoma and its malignant precursors represents one of the current challenges in clinical medicine. Laser induced autofluorescence spectra in cervical smear content were fitted to predict the cervical epithelium diagnosis as a lab off "optical biopsy" method. Liquid PAP supernatant sediment dried on Quartz plate spectroscopy was performed by 355 nm Nd YAG microlaser STA-1 (Standa, Ltd). For comparison a liquid supernatant spectroscopy was formed by laboratory "Perkin Elmer LS 50B spetrometer at 290, 300, 310 nm excitations. Analysis of spectrum was performed by approximation using the multi-peaks program with Lorentz functions for the liquid samples and with Gaussian functions for the dry samples. Ratio of spectral components area to the area under whole experimental curve (SPP) was calculated. The spectral components were compared by averages of SPP using Mann-Whitney U-test in histology groups. Results. Differentiation of Normal and HSIL/CIN2+ cases in whole supernatant could be performed by stationary laboratory lamp spectroscopy at excitation 290 nm and emission >379 nm with accuracy AUC 0,69, Sens 0,72, Spec 0,65. Differentiation Normal versus HSIL/CIN2+ groups in dried enriched supernatant could be performed by 355 nm microlaser excitation at emission 405-424 nm with accuracy (AUC 0,96, Sens 0,91, Spec 1.00). Diagnostic algorithm could be created for all histology groups differentiation under 355 nm excitation. Microlaser induced "optical biopsy "looks promising method for cervical screening at the point of care.

Calcium phosphates recovery from digester supernatant by fast precipitation and recrystallization

NASA Astrophysics Data System (ADS)

Vasenko, Liubov; Qu, Haiyan

2018-01-01

Conditional solubility of dicalcium phosphate dihydrate (DCPD) and hydroxyapatite (HAp) in digester supernatant was determined as a function of pH and was compared to its conditional solubility in distilled water. Conditional solubility of both substances in digester supernatant at pH >5-6 was higher than their conditional solubility in pure water due to the presence of impurities, and this influence is more significant for HAp. Amorphous CaP was precipitated through a fast precipitation process from digester supernatant with initial total phosphate concentration 0.008 mol/L and 0.015 mol/L and Ca/P ratios 2 and 5. The amorphous CaP can be subsequently recrystallized into crystalline CaP. Obtained amorphous products have Ca/P ratio > 1, which allow performing the recrystallization process without further Ca dosing into the system. Batch recrystallization of the amorphous products resulted in crystallization of HAp, DCPD or their mixture depending on the conditions of the process. Maximum achieved P-recovery was 69.5%. The increase of phosphate concentration and the addition of seeding decreased the yield of the process but promoted crystallization of DCPD. The increase of Ca/P ratio had a positive effect on the total P-recovery. Compared with the direct batch crystallization of CaP from digester supernatant, the two-step process with fast precipitation and recrystallization significantly improved the color of the obtained products.

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

NASA Astrophysics Data System (ADS)

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.

13 CFR 126.613 - How does a price evaluation preference affect the bid of a qualified HUBZone SBC in full and open...

Code of Federal Regulations, 2011 CFR

2011-01-01

... evaluation preference is not applied to change an offer and benefit a non-HUBZone SBC. Example 3: In a full... be awarded that is not greater than 25% of the total volume being procured for each commodity in a... than 25%, but not greater than 40%, of the total volume being procured for each commodity in a single...

Human platelet gel supernatant inactivates opportunistic wound pathogens on skin.

PubMed

Edelblute, Chelsea M; Donate, Amy L; Hargrave, Barbara Y; Heller, Loree C

2015-01-01

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants.

PubMed

Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

2017-01-16

To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (r s : 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (r s : 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.76), and PDGF-BB concentrations with activating substances (r s : 0.72). Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.

Synthesis of angiotensins by cultured granuloma macrophages in murine schistosomiasis mansoni

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weinstock, J.V.; Blum, A.M.

1986-03-01

Components of the angiotensin system are present in granulomas of murine schistosomiasis mansoni. Angiotensins may have immunoregulatory function. Granuloma macrophages cultured for up to 3 days generated substantial angiotensin I (AI) and angiotensin II (AII) which appeared in the culture supernatants. Macrophage monolayers were incubated with (/sup 3/H) amino acids, and culture supernatants were extracted with acetone and analyzed by HPLC. Radiolabeled products eluded at times corresponding to those of authentic angiotensins. Immunoadsorption of angiotensins with angiotensin antisera removed reputed radiolabeled angiotensins from the supernatants. Treatment of the elution fraction corresponding to that of authentic AI with angiotensin converting enzymemore » resulted in the generation of radiolabeled polypeptides which co-eluted with authentic AII and His-Leu. Similar experiments conducted with nonadherent granuloma cells devoid of macrophages failed to demonstrate angiotensin production. These results suggest that granuloma macrophages can synthesize angiotensin.« less

Global Profiling of Metabolite and Lipid Soluble Microbial Products in Anaerobic Wastewater Reactor Supernatant Using UPLC-MSE.

PubMed

Tipthara, Phornpimon; Kunacheva, Chinagarn; Soh, Yan Ni Annie; Wong, Stephen C C; Pin, Ng Sean; Stuckey, David C; Boehm, Bernhard O

2017-02-03

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

High rate manure supernatant digestion.

PubMed

Bergland, Wenche Hennie; Dinamarca, Carlos; Toradzadegan, Mehrdad; Nordgård, Anna Synnøve Røstad; Bakke, Ingrid; Bakke, Rune

2015-06-01

The study shows that high rate anaerobic digestion may be an efficient way to obtain sustainable energy recovery from slurries such as pig manure. High process capacity and robustness to 5% daily load increases are observed in the 370 mL sludge bed AD reactors investigated. The supernatant from partly settled, stored pig manure was fed at rates giving hydraulic retention times, HRT, gradually decreased from 42 to 1.7 h imposing a maximum organic load of 400 g COD L(-1) reactor d(-1). The reactors reached a biogas production rate of 97 g COD L(-1) reactor d(-1) at the highest load at which process stress signs were apparent. The yield was ∼0.47 g COD methane g(-1) CODT feed at HRT above 17 h, gradually decreasing to 0.24 at the lowest HRT (0.166 NL CH4 g(-1) CODT feed decreasing to 0.086). Reactor pH was innately stable at 8.0 ± 0.1 at all HRTs with alkalinity between 9 and 11 g L(-1). The first stress symptom occurred as reduced methane yield when HRT dropped below 17 h. When HRT dropped below 4 h the propionate removal stopped. The yield from acetate removal was constant at 0.17 g COD acetate removed per g CODT substrate. This robust methanogenesis implies that pig manure supernatant, and probably other similar slurries, can be digested for methane production in compact and effective sludge bed reactors. Denaturing gradient gel electrophoresis (DGGE) analysis indicated a relatively fast adaptation of the microbial communities to manure and implies that non-adapted granular sludge can be used to start such sludge bed bioreactors. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

PubMed

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.

Evaluation of Hanford Tank Supernatant Availability for Technetium Management Project Studies in FY16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rapko, Brian M.

2015-09-30

This report examines the need for actual Hanford tank waste solutions to support tasks in the Technetium Management Program in fiscal year (FY) 2016. One key need is to identify both samples where a majority of the soluble technetium is present as pertechnetate and samples where it is not. The total amount of tank supernatant needed from any given tank waste supernatant was determined by polling the tasks leaders for their technology testing needs in FY16 and then arbitrarily ascribing a 10% process loss associated with consolidation and the Cs-137 removal needed to reduce the dose to a level suitablemore » for testing in radiological fumehoods. These polling results identified a need for approximately 2.1 to 3.6 kg of any particular targeted Hanford tank waste supernatant.« less

NMR methods for metabolomics of mammalian cell culture bioreactors.

PubMed

Aranibar, Nelly; Reily, Michael D

2014-01-01

Metabolomics has become an important tool for measuring pools of small molecules in mammalian cell cultures expressing therapeutic proteins. NMR spectroscopy has played an important role, largely because it requires minimal sample preparation, does not require chromatographic separation, and is quantitative. The concentrations of large numbers of small molecules in the extracellular media or within the cells themselves can be measured directly on the culture supernatant and on the supernatant of the lysed cells, respectively, and correlated with endpoints such as titer, cell viability, or glycosylation patterns. The observed changes can be used to generate hypotheses by which these parameters can be optimized. This chapter focuses on the sample preparation, data acquisition, and analysis to get the most out of NMR metabolomics data from CHO cell cultures but could easily be extended to other in vitro culture systems.

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

PubMed

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.

The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions.

PubMed

Deftereos, Georgios; Finkelstein, Sydney D; Jackson, Sara A; Ellsworth, Eric M G; Krishnamurti, Uma; Liu, Yulin; Silverman, Jan F; Binkert, Candy R; Ujevich, Beth A; Mohanty, Alok

2014-04-01

Fine-needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. The aim of this study was to evaluate how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Twenty-three FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the microdissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in microdissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicates that supernatant fluid may be enriched with tumor DNA. Molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability and increase diagnostic yield

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

PubMed

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; P<0.01). The supernatant of oral 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.

Pseudomonas oleovorans Strain KBPF-004 Culture Supernatants Reduced Seed Transmission of Cucumber green mottle mosaic virus and Pepper mild mottle virus, and Remodeled Aggregation of 126 kDa and Subcellular Localization of Movement Protein of Pepper mild mottle virus

PubMed Central

Kim, Nam-Gyu; Seo, Eun-Young; Han, Sang-Hyuk; Gong, Jun-Su; Park, Cheol-Nam; Park, Ho-Seop; Domier, Leslie L; Hammond, John; Lim, Hyoun-Sub

2017-01-01

Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts of a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against plant viruses are poorly documented. Here we report isolation of a soil inhabiting bacterium, Pseudomonas oleovorans strain KBPF-004 (developmental code KNF2016) which showed antiviral activity against mechanical transmission of tobamoviruses. Antiviral activity was also evaluated in seed transmission of two tobamoviruses, Pepper mild mottle virus (PMMoV) and Cucumber green mottle mosaic virus (CGMMV), by treatment of seed collected from infected pepper and watermelon, respectively. Pepper and watermelon seeds were treated with culture supernatant of P. oleovorans strain KBPF-004 or control strain ATCC 8062 before planting. Seeds germinated after treatment with water or ATCC 8062 yielded about 60% CGMMV or PMMoV positive plants, whereas < 20% of KBPF-004-treated seeds were virus-infected, a significantly reduced seed transmission rate. Furthermore, supernatant of P. oleovorans strain KBPF-004 remodeled aggregation of PMMoV 126 kDa protein and subcellular localization of movement protein in Nicotiana benthamiana, diminishing aggregation of the 126 kDa protein and essentially abolishing association of the movement protein with the microtubule network. In leaves agroinfiltrated with constructs expressing the coat protein (CP) of either PMMoV or CGMMV, less full-size CP was detected in the presence of supernatant of P. oleovorans strain KBPF-004. These changes may contribute to the antiviral effects of P. oleovorans strain KBPF-004. PMID:28811756

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

PubMed

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.

Enhancing the culturability of bacteria from the gastrointestinal tract of farmed adult turbot Scophthalmus maximus

NASA Astrophysics Data System (ADS)

Xing, Mengxin; Hou, Zhanhui; Qu, Yanmei; Liu, Bin

2014-03-01

Eighteen agar media were tested for the culture of gut-associated bacteria from farmed adult turbot ( Scophthalmus maximus), including 16 agar media with or without 1% gastrointestinal (GI) supernatant, or with 2% or 4% GI supernatant. A total of 1 711 colonies were analyzed and 24 operational taxonomic units (OTUs) were identified. The greatest bacterial diversity was isolated on Zobell 2216E/Zobell 2216E+ agar media, whereas MRS/MRS+ agar media produced a low diversity of colonies. Agar media with GI supernatant (1%, 2%, or 4%) showed increased diversity and yielded different profiles of OTUs from the corresponding original media, suggesting that GI supernatant provides substances that enhance the culture efficiency of bacteria from the turbot GI tract. The large majority of the colonies (82%) were γ-Proteobacteria, whereas 15.6% and 2.4% of colonies were Firmicutes and Actinobacteria, respectively. At the genus level, 49.4% of all colonies were assigned to Vibrio. Other potential pathogens, including Pseudomonas, Photobacterium, and Enterobacter, and potential probiotics, including Bacillus, Paenibacillus, and Pseudomonas, were also isolated on agar media. Most cultured bacteria belonged to species that were first described in the turbot GI tract. The impact of these species on turbot physiology and health should be investigated further.

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

PubMed

Matsuda, Yoshiko; Imamura, Ryoichi; Takahara, Shiro

2017-01-01

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 × 10 5  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 × 10 5  cells/well), and the resulting in vitro analysis provided

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

PubMed Central

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

2018-01-01

Background & aims 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). Method 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. Results 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. Conclusion 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

Effective Trapping of Fruit Flies with Cultures of Metabolically Modified Acetic Acid Bacteria

PubMed Central

Ishii, Yuri; Akasaka, Naoki; Goda, Itsuko; Sakoda, Hisao

2015-01-01

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant. PMID:25595769

Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

USDA-ARS?s Scientific Manuscript database

Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

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

NASA Technical Reports Server (NTRS)

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

Yolk-shell structured Sb@C anodes for high energy Na-ion batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Junhua; Yan, Pengfei; Luo, Langli

Despite great advances in sodium-ion battery developments, the search for high energy and stable anode materials remains a challenge. Alloy or conversion-typed anode materials are attractive candidates of high specific capacity and low voltage potential, yet their applications are hampered by the large volume expansion and hence poor electrochemical reversibility and fast capacity fade. Here, we use antimony (Sb) as an example to demonstrate the use of yolk-shell structured anodes for high energy Na-ion batteries. The Sb@C yolk-shell structure prepared by controlled reduction and selective removal of Sb2O3 from carbon coated Sb2O3 nanoparticles can accommodate the Sb swelling upon sodiationmore » and improve the structural/electrical integrity against pulverization. It delivers a high specific capacity of ~554 mAh•g-1, good rate capability (315 mhA•g-1 at 10C rate) and long cyclability (92% capacity retention over 200 cycles). Full-cells of O3-Na0.9[Cu0.22Fe0.30Mn0.48]O2 cathodes and Sb@C-hard carbon composite anodes demonstrate a high specific energy of ~130 Wh•kg-1 (based on the total mass of cathode and anode) in the voltage range of 2.0-4.0 V, ~1.5 times energy of full-cells with similar design using hard carbon anodes.« less

PARTITIONING, DESORPTION, AND DECHLORINATION OF A PCB CONGENER IN SEDIMENT SLURRY SUPERNATANTS

EPA Science Inventory

Partitioning and desorption played specific roles in the dechlorination of 2-chlorobiphenyl (2-ClBP) in sediment slurry supernatants, which are suspensions of disssolved organic matter(DOM). In short-term experiments, the partition coefficient (K_p) was related to the a...

A novel three-dimensional bone chip organ culture.

PubMed

Kuttenberger, Johannes; Polska, Elzbieta; Schaefer, Birgit M

2013-07-01

The objective of this study was to develop a 3D bone chip organ culture model. We aimed to collect in vitro evidence of the ability of vital bone chips to promote new bone formation. We developed a 3D in vitro hypoxic bone chip organ culture model. Histology of the bone chips was performed before and after culture and immunohistochemistry after 3-week culture. The 3D culture supernatants were tested for the presence of pro-angiogenic growth factors, TGFβ1, GADPH, bone alkaline phosphatase, osteocalcin, osteonectin, osteopontin, bone sialoprotein and collagen type I. Histology after culture revealed bone chips in a matrix of fibrin remnants and a fibrous-appearing matter. Collagen type I- and IV-positive structures were also identified. Cells could be seen on the surface of the bone chips, with spindle-shaped cells bridging the bone chip particles. Pro-angiogenic growth factors and TGFβ1were detected in the 3D cell culture supernatants. The transcripts for osteocalcin, bone sialoprotein and collagen type I were revealed only via PCR. Our results indicate that bone chips in our 3D organ culture remain vital and may stimulate the growth of a bone-forming matrix. The use of autogenous bone chips for oral and maxillofacial bone augmentation procedures is widespread in clinical practice. The rationale for this is that if bone chips remain vital in vivo, they could provide an environment promoting new bone formation through growth factors and cells. This 3D culture method is an essential tool for investigating the behaviour of bone chips.

Yolk-shell structured Sb@C anodes for high energy Na-ion batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Junhua; Yan, Pengfei; Luo, Langli

Despite great advances in sodium-ion battery developments, the search for high energy and stable anode materials remains a challenge. Alloy or conversion-typed anode materials are attractive candidates of high specific capacity and low voltage potential, yet their applications are hampered by the large volume expansion and hence poor electrochemical reversibility and fast capacity fade. Here in this paper, we use antimony (Sb) as an example to demonstrate the use of yolk-shell structured anodes for high energy Na-ion batteries. The Sb@C yolk-shell structure prepared by controlled reduction and selective removal of Sb 2O 3 from carbon coated Sb 2O 3 nanoparticlesmore » can accommodate the Sb swelling upon sodiation and improve the structural/electrical integrity against pulverization. It delivers a high specific capacity of ~ 554 mAh g -1, good rate capability (315 mhA g-1 at 10 C rate) and long cyclability (92% capacity retention over 200 cycles). Full-cells of O3-Na 0.9[Cu0.22Fe 0.30Mn 0.48]O 2 cathodes and Sb@C-hard carbon composite anodes demonstrate a high specific energy of ~ 130 Wh kg-1 (based on the total mass of cathode and anode) in the voltage range of 2.0–4.0 V, ~ 1.5 times energy of full-cells with similar design using hard carbon anodes.« less

Yolk-shell structured Sb@C anodes for high energy Na-ion batteries

DOE PAGES

Song, Junhua; Yan, Pengfei; Luo, Langli; ...

2017-09-04

Despite great advances in sodium-ion battery developments, the search for high energy and stable anode materials remains a challenge. Alloy or conversion-typed anode materials are attractive candidates of high specific capacity and low voltage potential, yet their applications are hampered by the large volume expansion and hence poor electrochemical reversibility and fast capacity fade. Here in this paper, we use antimony (Sb) as an example to demonstrate the use of yolk-shell structured anodes for high energy Na-ion batteries. The Sb@C yolk-shell structure prepared by controlled reduction and selective removal of Sb 2O 3 from carbon coated Sb 2O 3 nanoparticlesmore » can accommodate the Sb swelling upon sodiation and improve the structural/electrical integrity against pulverization. It delivers a high specific capacity of ~ 554 mAh g -1, good rate capability (315 mhA g-1 at 10 C rate) and long cyclability (92% capacity retention over 200 cycles). Full-cells of O3-Na 0.9[Cu0.22Fe 0.30Mn 0.48]O 2 cathodes and Sb@C-hard carbon composite anodes demonstrate a high specific energy of ~ 130 Wh kg-1 (based on the total mass of cathode and anode) in the voltage range of 2.0–4.0 V, ~ 1.5 times energy of full-cells with similar design using hard carbon anodes.« less

Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures.

PubMed

Costa, Michael Rodrigues; Fischer, Nicole; Gulich, Barbara; Tönjes, Ralf R

2014-01-01

Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. Porcine endogenous retroviruses-A/C in

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

PubMed Central

Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

2017-01-01

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

Evaluation of the Antioxidative, Antibacterial, and Anti-Inflammatory Effects of the Aloe Fermentation Supernatant Containing Lactobacillus plantarum HM218749.1

PubMed Central

Deng, Kan; Jiang, Chunling; Fu, Mingui; Guo, Chunlan; Wang, Xiaolei; Wang, Xin; Meng, Fanjing; Yang, Shaoguo; Deng, Keyu

2016-01-01

Little work is done to develop Aloe vera (AV) using probiotics. To explore the potential benefits, the antioxidant effects and the antibacterial effects on foodborne pathogens of Aloe fermentation supernatant were evaluated in vitro. Our results indicated that the Aloe fermentation supernatant fermented by Lactobacillus plantarum HM218749.1 had very strong scavenging capacities of the DPPH (86%), O2 •− (85%), •OH (76%), and Fe2+ chelation (82%) and reducing powers (242.5 mg/L), and the inhibition zones for Salmonella typhimurium, Salmonella enteritidis, Shigella flexneri, Escherichia coli, Listeria monocytogenes, S. dysenteriae 301, Staphylococcus aureus Cowan1, and Propionibacterium acnes were 16, 15, 19, 20, 21, 20, and 27 mm. Moreover, the low concentration of Aloe fermentation supernatant had significantly reduced the production of IL-1β, TNF-α, and IL-6 in both mRNA and protein levels (P < 0.01). Therefore, the Aloe fermentation supernatant can be used as functional beverage or cosmetic ingredients to guard human intestinal health, delaying senescence, and prevent chronic diseases. PMID:27493450

Evaluation of the Antioxidative, Antibacterial, and Anti-Inflammatory Effects of the Aloe Fermentation Supernatant Containing Lactobacillus plantarum HM218749.1.

PubMed

Jiang, Meixiu; Deng, Kan; Jiang, Chunling; Fu, Mingui; Guo, Chunlan; Wang, Xiaolei; Wang, Xin; Meng, Fanjing; Yang, Shaoguo; Deng, Keyu; Chen, Tingtao; Xin, Hongbo

2016-01-01

Little work is done to develop Aloe vera (AV) using probiotics. To explore the potential benefits, the antioxidant effects and the antibacterial effects on foodborne pathogens of Aloe fermentation supernatant were evaluated in vitro. Our results indicated that the Aloe fermentation supernatant fermented by Lactobacillus plantarum HM218749.1 had very strong scavenging capacities of the DPPH (86%), O2 (•-) (85%), (•)OH (76%), and Fe(2+) chelation (82%) and reducing powers (242.5 mg/L), and the inhibition zones for Salmonella typhimurium, Salmonella enteritidis, Shigella flexneri, Escherichia coli, Listeria monocytogenes, S. dysenteriae 301, Staphylococcus aureus Cowan1, and Propionibacterium acnes were 16, 15, 19, 20, 21, 20, and 27 mm. Moreover, the low concentration of Aloe fermentation supernatant had significantly reduced the production of IL-1β, TNF-α, and IL-6 in both mRNA and protein levels (P < 0.01). Therefore, the Aloe fermentation supernatant can be used as functional beverage or cosmetic ingredients to guard human intestinal health, delaying senescence, and prevent chronic diseases.

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

PubMed

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

Batch Tests with IONSIV IE-911 and a Simulant of the Savannah River Site ''Average'' Supernatant: Distribution Ratios vs Time

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, K.K.; Collins, J.L.; Hunt, R.D.

1999-02-01

The Department of Energy (DOE) is required by law to treat and safely dispose of the radioactive wastes from its nuclear weapon production activities. The primary radionuclide in the DOE liquid wastes or supernatants is {sup 137}Cs. At the Savannah River Site (SRS), the In-Tank Precipitation (ITP) process was selected as the baseline technology to remove {sup 137}Cs from the supernatants, which are stored in underground storage tanks. In the ITP process, tetraphenylborate reacts with the water-soluble cesium to form a precipitant. The treated supernatant can then be immobilized in grout or saltstone and stored in vaults at the SRS.more » However, problems were encountered during the full-scale ITP processing. These difficulties have led to the evaluation of alternative technologies and/or concepts to the currently configured ITP process. The High-Level Waste Salt Disposition Team at the SRS is currently performing this assessment. After an initial screening of all potential alternatives, the Salt Disposition Team selected four primary options to evaluate further before the final down-selection. Crystalline silicotitanate (CST), an inorganic ion exchanger, was chosen as one of the leading alternatives. Since nearly all of the CST tests have been performed on supernatants from Hanford and Oak Ridge, the Salt Disposition Team has requested that personnel at the SRS and Oak Ridge National Laboratory (ORNL) determine the performance of the engineered form of CST, IONSIV{reg_sign} IE-911, with actual and simulated SRS supernatants.« less

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

PubMed

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.

Ada Compiler Validation Summary Report: Certificate Number 900726W1. 11022 Verdix Corporation, VADS VAX/VMS= Intel 386, VMS 5.2, VAda-110-03315, Version 6.0, MicroVAX 3100 = iSBC 386/32

DTIC Science & Technology

1991-01-22

iSBC 386/32, bare machine Customer Agreement Number: 90-05-29- VRX See Section 3.1 for any additional information about the testing environment. As a...Defense Avail and/or Washington DC 20301 -Dist Special ----- ---- i AVF Control Number: AVF-VSR-379.0191 22 January 1991 90-05-29- VRX Ada COMPILER

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

PubMed

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 <1kDa component of P was still the predominant fraction in the supernatant, and underestimated colloidal P accounted for 2.2%, 55.1%, 45.5%, and 38.7% of P adsorption onto the solid surface of FH, FH-HA, GE and GE-HA, respectively. Thus, the colloid P could not be neglected. Notably, it could be interpreted that Fe 3+ hydrolysis from the adsorbents followed by the formation of colloidal hydrous ferric 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.

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA.

PubMed

Takagi, M; Ilias, M; Yoshida, T

2000-01-01

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

Distinct galactofuranose antigens in the cell wall and culture supernatants as a means to differentiate Fusarium from Aspergillus species.

PubMed

Wiedemann, Annegret; Kakoschke, Tamara Katharina; Speth, Cornelia; Rambach, Günter; Ensinger, Christian; Jensen, Henrik Elvang; Ebel, Frank

2016-09-01

Detection of carbohydrate antigens is an important means for diagnosis of invasive fungal infections. For diagnosis of systemic Aspergillus infections, galactomannan is commonly used, the core antigenic structure of which consists of chains of several galactofuranose moieties. In this study, we provide evidence that Fusarium produces at least two distinct galactofuranose antigens: Smaller amounts of galactomannan and larger quantities of a novel antigen recognized by the monoclonal antibody AB135-8. In A. fumigatus, only minor amounts of the AB135-8 antigen are found in supernatants and in the apical regions of hyphae. A galactofuranose-deficient A. fumigatus mutant lacks the AB135-8 antigen, which strongly suggests that galactofuranose is an essential constituent of this antigen. Using a combination of AB135-8 and a galactomannan-specific antibody, we were able to unambiguously differentiate A. fumigatus and Fusarium hyphae in immunohistology. Moreover, since Fusarium releases the AB135-8 antigen, it appears to be a promising target antigen for a serological detection of Fusarium infections. Copyright © 2016 Elsevier GmbH. All rights reserved.

Far Ultraviolet Refractive Index of Optical Materials for Solar Blind Channel (SBC) Filters for HST Advanced Camera for Surveys

NASA Technical Reports Server (NTRS)

Leviton, Douglas B.; Madison, Timothy J.; Petrone, Peter

1998-01-01

Refractive index measurements using the minimum deviation method have been carried out for prisms of a variety of far ultraviolet optical materials used in the manufacture of Solar Blind Channel (SBC) filters for the HST Advanced Camera for Surveys (ACS). Some of the materials measured are gaining popularity in a variety of high technology applications including high power excimer lasers and advanced microlithography optics operating in a wavelength region where high quality knowledge of optical material properties is sparse. Our measurements are of unusually high accuracy and precision for this wavelength region owing to advanced instrumentation in the large vacuum chamber of the Diffraction Grating Evaluation Facility (DGEF) at Goddard Space Flight Center (GSFC). Index values for CaF2, BaF2, LiF, and far ultraviolet grades of synthetic sapphire and synthetic fused silica are reported and compared with values from the literature.

A preliminary study on using autologous and heterologous boar sperm supernatant from freezing processes as post-thawing solution: its effect on sperm motility.

PubMed

Kaeoket, Kampon; Chanapiwat, Panida; Tummaruk, Padet; Techakumphu, Mongkol; Kunavongkrit, Annop

2011-06-01

The aim of this study was to evaluate the effect of post-thawing dilution with autologous and heterologous sperm supernatant on motility of frozen-thawed boar spermatozoa. During the cryopreservation, sperm supernatant (a combination of seminal plasma and semen extender, 50% v/v) or seminal plasma from nine boars (Duroc, Large White, and Landrace; three in each) was collected by centrifugation and stored frozen until use as post-thawing solution. Sperm pellets were further processed and cryopreserved using control-rate freezer and was thawed at 50°C for 12 s. After thawing, frozen thawed semen samples were diluted with seminal plasma (group A), supernatant from Landrace (group B), supernatant from Large White (group C), supernatant from Duroc (group D), and Modena™ semen extender (group E). Post-thawing motility was evaluated using a phase-contrast microscope after thawing at 1, 10, 20, and 30 min. The present results show that at 1 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (53.3%) and C (53.9%) than the other groups. At 10 min, the highest (P ≤ 0.001) progressive motility was found in groups B (65%) and C (61%). At 20 and 30 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (58.9%), C (53.5%), and D (45.6%) than groups A (3.9%) and E (20.6%). It can be stated that supernatant from the freezing processes (consisting of seminal plasma and Modena™, 50% v/v) had a beneficial effect on post-thawing progressive motility of frozen boar semen.

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

PubMed

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.

Performance investigation of an innovative Vertical Axis Hydrokinetic Turbine – Straight Blade Cascaded (VAHT-SBC) for low current speed

NASA Astrophysics Data System (ADS)

Hantoro, R.; Prananda, J.; Mahmashani, A. W.; Septyaningru, E.; Imanuddin, F.

2018-05-01

Research on the development and innovation of Vertical Axis Hydrokinetic Turbine (VAHT) to improve performance has been done. One of the important indicator that affects VAHT’s performance is Coefficient of Performance (Cp). Theoretical Cp value for the VAT (Darrieus) turbine is 0.45. This paper presents the results of a performance investigation for an innovative Vertical Axis Hydrokinetic Turbine – Straight Blade Cascaded (VAHT-SBC) by modifying the number and the arrangement of blades using CFD simulation. Symmetrical NACA 0018 is used for this study, each model is simulated with current speed variation (U - m/s) of 0.5, 1 and 1.5. An increase in Cp value is shown in variation of 9 blades (3 blades cascaded in each arm) with Cp value of 0.396 at TSR of 2.27 which is reach 88% of the theoretical value. Furthermore, the streamline velocity of the pressure contour, velocity streamline and torque fluctuations are also presented in this paper to gain in deep information.

Separation of somatic and germ cells is required to establish primate spermatogonial cultures.

PubMed

Langenstroth, Daniel; Kossack, Nina; Westernströer, Birgit; Wistuba, Joachim; Behr, Rüdiger; Gromoll, Jörg; Schlatt, Stefan

2014-09-01

Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth

Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme.

PubMed

Ellis, T M; Gregory, A R; Logue, G D

1991-06-01

Investigations were conducted into an enterotoxaemia caused by Clostridium spiroforme responsible for significant losses in commercial rabbit farms in Western Australia. Two trials using laboratory and farm bred rabbits were performed to evaluate the protective value of a toxoid prepared from the supernatant of C. spiroforme cultures against intraperitoneal challenge with the trypsin-activated toxin of C. spiroforme. The trials showed clearly that a single vaccination at weaning (four weeks) was protective against toxin but more complete and lasting protection was conferred following a second vaccination administered 14 days after the first. Adults likewise showed similar levels of protective antibodies but did not appear to pass on this protection to their kits although ELISA results indicated levels of antibody in kits from unvaccinated mother to be lower than progeny from vaccinated mothers. However antibody levels in kits from vaccinated mothers were very low and did not protect against challenge with toxin.

Bias Properties of Extragalactic Distance Indicators. VIII. H0 from Distance-limited Luminosity Class and Morphological Type-Specific Luminosity Functions for SB, SBC, and SC Galaxies Calibrated Using Cepheids

NASA Astrophysics Data System (ADS)

Sandage, Allan

1999-12-01

Relative, reduced to absolute, magnitude distributions are obtained for Sb, Sbc, and Sc galaxies in the flux-limited Revised Shapley-Ames Catalog (RSA2) for each van den Bergh luminosity class (L), within each Hubble type (T). The method to isolate bias-free subsets of the total sample is via Spaenhauer diagrams, as in previous papers of this series. The distance-limited type and class-specific luminosity functions are normalized to numbers of galaxies per unit volume (105 Mpc3), rather than being left as relative functions, as in Paper V. The functions are calculated using kinematic absolute magnitudes, based on an arbitrary trial value of H0=50. Gaussian fits to the individual normalized functions are listed for each T and L subclass. As in Paper V, the data can be freed from the T and L dependencies by applying a correction of 0.23T+0.5L to the individual absolute magnitudes. Here, T=3 for Sb, 4 for Sbc, and 5 for Sc galaxies, and the L values range from 1 to 6 as the luminosity class changes from I to III-IV. The total luminosity function, obtained by combining the volume-normalized Sb, Sbc, and Sc individual luminosity functions, each corrected for the T and L dependencies, has an rms dispersion of 0.67 mag, similar to much of the Tully-Fisher parameter space. Absolute calibration of the trial kinematic absolute magnitudes is made using 27 galaxies with known T and L that also have Cepheid distances. This permits the systematic correction to the H0=50 kinematic absolute magnitudes of 0.22+/-0.12 mag, givingH0=55+/-3(internal) km s-1 Mpc-1 . The Cepheid distances are based on the Madore/Freedman Cepheid period-luminosity (PL) zero point that requires (m-M)0=18.50 for the LMC. Using the modern LMC modulus of (m-M)0=18.58 requires a 4% decrease in H0, giving a final value of H0=53+/-7 (external) by this method. These values of H0, based here on the method of luminosity functions, are in good agreement with (1) H0=55+/-5 by Theureau and coworkers from their bias

Induction of NKCF-like activity in mixed lymphocyte-tumor cell culture: direct involvement of mycoplasma infection of tumor cells.

PubMed

Wayner, E A; Brooks, C G

1984-04-01

Co-culture of CBA/J spleen cells and certain lines of YAC-1 stimulators resulted in the appearance of NKCF-like activity in 24- to 48-hr supernatants. Numerous other in vitro cell lines were effective stimulators of this splenic cytotoxic factor (SCF). The cells participating in SCF production were absent from normal thymocytes and were present in BALB/c nu/nu spleen, were nonadherent, asialo GM1+, and bore low levels of Thy-1.2. SCF could mediate lysis of certain NK-sensitive tumor targets in an 18-hr 51Cr-release assay. However, the induction of SCF was not correlated with the ability of a particular cell line to be lysed by NK cells, but showed an absolute correlation with the presence of mycoplasma contamination in cultured tumor cell lines. Mycoplasma negative cell lines, including an uninfected but NK-sensitive subline of YAC-1, were unable to induce SCF. Decontamination of mycoplasma-infected lines with antibiotics or by passage through syngeneic mice abrogated the ability of infected tumor cells to stimulate SCF. The ability to induce SCF could be restored by reinfection with mycoplasma. Tumor cell-free supernatants from contaminated cultures were mitogenic for CBA spleen cells and could themselves induce SCF activity in spleen cell supernatants. SCF production and the agent responsible could be removed by passing such supernatants through 0.1-micron filters. The organism apparently responsible for SCF induction from CBA spleen cells was typed and found to be Mycoplasma orale, a nonfermentative, arginine-dependent, common tissue culture contaminant. About 50 to 60% of SCF activity could be removed by 0.1-micron filters, suggesting that SCF is composed of two components: mycoplasma organisms themselves and a soluble cytotoxic factor produced in response to mycoplasma.

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

PubMed

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.

Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.

PubMed

Loganathan, G; Dawra, R K; Pugazhenthi, S; Wiseman, A C; Sanders, M A; Saluja, A K; Sutherland, D E R; Hering, B J; Balamurugan, A N

2010-01-01

Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT

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

PubMed

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.

A filterable lytic agent obtained from a red tide bloom that caused lysis of Karenia brevis (Gymnodinum breve) cultures

USGS Publications Warehouse

2002-01-01

A filterable lytic agent (FLA) was obtained from seawater in the southeastern Gulf of Mexico during a red tide bloom that caused lysis of Karenia brevis (formerly Gymnodinium breve) Piney Island. This agent was obtained from <0.2µ  filtrates that were concentrated by ultrafiltration using a 100 kDa filter. The FLA was propagated by passage on K. brevis cultures, and the filtered supernatants of such cultures resulted in K. brevis lysis when added to such cultures. The lytic activity was lost upon heating to 65°C or by 0.02 µm filtration. Epifluorescence and transmission electron microscopy (TEM) of supernatants of K. brevis cultures treated with the lytic agent indicated a high abundance of viral particles (4 × 109 to 7 × 109 virus-like particles [VLPs] ml–1) compared to control cultures (~107 ml–1). However, viral particles were seldom found in TEM photomicrograph thin sections of lysing K. brevis cells. Although a virus specific for K. brevis may have been the FLA, other explanations such as filterable bacteria or bacteriophages specific for bacteria associated with the K. brevis cultures cannot be discounted.

Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion

PubMed Central

Khodaii, Zohreh; Ghaderian, Sayyed Mohammad Hossein; Natanzi, Mahboobeh Mehrabani

2017-01-01

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion. PMID:29682490

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

PubMed

Silliman, Christopher C; Kelher, Marguerite R; Khan, Samina Y; West, F Bernadette; McLaughlin, Nathan J D; Elzi, David J; England, Kelly; Bjornsen, Jason; Kuldanek, Susan A; Banerjee, Anirban

2017-11-01

Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%] FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. The supernatants [10%-40%] FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%] FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of β-arrestin-1 with BLT2, protein kinase C (PKC)β I , and PKCδ and served as the first event in a two-event rodent model of ALI. Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients. © 2017 AABB.

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

PubMed

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

Evolution of composition of dairy manure supernatant in a controlled dung pit.

PubMed

Rico, C; García, H; Rico, J L; Fernández, J; Renedo, J

2009-12-01

Anaerobic conversion of dairy manure into biogas is an attractive way of managing this waste. It is well known that the hydrolysis of large molecules into small, directly biodegradable ones is the rate limiting step of the overall anaerobic process. The present work studies the development of the hydrolytic and acidogenic stages of dairy manure with different solid concentrations (40, 60 and 80 g VS/L) at ambient temperature (20 degrees C). The purpose was to determine the operational conditions that provide a liquid fraction with a high soluble chemical oxygen demand (COD) and a high volatile fatty acids (VFA) content in manure before the methanogenic stage starts up. At 20 degrees C, the evolution of the studied parameters showed that, in a controlled plug-flow dung pit, the hydrolytic and acidogenic stages progressed moderately in a continuous way during the 25 days that the experimentation lasted, whereas no methanization was observed. Supernatant COD and VFA concentrations increased 30% and 107%, respectively, for the 60 g VS/L samples. Manure was also operated at 35 degrees C with a similar increase in supernatant COD but a higher increase in VFA, 154%. For both operational temperatures, the predominant VFAs were, in this order, acetic, propionic and butyric acids. During the operation at 35 degrees C, the methanogenic stage started between days 20 and 25 for the samples with lower solids content, i.e. 40 and 60 g VS/L.

Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions?

PubMed

Buschmann, Johanna; Gao, Shuping; Härter, Luc; Hemmi, Sonja; Welti, Manfred; Werner, Clement M L; Calcagni, Maurizio; Cinelli, Paolo; Wanner, Guido A

2013-09-01

Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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.

PubMed

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.

Far-ultraviolet refractive index of optical materials for solar blind channel (SBC) filters for the HST advanced camera for surveys (ACS)

NASA Astrophysics Data System (ADS)

Leviton, Douglas B.; Madison, Timothy J.; Petrone, Peter

1998-10-01

Refractive index measurements using the minimum deviation method have been carried out for prisms of a variety of far ultraviolet optical materials used in the manufacture of Solar Blind Channel (SBC) filters for the HST Advanced Camera for Surveys (ACS). Some of the materials measured are gaining popularity in a variety of high technology applications including high power excimer lasers and advanced microlithography optics operating in a wavelength region where high quality knowledge of optical material properties is sparse yet critical. Our measurements are of unusually high accuracy and precision for this wavelength region owing to advanced instrumentation in the large vacuum chamber of the Diffraction Grating Evaluation Facility (DGEF) at Goddard Space Flight Center (GSFC) used to implement a minimum deviation method refractometer. Index values for CaF2, BaF2, LiF, and far ultraviolet grades of synthetic sapphire and synthetic fused silica are reported and compared with values from the literature.

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

PubMed

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

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

PubMed

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.

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

PubMed

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.

Effect of Microcystin-LR on Cultured Rat Endothelial Cells

DTIC Science & Technology

1990-02-26

protection, silymarin , and dithioerythritol 19. ABSTRACT (Continue on reverse if necessary and identify by block number) Primary cultureLs of adult rat...8217 22.g-Ldenine nucleotides, and a small reduction of cell density in endothelial cell monolayers._ Silymarin at 0.2 mM but not(dithioerythritol;at 2.5...monolayers. Silymarin at 0.2 mM but not dithioerythritol at 2.5 mM, partially protected changes in endothelial cells produced by supernatants derived

Tank 30 and 37 Supernatant Sample Cross-Check and Evaporator Feed Qualification Analysis-2012

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oji, L. N.

2013-03-07

This report summarizes the analytical data reported by the F/H and Savannah River National Laboratories for the 2012 cross-check analysis for high level waste supernatant liquid samples from SRS Tanks 30 and 37. The intent of this Tank 30 and 37 sample analyses was to perform cross-checks against routine F/H Laboratory analyses (corrosion and evaporator feed qualification programs) using samples collected at the same time from both tanks as well as split samples from the tanks.

Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

PubMed

Smith, Karen; Rajendran, Ranjith; Kerr, Stephen; Lappin, David F; Mackay, William G; Williams, Craig; Ramage, Gordon

2015-09-01

In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

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.

Analysis of tank 4 (FTF-4-15-22, 23) surface and subsurface supernatant samples in support of enrichment control, corrosion control and evaporator feed qualification programs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oji, L. N.

This report provides the results of analyses on Savannah River Site Tank 4 surface and subsurface supernatant liquid samples in support of the Enrichment Control Program (ECP), the Corrosion Control Program (CCP) and the Evaporator Feed Qualification (EFQ) Program. The purpose of the ECP sample taken from Tank 4 in August 2015 was to determine if the supernatant liquid would be “acceptable feed” to the 2H and 3H evaporator systems.

Recovery of urinary nanovesicles from ultracentrifugation supernatants.

PubMed

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.

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

PubMed

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.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum.

PubMed

Ren, Jiaqiang; Ward, Dawn; Chen, Steven; Tran, Katherine; Jin, Ping; Sabatino, Marianna; Robey, Pamela G; Stroncek, David F

2018-03-14

Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide.

PubMed

Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E; Bohórquez, Mabel E

2016-07-01

Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor kappa beta (NFkβ), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1) and cartilage oligomeric matrix protein (COMP) in normal cartilage explants (CEs) challenged with lipopolysaccharide (LPS). Overall, 25 % L-PRG supernatant (followed in order of importance by, 50 % P-PRG, 25 % P-PRG and 50 % L-PRG) represented the substance with the most important anti-inflammatory and anabolic effect. 25 % P-PRG supernatant presented important anabolic effects, but it induced a more severe chondrocyte apoptosis than the other evaluated substances. 25 % L-PRG supernatant presented the best therapeutic profile. Our results demonstrate that the biological variability of PRP preparations makes their application rather challenging. Additional in vivo research is necessary to know the effect of PRP preparations at different concentrations.

Small Bowel Carcinomas in Coeliac or Crohn's Disease: Clinico-pathological, Molecular, and Prognostic Features. A Study From the Small Bowel Cancer Italian Consortium.

PubMed

Vanoli, Alessandro; Di Sabatino, Antonio; Furlan, Daniela; Klersy, Catherine; Grillo, Federica; Fiocca, Roberto; Mescoli, Claudia; Rugge, Massimo; Nesi, Gabriella; Fociani, Paolo; Sampietro, Gianluca; Ardizzone, Sandro; Luinetti, Ombretta; Calabrò, Antonio; Tonelli, Francesco; Volta, Umberto; Santini, Donatella; Caio, Giacomo; Giuffrida, Paolo; Elli, Luca; Ferrero, Stefano; Latella, Giovanni; Ciardi, Antonio; Caronna, Roberto; Solina, Gaspare; Rizzo, Aroldo; Ciacci, Carolina; D'Armiento, Francesco P; Salemme, Marianna; Villanacci, Vincenzo; Cannizzaro, Renato; Canzonieri, Vincenzo; Reggiani Bonetti, Luca; Biancone, Livia; Monteleone, Giovanni; Orlandi, Augusto; Santeusanio, Giuseppe; Macciomei, Maria C; D'Incà, Renata; Perfetti, Vittorio; Sandri, Giancarlo; Silano, Marco; Florena, Ada M; Giannone, Antonino G; Papi, Claudio; Coppola, Luigi; Usai, Paolo; Maccioni, Antonio; Astegiano, Marco; Migliora, Paola; Manca, Rachele; Martino, Michele; Trapani, Davide; Cerutti, Roberta; Alberizzi, Paola; Riboni, Roberta; Sessa, Fausto; Paulli, Marco; Solcia, Enrico; Corazza, Gino R

2017-08-01

An increased risk of small bowel carcinoma [SBC] has been reported in coeliac disease [CD] and Crohn's disease [CrD]. We explored clinico-pathological, molecular, and prognostic features of CD-associated SBC [CD-SBC] and CrD-associated SBC [CrD-SBC] in comparison with sporadic SBC [spo-SBC]. A total of 76 patients undergoing surgical resection for non-familial SBC [26 CD-SBC, 25 CrD-SBC, 25 spo-SBC] were retrospectively enrolled to investigate patients' survival and histological and molecular features including microsatellite instability [MSI] and KRAS/NRAS, BRAF, PIK3CA, TP53, HER2 gene alterations. CD-SBC showed a significantly better sex-, age-, and stage-adjusted overall and cancer-specific survival than CrD-SBC, whereas no significant difference was found between spo-SBC and either CD-SBC or CrD-SBC. CD-SBC exhibited a significantly higher rate of MSI and median tumour-infiltrating lymphocytes [TIL] than CrD-SBC and spo-SBC. Among the whole SBC series, both MSI─which was the result of MLH1 promoter methylation in all but one cases─and high TIL density were associated with improved survival at univariable and stage-inclusive multivariable analysis. However, only TILs retained prognostic power when clinical subgroups were added to the multivariable model. KRAS mutation and HER2 amplification were detected in 30% and 7% of cases, respectively, without prognostic implications. In comparison with CrD-SBC, CD-SBC patients harbour MSI and high TILs more frequently and show better outcome. This seems mainly due to their higher TIL density, which at multivariable analysis showed an independent prognostic value. MSI/TIL status, KRAS mutations and HER2 amplification might help in stratifying patients for targeted anti-cancer therapy. Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

Concentration of adipogenic and proinflammatory cytokines in the bone marrow supernatant fluid of osteoporotic women.

PubMed

Pino, Ana María; Ríos, Susana; Astudillo, Pablo; Fernández, Mireya; Figueroa, Paula; Seitz, Germán; Rodríguez, J Pablo

2010-03-01

Osteoporosis is characterized by low bone mass, microarchitectural deterioration of bone tissue leading to increased bone fragility, and a resulting susceptibility to fractures. Distinctive environmental bone marrow conditions appear to support the development and maintenance of the unbalance between bone resorption and bone formation; these complex bone marrow circumstances would be reflected in the fluid surrounding bone marrow cells. The content of regulatory molecules in the extracellular fluid from the human bone marrow is practically unknown. Since the content of cytokines such as adiponectin, leptin, osteoprogeterin (OPG), soluble receptor activator of nuclear factor kappaB ligand (s-RANKL), tumor necrosis factor alpha, and interleukin 6 (IL-6) may elicit conditions promoting or sustaining osteoporosis, in this work we compared the concentrations of the above-mentioned cytokines and also the level of the soluble receptors for both IL-6 and leptin in the extracellular fluid from the bone marrow of nonosteoporotic and osteoporotic human donors. A supernatant fluid (bone marrow supernatant fluid [BMSF]) was obtained after spinning the aspirated bone marrow samples; donors were classified as nonosteoporotic or osteoporotic after dual-energy X-ray absorptiometry (DXA) measuring. Specific commercially available kits were used for all measurements. The cytokines' concentration in BMSF showed differently among nonosteoporotic and osteoporotic women; this last group was characterized by higher content of proinflammatory and adipogenic cytokines. Also, osteoporotic BMSF differentiated by decreased leptin bioavailability, suggesting that insufficient leptin action may distinguish the osteoporotic bone marrow. Copyright 2010 American Society for Bone and Mineral Research.

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

PubMed

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

2017-03-06

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 integrity. In vivo experiments revealed that pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E. coli K1 infection as reflected by reduced bacterial intestinal colonization, translocation, dissemination and systemic infections. Further, we found that LCS treated neonatal rats have higher intestinal expressions of Ki67, MUC2, ZO-1, IgA, mucin and lower barrier permeability than those in untreated rats. These results indicated that LCS could enhance neonatal resistance to systemic E. coli K1 infection via promoting maturation of neonatal intestinal defense. In conclusions, our findings suggested that LCS has a prophylactic effect against systemic E. coli K1 infection in neonates. Future studies aimed at identifying the specific active ingredients in LCS will be helpful in developing effective pharmacological strategies for preventing neonatal E. coli K1 infection.

3 CFR 8803 - Proclamation 8803 of April 20, 2012. Establishment of the Fort Ord National Monument

Code of Federal Regulations, 2013 CFR

2013-01-01

... of the United States of America A Proclamation In the heart of California’s Central Coast, the former.... From World War I through the early 1990s, the area’s rugged terrain served as a military training... landscape’s unique array of plant and wildlife communities. The area is also notable for its historical...

A combination turbidity and supernatant microplate assay to rank-order the supersaturation limits of early drug candidates.

PubMed

Morrison, John S; Nophsker, Michelle J; Haskell, Roy J

2014-10-01

A unique opportunity exists at the drug discovery stage to overcome inherently poor solubility by selecting drug candidates with superior supersaturation propensity. Existing supersaturation assays compare either precipitation-resistant or precipitation-inhibiting excipients, or higher-energy polymorphic forms, but not multiple compounds or multiple concentrations. Furthermore, these assays lack sufficient throughput and compound conservation necessary for implementation in the discovery environment. A microplate-based combination turbidity and supernatant concentration assay was therefore developed to determine the extent to which different compounds remain in solution as a function of applied concentration in biorelevant media over a specific period of time. Dimethyl sulfoxide stock solutions at multiple concentrations of four poorly soluble, weak base compounds (Dipyridamole, Ketoconazole, Albendazole, and Cinnarizine) were diluted with pH 6.5 buffer as well as FaSSIF. All samples were monitored for precipitation by turbidity at 600 nm over 1 h and the final supernatant concentrations were measured. The maximum supersaturation ratio was calculated from the supersaturation limit and the equilibrium solubility in each media. Compounds were rank-ordered by supersaturation ratio: Ketoconazole > Dipyridamole > Cinnarizine ∼ Albendazole. These in vitro results correlated well with oral AUC ratios from published in vivo pH effect studies, thereby confirming the validity of this approach. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

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

PubMed

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.

Inhibition of biofilm development and spoilage potential of Shewanella baltica by quorum sensing signal in cell-free supernatant from Pseudomonas fluorescens.

PubMed

Zhao, Aifei; Zhu, Junli; Ye, Xiaofeng; Ge, Yangyang; Li, Jianrong

2016-08-02

The objective of this study was to in vitro evaluate the effect of a cell-free supernatant (CFS) containing quorum sensing (QS) signal of Pseudomonas fluorescens on the growth, biofilm development and spoilage potential of Shewanella baltica, and preliminarily assess the interactive influences of various chemically synthesized autoinducers on spoilage phenotypes of S. baltica. PF01 strain isolated from spoiled Pseudosciaen crocea was identified P. fluorescens. The addition of 25% and 50% CFS to S. baltica culture had no effect on the growth rate during the lag and exponential phase, however, caused cell decline during the stationary phase. The presence of CFS from P. fluorescens significantly inhibited biofilm development, and greatly decreased the production of trimethylamine (TMA) and biogenic amino in S. baltica. Various signal molecules of QS in the CFS of P. fluorescens culture were detected, including seven N-acyl-l-homoserine lactones (AHLs), autoinducer-2 (AI-2) and two diketopiperazines (DKPs). Exogenous supplement of synthesized seven AHLs containing in the CFS decreased biofilm formation and TMA production in S. baltica, while exposure to exogenous cyclo-(l-Pro-l-Leu) was showed to promote spoilage potential, which revealed that S. baltica also sense the two QS molecules. Furthermore, the stimulating effect of cyclo-(l-Pro-l-Leu) was affected when AHL was simultaneously added, suggesting that the inhibitory activity of spoilage phenotypes in S. baltica might be attributed to a competitive effect of these QS compounds in the CFS of P. fluorescens. The present studies provide a good basis for future research on the role of QS in the regulation of spoilage microbial flora. Copyright © 2016 Elsevier B.V. All rights reserved.

A simple and rapid latex fixation test for measuring immunoglobulins produced in cell cultures.

PubMed

Kasahara, T; Harada, H; Enomoto, H; Itoh, Y; Kawai, T; Shioiri-Nakano, K

1981-01-01

A rapid and simple latex fixation test (LFT), which quantifies immunoglobulin (Ig) released into culture supernatants is described. Latex particles are coated with rabbit anti-human IgG, IgA or IgM antibodies. With this LFT technique the concentration of Ig is determined within a few minutes. The LFT is as sensitive and quantitative as double-antibody radioimmunoassay and is capable of detecting 35, 68 and 225 ng/ml of IgG, IgA and IgM, respectively.

Growing Bacteriophage M13 in Liquid Culture.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼10 12 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque. © 2017 Cold Spring Harbor Laboratory Press.

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

PubMed

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.

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

PubMed Central

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

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 integrity. In vivo experiments revealed that pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E. coli K1 infection as reflected by reduced bacterial intestinal colonization, translocation, dissemination and systemic infections. Further, we found that LCS treated neonatal rats have higher intestinal expressions of Ki67, MUC2, ZO-1, IgA, mucin and lower barrier permeability than those in untreated rats. These results indicated that LCS could enhance neonatal resistance to systemic E. coli K1 infection via promoting maturation of neonatal intestinal defense. In conclusions, our findings suggested that LCS has a prophylactic effect against systemic E. coli K1 infection in neonates. Future studies aimed at identifying the specific active ingredients in LCS will be helpful in developing effective pharmacological strategies for preventing neonatal E. coli K1 infection. PMID:28262688

Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

PubMed

Tour, Gregory; Wendel, Mikael; Tcacencu, Ion

2013-10-01

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alterations in cultured human brains.

PubMed Central

Pulliam, L; Herndier, B G; Tang, N M; McGrath, M S

1991-01-01

We wanted to establish an in vitro human model for AIDS-associated dementia and pursue the hypothesis that this disease process may be a result of soluble factors produced by HIV-infected macrophages. Human brain aggregates were prepared from nine different brain specimens, and were treated with supernatants from in vitro HIV-infected macrophages (SI), uninfected macrophages (SU), infected T cells, or macrophage-conditioned media from four AIDS patients. Seven of nine treated brains exposed to SI showed peripheral rarefaction after 1 wk of incubation that by ultrastructural analysis showed cytoplasmic vacuolation. Aggregates from two of three brain cultures treated with SI for 3 wk became smaller, an approximately 50% decrease in size. The degree of apparent toxicity in brains exposed to patient-derived macrophage supernatants paralleled the proportion of macrophages found to be expressing HIV p24. Ultrastructural abnormalities were not observed in brains treated with supernatants from HIV-infected T cells, uninfected macrophages, or LPS-activated macrophages. Levels of five neurotransmitter amino acids were decreased in comparison to the structural amino acid leucine. These findings suggest that HIV-infected macrophages, infected both in vitro as well as derived from AIDS patients' peripheral blood, produce factors that cause reproducible histochemical, ultrastructural, and functional abnormalities in human brain aggregates. Images PMID:1671392

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

PubMed

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.

Levels of IL-32 in Serum, Induced Sputum Supernatant, and Bronchial Lavage Fluid of Patients with Chronic Obstructive Pulmonary Disease.

PubMed

Gasiuniene, Edita; Lavinskiene, Simona; Sakalauskas, Raimundas; Sitkauskiene, Brigita

2016-10-01

Interleukin-32 (IL-32) is a newly described cytokine which is expected to have an important role in autoimmune disorders. It was shown that chronic obstructive pulmonary disease (COPD) has a component of autoimmunity, though the role of IL-32 in its pathogenesis is not known. The aim of this study was to estimate IL-32 concentrations in serum, induced sputum (IS) supernatant and bronchoalveolar lavage (BAL) fluid from patients with COPD, and to compare asthma patients with and healthy subjects. Outpatients with COPD (63.7 ± 8.4 years, n = 51), asthma (58.3 ± 12.4 years, n = 31), and healthy subjects (59.8 ± 8.2 years, n = 9) were studied. The levels of IL-32 in serum, BAL fluid, and IS supernatant samples were analyzed by ELISA. Concentrations of IL-32 were higher in all the studied materials from patients with COPD (BAL 22.46 ± 2.48 pg/ml, IS 19.66 ± 1.69 pg/ml, serum 26.77 ± 2.56 pg/ml) in comparison with patients with asthma (BAL 6.25 ± 1.08 pg/ml, IS 5.82 ± 1.15 pg/ml, serum 6.09 ± 1.16 pg/ml, p < 0.05 respectively) as well as healthy subjects (BAL 4.21 ± 1.13 pg/ml, IS 3.59 ± 0.66 pg/ml, serum 4.63 ± 1.03 pg/ml, p < 0.05 respectively). Moreover, the level of IL-32 was higher in COPD smokers than in COPD ex-smokers in investigated respiratory tissue compartments and serum, and correlated with smoking history. Increased level of IL-32 in serum, IS supernatant, and BAL fluid from patients with COPD in comparison with asthma patients and healthy subjects suggest that IL-32 may play an important role in the pathogenesis of COPD, which depends on the smoking history.

Nutritive value of extruded or multi-enzyme supplemented cold-pressed soybean cake for pigs.

PubMed

Woyengo, T A; Patterson, R; Levesque, C L

2016-12-01

The objectives were to determine the standardized ileal digestibility (SID) of AA and NE value of cold-pressed soybean cake (CP-SBC), and the effect of extrusion or adding multi-enzyme to CP-SBC diet for growing pigs. Eight ileal-cannulated pigs (initial BW = 79.7 ± 3.97 kg) were fed 4 diets in a replicated 4 × 4 Latin square design to give 8 replicates per diet. Diets included a cornstarch-based diet with CP-SBC, extruded CP-SBC, and SBC plus multi-enzyme (1,200 U of xylanase, 150 U of glucanase, 500 U of cellulase, 60 U of mannanase, 700 U of invertase, 5,000 U of protease, and 12,000 U of amylase/kilogram of diet; Superzyme-CS, 0.5 g/kg); and a N-free diet. The CP-SBC was the sole source of protein in the CP-SBC-containing diets. The ratio of cornstarch to sugar and soybean oil in CP-SBC-containing diets was identical to the N-free diet to allow calculation of energy digestibility of CP-SBC by the difference method. The evaluated CP-SBC had been produced by heating the soybean seed at 105°C for 60 min followed by pressing of the heated soybean seeds at less than 42°C (barrel temperature). On a DM basis, CP-SBC and extruded CP-SBC contained 47.8 and 47.1% CP, 15.6 and 10.5% ADF, 7.23 and 8.85% ether extract, 3.11 and 3.08% Lys, and 2.25 and 3.70 trypsin inhibitor units per mg, respectively. Extrusion increased ( < 0.001) the SID of AA for the CP-SBC by an average of 12%. Also, extrusion increased ( < 0.001) the NE value of the CP-SBC from 2,743 to 2,853 kcal/kg of DM. Supplementation of CP-SBC diet with the multi-enzyme increased ( < 0.05) the SID of Arg and Pro, and tended to increase ( < 0.1) the SID of Ile and Tyr. However, the multi-enzyme supplementation did not affect the NE value of CP-SBC. In conclusion, the CP-SBC evaluated in the present study could be an alternative source of AA and energy in swine diets, and its nutritive value can be increased by extrusion following cold-pressing. The multi-enzyme used in this study improved the digestibility of

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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.more » 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.« less

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

PubMed

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.

Ex-vivo dynamic 3-D culture of human tissues in the RCCS™ bioreactor allows the study of Multiple Myeloma biology and response to therapy.

PubMed

Ferrarini, Marina; Steimberg, Nathalie; Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy

PubMed Central

Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy. PMID:23990965

Suicide by cop in film and society: dangerousness, depression, and justice.

PubMed

Stack, Steven; Bowman, Barbara; Lester, David

2012-08-01

Provoking police officers to kill oneself or "suicide by cop" (SBC) has received scholarly as well as public attention. The extent to which film representations of SBC reflect SBC in society in relation to danger, depression, and other features was assessed in this study. Data on cinematic portrayals of SBC are from 16 American films; corresponding data on SBC in the real world are taken from 19 empirical studies. While the demographic features of SBC in the real world are largely reflected in film, major differences exist on motivations and incident characteristics. Motives for cinematic SBC were unlike SBC in society, wherein, respectively, 0% vs. 35% had attempted suicide in the past, 6% vs. 47% were chronically depressed, and 77% vs. 7% had recently killed someone. Cinematic SBC events disproportionately involve murderous, dangerous persons who are not depressed, and their deaths convey a sense of justice. While such media distortions can be profitable and entertain the audience, they also contribute to public misunderstandings of the nature of deviance and hinder the development of effective suicide and violence prevention programs. © 2012 The American Association of Suicidology.

The optimization of microalgal culturing in liquid digestate after struvite precipitation using gray relational analysis.

PubMed

Jiang, Yiqi; Wang, Hong; Wang, Wannan; Deng, Liangwei; Wang, Wenguo

2018-05-01

Liquid digestate (LD) is highly turbid and contains ammonium (NH 4 + -N), which negatively influences microalgal growth. Therefore, a method of reducing LD turbidity and NH 4 + -N content is proposed, using struvite precipitation. To obtain struvite precipitation supernatant with an ideal UV transmittance, NH 4 + -N concentration, and N/P ratio for microalgal growth, the effects of pH and the molar ratio of NH 4 + /Mg 2+ /PO 4 3- were studied. Results show that the optimal NH 4 + /Mg 2+ /PO 4 3- molar ratio was 1:1.5:1.5, with a pH value of 8.5, following NaOH addition. Gray relational analysis (GRA) was applied to obtain a relative gray scale for the evaluation of multiple outputs. Results show that Chlorella regularis FACHB-1068 was the optimal microalgae species to support growth in the struvite precipitation supernatant. Using struvite precipitation and treatment with cultured C. regularis FACHB-1068 for 7 days, the removal efficiencies of NH 4 + -N, PO 4 3- -P, and COD in LD were 96.52, 99.33, and 35.30%, respectively.

Child-targeted TV advertising and preschoolers' consumption of high-sugar breakfast cereals.

PubMed

Longacre, Meghan R; Drake, Keith M; Titus, Linda J; Harris, Jennifer; Cleveland, Lauren P; Langeloh, Gail; Hendricks, Kristy; Dalton, Madeline A

2017-01-01

Breakfast cereals represent the most highly advertised packaged food on child-targeted television, and most ads are for cereals high in sugar. This study examined whether children's TV exposure to child-targeted, high-sugar breakfast cereal (SBC) ads was associated with their consumption of those SBC brands. Parents of 3- to 5-year-old children were recruited from pediatric and Women, Infants, and Children (WIC) clinics in Southern New Hampshire, USA, and completed a cross-sectional survey between April-December 2013. Parents reported their child's consumption of SBC brands; whether their child had watched any of 11 kids' channels in the past week; their child's TV viewing time; and socio-demographics. Children's exposure to child-targeted SBC TV ads was calculated by combining TV channel and viewing time with advertising data for SBC ads aired on kids' TV channels during the same timeframe. Five hundred forty-eight parents completed surveys; 52.7% had an annual household income of $50,000 or less. Children's mean age was 4.4 years, 51.6% were female, and 72.5% were non-Hispanic white. In the past week, 56.9% (N = 312) of children ate SBCs advertised on kids' channels. Overall, 40.6% of children were exposed to child-targeted SBC TV ads in the past week. In fully adjusted analyses, the number of SBC brands children consumed was positively associated with their exposure to child-targeted SBC ads. Children consumed 14% (RR = 1.14, 95% CI: 1.02, 1.27) more SBC brands for every 10 SBC ads seen in the past 7 days. Exposure to child-targeted SBC TV advertising is positively associated with SBC brand consumption among preschool-aged children. These findings support recommendations to limit the marketing of high-sugar foods to young children. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suicide by Cop in Film and Society: Dangerousness, Depression, and Justice

ERIC Educational Resources Information Center

Stack, Steven; Bowman, Barbara; Lester, David

2012-01-01

Provoking police officers to kill oneself or "suicide by cop" (SBC) has received scholarly as well as public attention. The extent to which film representations of SBC reflect SBC in society in relation to danger, depression, and other features was assessed in this study. Data on cinematic portrayals of SBC are from 16 American films;…

Release of Black Carbon From Thawing Permafrost Estimated by Sequestration Fluxes in the East Siberian Arctic Shelf Recipient

NASA Astrophysics Data System (ADS)

Salvadó, Joan A.; Bröder, Lisa; Andersson, August; Semiletov, Igor P.; Gustafsson, Örjan

2017-10-01

Black carbon (BC) plays an important role in carbon burial in marine sediments globally. Yet the sequestration of BC in the Arctic Ocean is poorly understood. Here we assess the concentrations, fluxes, and sources of soot BC (SBC)—the most refractory component of BC—in sediments from the East Siberian Arctic Shelf (ESAS), the World's largest shelf sea system. SBC concentrations in the contemporary shelf sediments range from 0.1 to 2.1 mg g-1 dw, corresponding to 2-12% of total organic carbon. The 210Pb-derived fluxes of SBC (0.42-11 g m-2 yr-1) are higher or in the same range as fluxes reported for marine surface sediments closer to anthropogenic emissions. The total burial flux of SBC in the ESAS ( 4,000 Gg yr-1) illustrates the great importance of this Arctic shelf in marine sequestration of SBC. The radiocarbon signal of the SBC shows more depleted yet also more uniform signatures (-721 to -896‰; average of -774 ± 62‰) than of the non-SBC pool (-304 to -728‰; average of -491 ± 163‰), suggesting that SBC is coming from an, on average, 5,900 ± 300 years older and more specific source than the non-SBC pool. We estimate that the atmospheric BC input to the ESAS is negligible ( 0.6% of the SBC burial flux). Statistical source apportionment modeling suggests that the ESAS sedimentary SBC is remobilized by thawing of two permafrost carbon (PF/C) systems: surface soil permafrost (topsoil/PF; 25 ± 8%) and Pleistocene ice complex deposits (ICD/PF; 75 ± 8%). The SBC contribution to the total mobilized permafrost carbon (PF/C) increases with increasing distance from the coast (from 5 to 14%), indicating that the SBC is more recalcitrant than other forms of translocated PF/C. These results elucidate for the first time the key role of permafrost thaw in the transport of SBC to the Arctic Ocean. With ongoing global warming, these findings have implications for the biogeochemical carbon cycle, increasing the size of this refractory carbon pool in the Arctic

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.

PubMed

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.

Nutritional evaluation of Zambia indigenous soy bean (Glycine max) and sunflower (Helianthus annus) as protein sources in poultry and pigs diets.

PubMed

Aregheore, E M

1998-10-01

Two trials were carried out to compare the nutritional values of two Zambian indigenous plant protein sources--soy bean cake (SBC) and sunflower meal (SFM) in the diets of broilers and growing pigs. In trial 1, 120 one week old chickens (Abbor acre strain) were used. There were no differences (P > 0.05) between chickens on SBC and SFM in voluntary feed intake. Average daily gain of SBC chickens differed (P < 0.05) from those of SFM. The protein source had an effect (P < 0.05) on N retained [g/day]. Carcasses dry matter and crude protein were higher (P < 0.05) in SBC chickens, but ash, ether extract, Ca and P were the same as SFM. In trial 2, 12 Large white x Landrace growing barrows 1-2 months old were used. In this trial, SBC diet was consumed more than SFM. Pigs on SBC and SFM gained 0.526 and 0.284 g/head/day, respectively (P < 0.05). Nutrient digestibility was higher (P < 0.05) in SBC diet. Trial 1, demonstrated that SBC and SFM could be used for broilers without adverse effect on growth rate and body conformation. However, for growing pigs SBC is a better protein source than SFM in the tropical environment of Zambia. Finally, results obtained seem to suggest that SBC and SFM can be used as plant protein sources, but SFM is not an ideal plant protein source for growing pigs.

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.

PubMed

Sánchez-Reyes, Karina; Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Jave-Suárez, Luis Felipe; Gómez-Lomelí, Paulina; de Celis, Ruth; Aguilar-Lemarroy, Adriana; Domínguez-Rodríguez, Jorge Ramiro; Ortiz-Lazareno, Pablo Cesar

2014-01-01

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. 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. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

Suppression of bactericidal activity of human polymorphonuclear leukocytes by Bacteroides gingivalis.

PubMed Central

Yoneda, M; Maeda, K; Aono, M

1990-01-01

The direct effects of the culture supernatant of oral microorganisms on the bactericidal activity of human polymorphonuclear leukocytes (PMNs) were investigated. The bactericidal activity of PMNs, which were preincubated with the supernatant of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides melaninogenicus or phosphate-buffered saline, was examined by counting the surviving bacteria. B. gingivalis-treated PMNs were found to have a diminished ability for killing bacteria in the presence or absence of serum. The chemiluminescence response of PMNs, which were preincubated with the culture supernatant of B. gingivalis, was strikingly reduced compared with that of PMNs preincubated with phosphate-buffered saline or other bacterial culture supernatants. The production of superoxide anion (O2-) by PMNs stimulated with either formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate was reduced in both cases after the PMNs were preincubated with the culture supernatant of B. gingivalis. However, it was observed that there was more reduction in superoxide anion (O2-) production stimulated with formyl-methionyl-leucyl-phenylalanine compared with that stimulated with phorbol myristate acetate. These results suggest that B. gingivalis releases a factor which interferes with the bactericidal activity of PMNs by modulating the generation of reactive oxygen species. These suppressive effects on bactericidal activity may be important in the pathogenesis of this microorganism. PMID:2153632

Evaluation of the prototype surface bypass for salmonid smolts in Spring 1996 and 1997 at Lower Granite Dam on the Snake River, Washington

USGS Publications Warehouse

Johnson, G.E.; Adams, N.S.; Johnson, Robert L.; Rondorf, D.W.; Dauble, D.D.; Barila, T.Y.

2000-01-01

In spring 1996 and 1997, we studied the prototype surface bypass and collector (SBC) at Lower Granite Dam on the Snake River in Washington. Our objectives were to determine the most efficient SBC configuration and to describe smolt movements and swimming behavior in the forebay. To do this, we used hydroacoustic and radiotelemetry techniques. The SBC was retrofitted onto the upstream face of the north half of the powerhouse to test the surface bypass method of diverting smolts from turbines. The SBC had three entrances, with mean velocities ranging from 0.37 to 1.92 m/s, and it discharged 113 m3/s through its outlet at Spill Bay 1, which was adjacent to the powerhouse. Different SBC configurations were created by altering the size and shape of entrances. During spring 1996 and 1997, river discharge was well above normal (123 and 154% of average, respectively). Powerhouse operations caused a strong downward component of flow upstream of the SBC. Many smolts (primarily steelhead and secondarily chinook salmon) were observed actively swimming upward in the water column. There were four times as many smolts diverted from turbines per unit volume of water with SBC flow than with spill flow, which indicated that the SBC may be an especially important bypass consideration in moderate- or low-flow years. The highest SBC efficiency (the proportion of total fish passing through the north half of the powerhouse by all routes that passed through the SBC) for any configuration tested was about 40%. Although no single SBC configuration stood out as the most efficient, the horizontal surface and maximum area configurations, or some combination of the two, are worth further investigation because they were moderately efficient.

Analysis of tank 7 surface supernatant sample (FTF-7-15-26) in support of corrosion control program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oji, L. N

2015-10-01

This report provides the results of analyses on Savannah River Site Tank 7 surface supernatant liquid sample in support of the Corrosion Control Program (CCP). The measured nitrate, nitrite and free-hydroxide concentrations for the Tank 7 surface sample averaged, 3.74E-01 ± 1.88E-03, 4.17E-01 ± 9.01E-03 and 0.602 ± 0.005 M, respectively. The Tank 7 surface cesium-137, sodium and silicon concentrations were, respectively, 3.99E+08, ± 3.25E+06 dpm/mL, 2.78 M and <3.10 mg/L. The measured aluminum concentration in the Tank 7 surface sample averaged 0.11 M.

Comprehensive Evaluation of Neuroprotection Achieved by Extended Selective Brain Cooling Therapy in a Rat Model of Penetrating Ballistic-Like Brain Injury

PubMed Central

Shear, Deborah A.; Deng-Bryant, Ying; Leung, Lai Yee; Wei, Guo; Chen, Zhiyong; Tortella, Frank C.

2016-01-01

Brain hypothermia has been considered as a promising alternative to whole-body hypothermia in treating acute neurological disease, for example, traumatic brain injury. Previously, we demonstrated that 2-hours selective brain cooling (SBC) effectively mitigated acute (≤24 hours postinjury) neurophysiological dysfunction induced by a penetrating ballistic-like brain injury (PBBI) in rats. This study evaluated neuroprotective effects of extended SBC (4 or 8 hours in duration) on sub-acute secondary injuries between 3 and 21 days postinjury (DPI). SBC (34°C) was achieved via extraluminal cooling of rats' bilateral common carotid arteries (CCA). Depending on the experimental design, SBC was introduced either immediately or with a 2- or 4-hour delay after PBBI and maintained for 4 or 8 hours. Neuroprotective effects of SBC were evaluated by measuring brain lesion volume, axonal injury, neuroinflammation, motor and cognitive functions, and post-traumatic seizures. Compared to untreated PBBI animals, 4 or 8 hours SBC treatment initiated immediately following PBBI produced comparable neuroprotective benefits against PBBI-induced early histopathology at 3 DPI as evidenced by significant reductions in brain lesion volume, axonal pathology (beta-amyloid precursor protein staining), neuroinflammation (glial fibrillary acetic protein stained-activated astrocytes and rat major histocompatibility complex class I stained activated microglial cell), and post-traumatic nonconvulsive seizures. In the later phase of the injury (7–21 DPI), significant improvement on motor function (rotarod test) was observed under most SBC protocols, including the 2-hour delay in SBC initiation. However, SBC treatment failed to improve cognitive performance (Morris water maze test) measured 13–17 DPI. The protective effects of SBC on delayed axonal injury (silver staining) were evident out to 14 DPI. In conclusion, the CCA cooling method of SBC produced neuroprotection measured across multiple

Effect of Pseudomonas sp. P7014 on the growth of edible mushroom Pleurotus eryngii in bottle culture for commercial production.

PubMed

Kim, Min Keun; Math, Renukaradhya K; Cho, Kye Man; Shin, Ki Jae; Kim, Jong Ok; Ryu, Jae San; Lee, Young Han; Yun, Han Dae

2008-05-01

Addition of bacterial culture strain P7014 and its supernatant to the mushroom growing media resulted in mushroom mycelia run faster. Mycelial growth rate of Pleurotus eryngii was increased up to 1.6 fold and primordial formation was induced one day earlier. Moreover, it was supposed that addition of bacteria had beneficial applications for commercial mushroom production, which appreciably reduced total number of days for cultivation of about 5+/-2 days compared with uninoculated, which took 55+/-2 days.

Plasmodium falciparum polypeptides released during in vitro cultivation*

PubMed Central

Da Silva, L. Rodriguez; Loche, M.; Dayal, R.; Perrin, L. H.

1983-01-01

Synchronous cultures of Plasmodium falciparum were successively labelled with (35S)-methionine and both the supernatants and the pellets of infected red blood cells were collected. The release of TCA-precipitable material in the culture supernatants was low during the development of ring forms and trophozoites, increased during schizogony, and was maximum at the time of schizont rupture and merozoite reinvasion. Analysis of the supernatants by SDS — PAGE and autoradiography showed that both polypeptides common to the various developmental stages of the parasite and schizont/merozoite-specific polypeptides were released. Polypeptides of relative molecular mass 140 000, 82 000 and, to a lower degree, 41 000 were present in high amounts in the culture supernatants. These polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the growth of P. falciparum cultures, and may be involved in protective immunity. The released polypeptides may also be used as target antigens in immunodiagnostic tests aiming at the detection of malaria infection. ImagesFig. 2AFig. 2BFig. 3 PMID:6340846

Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants

PubMed Central

De Nardi, Frédéric; Lefort, Claudie; Bréard, Dimitri; Richomme, Pascal; Legros, Christian; Guérineau, Nathalie C.

2017-01-01

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response. PMID:28993760

Characterization of Ferroplasma acidiphilum growing in pure and mixed culture with Leptospirillum ferriphilum.

PubMed

Merino, M P; Andrews, B A; Parada, P; Asenjo, J A

2016-11-01

Biomining is defined as biotechnology for metal recovery from minerals, and is promoted by the concerted effort of a consortium of acidophile prokaryotes, comprised of members of the Bacteria and Archaea domains. Ferroplasma acidiphilum and Leptospirillum ferriphilum are the dominant species in extremely acid environments and have great use in bioleaching applications; however, the role of each species in this consortia is still a subject of research. The hypothesis of this work is that F. acidiphilum uses the organic matter secreted by L. ferriphilum for growth, maintaining low levels of organic compounds in the culture medium, preventing their toxic effects on L. ferriphilum. To test this hypothesis, a characterization of Ferroplasma acidiphilum strain BRL-115 was made with the objective of determining its optimal growth conditions. Subsequently, under the optimal conditions, L. ferriphilum and F. acidiphilum were tested growing in each other's supernatant, in order to define if there was exchange of metabolites between the species. With these results, a mixed culture in batch cyclic operation was performed to obtain main specific growth rates, which were used to evaluate a mixed metabolic model previously developed by our group. It was observed that F. acidiphilum, strain BRL-115 is a chemomixotrophic organism, and its growth is maximized with yeast extract at a concentration of 0.04% wt/vol. From the experiments of L. ferriphilum growing on F. acidiphilum supernatant and vice versa, it was observed that in both cases cell growth is favorably affected by the presence of the filtered medium of the other microorganism, proving a synergistic interaction between these species. Specific growth rates were obtained in cyclic batch operation of the mixed culture and were used as input data for a Flux Balance Analysis of the mixed metabolic model, obtaining a reasonable behavior of the metabolic fluxes and the system as a whole, therefore consolidating the model

Engineering of an MBR supernatant fouling layer by fine particles addition: a possible way to control cake compressibility.

PubMed

Teychene, Benoît; Guigui, Christelle; Cabassud, Corinne

2011-02-01

For membrane bioreactors (MBR) applied to wastewater treatment membrane fouling is still the prevalent issue. The main limiting phenomena related to fouling is a sudden jump of the transmembrane pressure (TMP) often attributed to the collapse of the fouling layer. Among existing techniques to avoid or to delay this collapse, the addition of active particles membrane fouling reducers (polymer, resins, powdered activated carbon (PAC), zeolithe...) showed promising results. Thus the main objective of this work is to determine if fouling can be reduced by inclusion of inert particles (500 nm and inert compared to other fouling reducers) and which is the impact on filtration performances of the structuring of the fouling. Those particles were chosen for their different surface properties and their capability to form well structured layer. Results, obtained at constant pressure in dead end mode, show that the presence of particles changes foulant deposition and induces non-compressible fouling (in the range of 0.5-1 bar) and higher rejection values compared to filtration done on supernatant alone. Indeed dead end filtration tests show that whatever interactions between biofluid and particles, the addition of particles leads to better filtration performances (in terms of rejection, and fouling layer compressibility). Moreover results confirm the important role played by macromolecular compounds, during supernatant filtration, creating highly compressible and reversible fouling. In conclusion, this study done at lab-scale suggests the potential benefit to engineer fouling structure to control or to delay the collapse of the fouling layer. Finally this study offers the opportunities to enlarge the choice of membrane fouling reducers by taking into consideration their ability to form more consistent fouling (i.e. rigid, structured fouling). Copyright © 2010 Elsevier Ltd. All rights reserved.

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

PubMed

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.

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

PubMed Central

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

The speciation of soluble sulphur compounds in bacterial culture fluids by X-ray absorption near edge structure spectroscopy.

PubMed

Franz, Bettina; Lichtenberg, Henning; Hormes, Josef; Dahl, Christiane; Prange, Alexander

2009-11-01

Over the last decade X-ray absorption near edge structure (XANES) spectroscopy has been used in an increasing number of microbiological studies. In addition to other applications it has served as a valuable tool for the investigation of the sulphur globules deposited intra- or extracellularly by certain photo- and chemotrophic sulphur-oxidizing (Sox) bacteria. For XANES measurements, these deposits can easily be concentrated by filtration or sedimentation through centrifugation. However, during oxidative metabolism of reduced sulphur compounds, such as sulphide or thiosulphate, sulphur deposits are not the only intermediates formed. Soluble intermediates such as sulphite may also be produced and released into the medium. In this study, we explored the potential of XANES spectroscopy for the detection and speciation of sulphur compounds in culture supernatants of the phototrophic purple sulphur bacterium Allochromatium vinosum. More specifically, we investigated A. vinosum DeltasoxY, a strain with an in frame deletion of the soxY gene. This gene encodes an essential component of the thiosulphate-oxidizing Sox enzyme complex. Improved sample preparation techniques developed for the DeltasoxY strain allowed for the first time not only the qualitative but also the quantitative analysis of bacterial culture supernatants by XANES spectroscopy. The results thus obtained verified and supplemented conventional HPLC analysis of soluble sulphur compounds. Sulphite and also oxidized organic sulphur compounds were shown by XANES spectroscopy to be present, some of which were not seen when standard HPLC protocols were used.

Effect of localizing fruit and vegetable consumption on greenhouse gas emissions and nutrition, Santa Barbara County.

PubMed

Cleveland, David A; Radka, Corie N; Müller, Nora M; Watson, Tyler D; Rekstein, Nicole J; Wright, Hannah Van M; Hollingshead, Sydney E

2011-05-15

The US agrifood system is very productive, but highly centralized and resource intensive with very weak links between production and consumption. This contributes to high levels of malnutrition and greenhouse gas emissions (GHGE). A popular approach to improvement is localization-reducing direct transport (farm to retail distance, or "food miles"). We examined Santa Barbara County (SBC) California, which mirrors the high production, nutritional and environmental problems, and growing localization movement of California. SBC ranks in the top 1% of US counties in value of agricultural products, and >80% of this value is produce (fruits and vegetables). We calculated the amount of produce grown in and consumed in SBC and estimated that >99% of produce grown in SBC is exported from the county, and >95% of produce consumed in SBC is imported. If all produce consumed in SBC was grown in the county (100% localization), it would reduce GHGE from the agrifood system <1%, and not necessarily affect nutrition. While food miles capture only a portion of the environmental impact of agrifood systems, localization could be done in ways that promote synergies between improving nutrition and reducing GHGE, and many such efforts exist in SBC.

A novel suction/coagulation integrated probe for achieving better hemostasis: development and clinical use.

PubMed

Takahashi, Hidekazu; Haraguchi, Naotsugu; Nishimura, Junichi; Hata, Taishi; Matsuda, Chu; Yamamoto, Hirofumi; Mizushima, Tsunekazu; Mori, Masaki; Doki, Yuichiro; Nakajima, Kiyokazu

2018-06-01

Modern electrosurgical tools have a specific coagulation mode called "soft coagulation". However, soft coagulation has not been widely accepted for surgical operations. To optimize the soft coagulation environment, we developed a novel suction device integrated with an electrosurgical probe, called the "Suction ball coagulator" (SBC). In this study, we aimed to optimize the SBC design with a prototyping process involving a bench test and preclinical study; then, we aimed to demonstrate the feasibility, safety, and potential effectiveness of the SBC for laparoscopic surgery in clinical settings. SBC prototyping was performed with a bench test. Device optimization was performed in a preclinical study with a domestic swine bleeding model. Then, SBC was tested in a clinical setting during 17 clinical laparoscopic colorectal surgeries. In the bench tests, two tip hole sizes and patterns showed a good suction capacity. The preclinical study indicated the best tip shape for accuracy. In clinical use, no device-related adverse event was observed. Moreover, the SBC was feasible for prompt hemostasis and blunt dissections. In addition, SBC could evacuate vapors generated by tissue ablation using electroprobe during laparoscopic surgery. We successfully developed a novel, integrated suction/coagulation probe for hemostasis and commercialized it.

The effects of cetrorelix and triptorelin on the viability and steroidogenesis of cultured human granulosa luteinized cells.

PubMed

Metallinou, Chryssa; Köster, Frank; Diedrich, Klaus; Nikolettos, Nikos; Asimakopoulos, Byron

2012-01-01

We investigated the effects of the gonadotropin-releasing hormone (GnRH) agonist triptorelin as well the GnRH antagonist cetrorelix those of on the viability and steroidogenesis in human granulosa luteinized (hGL) cell cultures. The hGL cells were obtained from 34 women undergoing ovarian stimulation for IVF treatment. The cells were cultured for 48 h with or without 1 nM or 3 nM of cetrorelix or triptorelin in serum-free media. The cell viability was evaluated by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The concentrations of estradiol and progesterone in culture supernatants were measured by ELISA. Treatment with triptorelin slightly increased cell viability, whereas treatment with 3 nM cetrorelix led to a significant decrease. Estradiol concentrations were reduced with 3 nM triptorelin. Cultures treated with high-dose of either cetrorelix or triptorelin tended to secrete less progesterone than controls. Cetrorelix significantly reduces the viability of hGL cells. Triptorelin and cetrorelix may have minor effects on steroidogenesis. These results suggest that GnRH analogues may influence ovarian functions.

Identification of a Serratia marcescens virulence factor that promotes hemolymph bleeding in the silkworm, Bombyx mori.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Hara, Takashi; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-03-01

Injection of culture supernatant of Serratia marcescens, a Gram-negative bacterium pathogenic to a wide range of host animals including insects and mammals, into the hemolymph of silkworm (Bombyx mori) larvae led to continuous flow of the hemolymph (blood of insects) from the injection site. The amount of hemolymph lost within 60 min reached 15-20% of the total larval weight. Using a bioassay with live silkworms, we purified Serralysin, a metalloprotease that requires divalent cations for its activity, as the factor responsible for the promotion of hemolymph bleeding from the culture supernatant of S. marcescens. Recombinant protein also induced hemolymph bleeding in silkworms. Moreover, the culture supernatant of an S. marcescens disruption mutant of the ser gene showed attenuated ability to promote hemolymph bleeding. In addition, this bleeding-promoting activity of the S. marcescens culture supernatant was attenuated by disruption of the wecA gene, which is involved in the biosynthesis of the lipopolysaccharide O-antigen. These findings suggest that Serralysin metalloprotease contributes to the pathogenesis of S. marcescens by inhibiting wound healing, which leads to a massive loss of hemolymph from silkworm larvae. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed Central

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

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

PubMed

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.

Synthesis and self-assembly behavior of a biodegradable and sustainable soybean oil-based copolymer nanomicelle

NASA Astrophysics Data System (ADS)

Bao, Lixia; Bian, Longchun; Zhao, Mimi; Lei, Jingxin; Wang, Jiliang

2014-08-01

Herein, we report a novel amphiphilic biodegradable and sustainable soybean oil-based copolymer (SBC) prepared by grafting hydrophilic and biocompatible hydroxyethyl acrylate (HEA) polymeric segments onto the natural hydrophobic soybean oil chains. FTIR, H1-NMR, and GPC measurements have been used to investigate the molecular structure of the obtained SBC macromolecules. Self-assembly behaviors of the prepared SBC in aqueous solution have also been extensively evaluated by fluorescence spectroscopy and transmission electron microscopy. The prepared SBC nanocarrier with the size range of 40 to 80 nm has a potential application in the biomedical field.

Miniaturized Mass-Spectrometry-Based Analysis System for Fully Automated Examination of Conditioned Cell Culture Media

PubMed Central

Weber, Emanuel; Pinkse, Martijn W. H.; Bener-Aksam, Eda; Vellekoop, Michael J.; Verhaert, Peter D. E. M.

2012-01-01

We present a fully automated setup for performing in-line mass spectrometry (MS) analysis of conditioned media in cell cultures, in particular focusing on the peptides therein. The goal is to assess peptides secreted by cells in different culture conditions. The developed system is compatible with MS as analytical technique, as this is one of the most powerful analysis methods for peptide detection and identification. Proof of concept was achieved using the well-known mating-factor signaling in baker's yeast, Saccharomyces cerevisiae. Our concept system holds 1 mL of cell culture medium and allows maintaining a yeast culture for, at least, 40 hours with continuous supernatant extraction (and medium replenishing). The device's small dimensions result in reduced costs for reagents and open perspectives towards full integration on-chip. Experimental data that can be obtained are time-resolved peptide profiles in a yeast culture, including information about the appearance of mating-factor-related peptides. We emphasize that the system operates without any manual intervention or pipetting steps, which allows for an improved overall sensitivity compared to non-automated alternatives. MS data confirmed previously reported aspects of the physiology of the yeast-mating process. Moreover, matingfactor breakdown products (as well as evidence for a potentially responsible protease) were found. PMID:23091722

Analysis of Tank 38H (HTF-38-15-47, 49) and Tank 43H (HTF-43-15-51, 53) surface and subsurface supernatant samples in support of enrichment and corrosion control programs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oji, L. N.

This report provides the results of analyses on Tanks 38H and 43H surface and subsurface supernatant liquid samples in support of the Enrichment Control Program (ECP) and the Corrosion Control Program (CCP).

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

PubMed

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.

Metabolism of 13-cis-retinoic acid by a rat liver 9000g supernatant preparation.

PubMed

Vane, F M; Buggé, C J; Williams, T H

1982-01-01

The in vitro metabolites formed on incubation of 13-cis-retinoic acid (13-cis-RA, isotretinoin) with a 9000g rat liver supernatant system were isolated by HPLC and identified by their mass and NMR spectra. The major metabolic pathway was hydroxylation at C4 to give 4-hydroxy-13-cis-RA, which was rapidly oxidized to 4-oxo-13-cis-RA, the major isolated metabolite. Further metabolism of this 4-oxo metabolite led to two novel compounds, 2-hydroxy-4-oxo-13-cis-RA and 3-hydroxy-4-oxo-13-cis-RA. In addition, small amounts of 13-cis-RA and 4-oxo-13-cis-RA were enzymatically converted to their all-trans isomers. Support for these pathways was obtained by the metabolism of reference samples of 4-hydroxy-13-cis-RA, 4-oxo-13-cis-RA, all-trans-RA, and 4-oxo-all-trans-RA. The predominant formation of 4-oxo metabolites of 13-cis-RA in this in vitro rat system and the results from previously reported in vivo metabolism studies suggest that oxidation at C4 is a major metabolic pathway of 13-cis-RA in both rats and humans.

Effect of adsorbants on in vitro biohydrogenation of 22:6n-3 by mixed cultures of rumen microorganisms.

PubMed

Escobar, M; Vlaeminck, B; Jeyanathan, J; Thanh, L P; Shingfield, K J; Wallace, R J; Fievez, V

2016-09-01

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.

Oil cakes - a by-product of agriculture industry as a fortificant in bakery products.

PubMed

Behera, Satyabadi; Indumathi, K; Mahadevamma, S; Sudha, M L

2013-11-01

Groundnut cake (GNC) and soybean cake (SBC) by-product of agriculture industry had protein and protein digestibility in the range of 42.7-50.5 and 71.3-76.8%, respectively. Polyphenols present in GNC and SBC were cholorogenic acid, syringic acid and p-coumaric acid. The number of bands separated in soybean meal was greater than the bands observed in GNC flour as seen in SDS-PAGE pattern, respectively. SEM of groundnut flour showed distension of protein bodies due to roasting of the oil cakes. The water absorption of wheat flour GNC blends decreased from 59.2 to 57.3% and increased in wheat flour SBC blends from 59.2 to 68.3% with an increase in oil cake from 0 to 20%. With increase in either GNC or SBC, the biscuits became harder. Addition of glycerol monostearate and sodium stearoyl lactylate in combination with 20% blend of GNC/SBC decreased the breaking strength values and increased the sensory parameters of the biscuits. Nutritionally rich biscuits were thus prepared by incorporating GNC/SBC.

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

PubMed Central

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) against Pseudomonas aeruginosa PTCC 1430 was evaluated. Materials and Methods: 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. Results: 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). Conclusions: Our findings indicate that probiotic bacterial

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

PubMed

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

78 FR 12062 - Nominations to the Advisory Committee on Blood and Tissue Safety and Availability

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-21

... Committee is the responsibility of the OASH. The qualified individuals will be nominated to the Secretary of... Mr. James Berger, Senior Advisor for Blood Policy; Division of Blood and Tissue Safety and...: (240) 453-8803. FOR FURTHER INFORMATION CONTACT: Mr. James Berger, Senior Advisor for Blood Policy...

Experimental Clostridium perfringens type D enterotoxemia in goats.

PubMed

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.

Increased expression of Fas receptor and Fas ligand in the culture of the peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato.

PubMed

Grygorczuk, Sambor; Osada, Joanna; Moniuszko, Anna; Świerzbińska, Renata; Kondrusik, Maciej; Zajkowska, Joanna; Dunaj, Justyna; Dąbrowska, Milena; Pancewicz, Sławomir

2015-03-01

Apoptosis of the lymphocytes plays an essential role in the regulation of inflammatory/immune responses and its abnormalities may contribute to a chronic infection, persistent inflammation and autoimmunity. Its role in the pathogenesis of the late Lyme borreliosis manifestations has not been studied so far. We have measured Th lymphocyte apoptosis rate, membrane expression of pro-apoptotic Fas receptor, and supernatant concentrations of selected soluble pro- and anti-apoptotic mediators in cultures of peripheral blood mononuclear cells from 16 patients with disseminated Lyme borreliosis (6 with osteoarticular symptoms, 7 with neuroborreliosis and 3 with acrodermatitis chronica atrophicans) and 8 healthy controls. The cultures stimulated for 48h with live Borrelia burgdorferi sensu stricto, B. garinii or B. afzelii spirochetes. Fraction of the apoptotic Th (CD3+CD4+) lymphocytes and expression of Fas in this cell population was measured cytometrically and concentrations of soluble Fas, soluble Fas ligand, IL-10, IL-12 and TGF-β in culture supernatant with ELISA assays. The expression of IL-10, soluble and membrane Fas and soluble Fas ligand was increased under stimulation and higher in the presence of B. burgdorferi sensu stricto than the other species. Apoptosis rate was not affected. There was no difference between Lyme borreliosis patients and controls. IL-10 concentration correlated negatively with the membrane Fas expression and apoptosis under stimulation with B. afzelii and B. garinii. Expression of Fas/FasL system is up-regulated under stimulation with B. burgdorferi, but without corresponding increase in lymphocyte apoptosis. Variable responses observed with different B. burgdorferi species may reflect differences in the pathogenesis of the infection in vivo. Copyright © 2014 Elsevier GmbH. All rights reserved.

Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

PubMed Central

Lee, Sun-og; Kato, Junichi; Takiguchi, Noboru; Kuroda, Akio; Ikeda, Tsukasa; Mitsutani, Atsushi; Ohtake, Hisao

2000-01-01

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-Mw-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium. PMID:11010878

Production stability of active polysaccharides of Dendrobium huoshanense using long-term cultures of protocorm-like bodies.

PubMed

Zha, Xue-Qiang; Luo, Jian-Ping

2008-01-01

In this study, the production stability of active polysaccharides in protocorm-like bodies (PLBs) induced from the seedling segments of Dendrobium huoshanense C. Z. Tang et S. J. Cheng was investigated during long-term subculture. Subcultures were conducted once every 30 days. With an average inoculum of 39 g/L fresh PLBs, the increase in biomass ranged from 95.7 g/L to 103.9 g/L in fresh weight and 3.2 g/L to 3.4 g/L in dry weight during eighteen continuous subcultures while polysaccharide content in PLBs was from 0.8 mg/g Fw (mg polysaccharide per gram PLBs in fresh weight) to 1.0 mg/g Fw. In addition, polysaccharides from all cultures showed a similar potential of stimulating interferon gamma (IFN-gamma) release in the supernatant of splenocytes and tumor necrosis factor alpha (TNF-alpha) release in the supernatant of peritoneal macrophages. To elucidate the genetic basis of polysaccharide production stability in long-term subculture of PLBs, the genetic fingerprints by RAPD were further analyzed using plantlets from PLB development. Results showed that there is no evidence of genetic variation both within the plantlets from the different subcultures of PLBs and between long-term subcultures and the donor plants.

Effects of lactoferrin on the production of interferon-λ by the human intestinal epithelial cell line HT-29.

PubMed

Shin, Kouichirou; Oda, Hirotsugu; Wakabayashi, Hiroyuki; Yamauchi, Koji; Abe, Fumiaki

2017-02-01

We examined the in-vitro effects of bovine lactoferrin (LF) on the production of interferon-λ (IFN-λ), an antiviral cytokine important for the defense of enterocytes, using the human intestinal epithelial cell line HT-29. HT-29 cell cultures were treated with LF for 1 h, and the cultures were stimulated with polyinosinic-polycytidylic acid (poly I:C). LF increased the concentration of IFN-λ in the culture supernatant after stimulation in a dose-dependent manner. A similar increase in the concentration of IFN-λ was observed in the supernatant of cells washed between treatment with LF and stimulation with poly I:C. At 6 and 24 h after stimulation with poly I:C (early and late phases, respectively) treated cultures contained significantly higher concentrations of IFN-λ1 in the culture supernatant, and significantly higher IFN-λ1 and IFN-λ2 mRNA levels, than controls. These results suggest that LF activates the innate cellular immunity of the enterocytes to double-stranded RNA and increases the production of IFN-λ.

[The specific bronchial provocation test in the diagnosis of occupational asthma].

PubMed

Fraj, J; Duce, F; Lezaun, A; Colás, C; Domínguez, M A; Abadía, M C

1997-10-01

Specific bronchial challenge (SBC) testing is a key technique for diagnosing the origin of occupational asthma (OA). SBC is indicated in specific circumstances, including whenever several agents present in the work environment may be the cause of OA, when new or unusual occupational agents need to be identified, when evidence for legal action is required, or when research is conducted. SBC procedures are not standardized, because of the great diversity of occupational agents and the variety of physical and chemical properties involved. Thus, SBC testing with agents found in fumes, gases or vapors can be administered in special cabins or in closed circuits with continuous monitoring of sub-irritant concentrations. Agents found in dust, most but not all of which have high molecular weights, may be appropriate for routine SBC testing in an allergy laboratory. This paper will treat only these cases. SBC must be formed in specialized centers by experienced personnel, as it is a sophisticated and potentially dangerous technique. We describe a series of 20 patients diagnosed of OA in our unit over the past two years in whom SBC provided an etiologic diagnosis. All were exposed to dust or aerosols at work. The cause was a substance of high molecular weight in 17 cases, and low molecular weight in 3. The procedure used is described and models of bronchial response are discussed.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.

1985-08-01

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. (/sup 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 inducedmore » a concentration-dependent inhibition of (/sup 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.« less

Differential effects of catecholamines on in vitro growth of pathogenic bacteria

NASA Technical Reports Server (NTRS)

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.

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

PubMed

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.

The Reproducibility of Blood Acid Base Responses in Male Collegiate Athletes Following Individualised Doses of Sodium Bicarbonate: A Randomised Controlled Crossover Study.

PubMed

Gough, Lewis A; Deb, Sanjoy K; Sparks, Andy S; McNaughton, Lars R

2017-10-01

Current evidence suggests sodium bicarbonate (NaHCO 3 ) should be ingested based upon the individualised alkalotic peak of either blood pH or bicarbonate (HCO 3 - ) because of large inter-individual variations (10-180 min). If such a strategy is to be practical, the blood analyte response needs to be reproducible. This study aimed to evaluate the degree of reproducibility of both time to peak (TTP) and absolute change in blood pH, HCO 3 - and sodium (Na + ) following acute NaHCO 3 ingestion. Male participants (n = 15) with backgrounds in rugby, football or sprinting completed six randomised treatments entailing ingestion of two doses of 0.2 g·kg -1 body mass (BM) NaHCO 3 (SBC2a and b), two doses of 0.3 g·kg -1 BM NaHCO 3 (SBC3a and b) or two control treatments (CON1a and b) on separate days. Blood analysis included pH, HCO 3 - and Na + prior to and at regular time points following NaHCO 3 ingestion over a 3-h period. HCO 3 - displayed greater reproducibility than pH in intraclass correlation coefficient (ICC) analysis for both TTP (HCO 3 - SBC2 r = 0.77, P = 0.003; SBC3 r = 0.94, P < 0.001; pH SBC2 r = 0.62, P = 0.044; SBC3 r = 0.71, P = 0.016) and absolute change (HCO 3 - SBC2 r = 0.89, P < 0.001; SBC3 r = 0.76, P = 0.008; pH SBC2 r = 0.84, P = 0.001; SBC3 r = 0.62, P = 0.041). Our results indicate that both TTP and absolute change in HCO 3 - is more reliable than pH. As such, these data provide support for an individualised NaHCO 3 ingestion strategy to consistently elicit peak alkalosis before exercise. Future work should utilise an individualised NaHCO 3 ingestion strategy based on HCO 3 - responses and evaluate effects on exercise performance.

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi.

PubMed Central

Schirmböck, M; Lorito, M; Wang, Y L; Hayes, C K; Arisan-Atac, I; Scala, F; Harman, G E; Kubicek, C P

1994-01-01

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens. Images PMID:7811076

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.

PubMed

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.

Dynamic study of Cr(VI) removal performance and mechanism from water using multilayer material coated nanoscale zerovalent iron.

PubMed

Wu, Bin; Peng, Dinghua; Hou, Siyu; Tang, Bicong; Wang, Can; Xu, Heng

2018-05-16

In this study, the dynamic Cr(VI) removal process from water by the synthesized multilayer material coated nanoscale zerovalent iron (SBC-nZVI) was systematically discussed at different treatment conditions. The results showed that initial pH, contact time, Cr(VI) concentration and the dosage of SBC-nZVI were important parameters that influenced the Cr(VI) removal efficiency. The major Cr(VI) removal occurred within 60 min and gradually tend to equilibrium with consistent treatment. The removal efficiency was highly depended on pH values and the adsorption kinetics agreed well with the pseduo-second-order model (PSO). When the initial Cr(VI) concentration was below 15 mg/L, the removal rate could reach to about 100%. Moreover, the removal efficiency increased with the increase of SBC-nZVI dosage, which related to the increase of reactive sites. To understand the removal mechanism, SBC-nZVI before and after reaction with Cr(VI) were characterized by fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), and X-ray photoelectron spectroscopy (XPS). These analysis showed that the interaction of SBC-nZVI with Cr(VI) was mainly controlled by reduction and electrostatic attraction. Therefore, these results explained the interaction between Cr(VI) and SBC-nZVI material in detail, and further proved that SBC-nZVI could be an effective material to remove Cr(VI) from water. Copyright © 2018. Published by Elsevier Ltd.

Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

PubMed Central

Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q.; Chen, Wei

2015-01-01

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. PMID:25789875

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

PubMed

de Michele, F; Poels, J; Weerens, L; Petit, C; Evrard, Z; Ambroise, J; Gruson, D; Wyns, C

2017-01-01

Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm 3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either

Secondary Breast Cancer Risk by Radiation Volume in Women With Hodgkin Lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conway, Jessica L.; Department of Surgery, University of British Columbia, Vancouver, British Columbia; Connors, Joseph M.

Purpose: To determine whether the risk of secondary breast cancer (SBC) is reduced in women with Hodgkin lymphoma (HL) treated with smaller field radiation therapy (SFRT) versus mantle field radiation therapy (MRT). Methods and Materials: We used the BC Cancer Agency (BCCA) Lymphoid Cancer Database to identify female patients treated for HL between January 1961 and December 2009. Radiation therapy volumes were categorized as MRT or SFRT, which included involved field, involved site, or involved nodal radiation therapy. SBC risk estimates were compared using competing risk analysis and Fine and Gray multivariable model: MRT ± chemotherapy, SFRT ± chemotherapy, or chemotherapy-only. Results: Of 734 eligible patients,more » 75% of the living patients have been followed up for more than 10 years, SBC has developed in 54, and 15 have died of breast cancer. The 20-year estimated risks (competing risk cumulative incidence) for SBC differed significantly: MRT 7.5% (95% confidence interval [CI] 4.4%-11.5%), SFRT 3.1% (95% CI 1.0%-7.7%), and chemotherapy-only 2.2% (95% CI 1.0%-4.8%) (P=.01). Using a Fine and Gray model to control for death and patients lost to follow-up, MRT was associated with a higher risk of SBC (hazard ratio [HR] = 2.9; 95% CI 1.4%-6.0%; P=.004) compared with chemotherapy-only and with SFRT (HR = 3.3; 95% CI 1.3%-8.4%; P=.01). SFRT was not associated with a greater risk of SBC compared with chemotherapy-only (HR = 0.87; 95% CI 0.28%-2.66%; P=.80). Conclusion: This study confirms that large-volume MRT is associated with a markedly increased risk of SBC; however, more modern small-volume RT is not associated with a greater risk of SBC than chemotherapy alone.« less

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

PubMed Central

Ontweka, Lameck N.; Deng, Lul O.; Rauzier, Jean; Debes, Amanda K.; Tadesse, Fisseha; Parker, Lucy A.; Wamala, Joseph F.; Bior, Bior K.; Lasuba, Michael; But, Abiem Bona; Grandesso, Francesco; Jamet, Christine; Cohuet, Sandra; Ciglenecki, Iza; Serafini, Micaela; Sack, David A.; Quilici, Marie-Laure; Azman, Andrew S.; Luquero, Francisco J.

2016-01-01

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited. PMID:27992488

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

PubMed Central

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

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.

PubMed

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mokhtari, Narges; Daneshpajouh, Shahram; Seyedbagheri, Seyedali

This study has investigated different visible-light irradiation's effect on the formation of silver nanoparticles from silver nitrate using the culture supernatant of Klebsiella pneumonia. Our study shows that visible-light emission can significantly prompt the synthesis of silver nanoparticles. Also, the study experimentally investigated the liquid mixing process effect on silver nanoparticle synthesis by visible-light irradiation. This study successfully synthesized uniformly dispersed silver nanoparticles with a uniform size and shape in the range of 1-6 nm with an average size of 3 nm. Furthermore, the study investigated the mechanism of the reduction of silver ions by culture supernatant of K. pneumonia,more » and used X-ray diffraction to characterize silver chloride as an intermediate compound. Silver chloride was prepared synthetically and used as a substrate for the synthesis of silver nanoparticles by culture supernatant of K. pneumonia. The silver nanoparticles have been prepared from silver chloride during this investigation for the first time.« less

Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe.

PubMed

Hernandez, Frank J; Huang, Lingyan; Olson, Michael E; Powers, Kristy M; Hernandez, Luiza I; Meyerholz, David K; Thedens, Daniel R; Behlke, Mark A; Horswill, Alexander R; McNamara, James O

2014-03-01

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2'-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens.

Determining the best treatment for simple bone cyst: a decision analysis.

PubMed

Lee, Seung Yeol; Chung, Chin Youb; Lee, Kyoung Min; Sung, Ki Hyuk; Won, Sung Hun; Choi, In Ho; Cho, Tae-Joon; Yoo, Won Joon; Yeo, Ji Hyun; Park, Moon Seok

2014-03-01

The treatment of simple bone cysts (SBC) in children varies significantly among physicians. This study examined which procedure is better for the treatment of SBC, using a decision analysis based on current published evidence. A decision tree focused on five treatment modalities of SBC (observation, steroid injection, autologous bone marrow injection, decompression, and curettage with bone graft) were created. Each treatment modality was further branched, according to the presence and severity of complications. The probabilities of all cases were obtained by literature review. A roll back tool was utilized to determine the most preferred treatment modality. One-way sensitivity analysis was performed to determine the threshold value of the treatment modalities. Two-way sensitivity analysis was utilized to examine the joint impact of changes in probabilities of two parameters. The decision model favored autologous bone marrow injection. The expected value of autologous bone marrow injection was 0.9445, while those of observation, steroid injection, decompression, and curettage and bone graft were 0.9318, 0.9400, 0.9395, and 0.9342, respectively. One-way sensitivity analysis showed that autologous bone marrow injection was better than that of decompression for the expected value when the rate of pathologic fracture, or positive symptoms of SBC after autologous bone marrow injection, was lower than 20.4%. In our study, autologous bone marrow injection was found to be the best choice of treatment of SBC. However, the results were sensitive to the rate of pathologic fracture after treatment of SBC. Physicians should consider the possibility of pathologic fracture when they determine a treatment method for SBC.

Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures.

PubMed

Heinrich, A; Balszuweit, F; Thiermann, H; Kehe, K

2009-12-15

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180-200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISA(plus) (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant. To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISA(plus) and the ToxiLight Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300 microM for 30 min, Lewisite (L) 60 microM for 5 min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent. This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.

Analysis of the Anti-Cancer Effects of Cincau Extract (Premna oblongifolia Merr) and Other Types of Non-Digestible Fibre Using Faecal Fermentation Supernatants and Caco-2 Cells as a Model of the Human Colon.

PubMed

Nurdin, Samsu U; Le Leu, Richard K; Young, Graeme P; Stangoulis, James C R; Christophersen, Claus T; Abbott, Catherine A

2017-04-03

Green cincau ( Premna oblongifolia Merr) is an Indonesian food plant with a high dietary fibre content. Research has shown that dietary fibre mixtures may be more beneficial for colorectal cancer prevention than a single dietary fibre type. The aim of this study was to investigate the effects of green cincau extract on short chain fatty acid (SCFA) production in anaerobic batch cultures inoculated with human faecal slurries and to compare these to results obtained using different dietary fibre types (pectin, inulin, and cellulose), singly and in combination. Furthermore, fermentation supernatants (FSs) were evaluated in Caco-2 cells for their effect on cell viability, differentiation, and apoptosis. Cincau increased total SCFA concentration by increasing acetate and propionate, but not butyrate concentration. FSs from all dietary fibre sources, including cincau, reduced Caco-2 cell viability. However, the effects of all FSs on cell viability, cell differentiation, and apoptosis were not simply explainable by their butyrate content. In conclusion, products of fermentation of cincau extracts induced cell death, but further work is required to understand the mechanism of action. This study demonstrates for the first time that this Indonesian traditional source of dietary fibre may be protective against colorectal cancer.

Analysis of the Anti-Cancer Effects of Cincau Extract (Premna oblongifolia Merr) and Other Types of Non-Digestible Fibre Using Faecal Fermentation Supernatants and Caco-2 Cells as a Model of the Human Colon

PubMed Central

Nurdin, Samsu U.; Le Leu, Richard K.; Young, Graeme P.; Stangoulis, James C. R.; Christophersen, Claus T.; Abbott, Catherine A.

2017-01-01

Green cincau (Premna oblongifolia Merr) is an Indonesian food plant with a high dietary fibre content. Research has shown that dietary fibre mixtures may be more beneficial for colorectal cancer prevention than a single dietary fibre type. The aim of this study was to investigate the effects of green cincau extract on short chain fatty acid (SCFA) production in anaerobic batch cultures inoculated with human faecal slurries and to compare these to results obtained using different dietary fibre types (pectin, inulin, and cellulose), singly and in combination. Furthermore, fermentation supernatants (FSs) were evaluated in Caco-2 cells for their effect on cell viability, differentiation, and apoptosis. Cincau increased total SCFA concentration by increasing acetate and propionate, but not butyrate concentration. FSs from all dietary fibre sources, including cincau, reduced Caco-2 cell viability. However, the effects of all FSs on cell viability, cell differentiation, and apoptosis were not simply explainable by their butyrate content. In conclusion, products of fermentation of cincau extracts induced cell death, but further work is required to understand the mechanism of action. This study demonstrates for the first time that this Indonesian traditional source of dietary fibre may be protective against colorectal cancer. PMID:28368356

Develoment and Evaluation of Immunomodulators of Hemopoietic and Immunologic Mechanisms.

DTIC Science & Technology

1991-07-02

secretion was much more marked : in the presence of IL-4 cultuie supernatants. these effects were dissociated to some culture supernatants had average IgE...required to induce IgE secretion. and 25. Cobbold . S.. Hale. G. & Waldman. H. 11987) in Leukocvte Tvpine 4 iii) these effects are not inhibited by a

Suicide by cop: clinical risks and subtypes.

PubMed

Dewey, Lauren; Allwood, Maureen; Fava, Joanna; Arias, Elizabeth; Pinizzotto, Anthony; Schlesinger, Louis

2013-01-01

This study examines whether clinical classification schemes from general suicide research are applicable for cases of suicide by cop (SbC) and whether there are indicators as to why the police might be engaged in the suicide. Using archival law enforcement data, 13 clinical risks were examined among 68 cases of SbC using exploratory factor analysis and k-means cluster analysis. Three subtypes of SbC cases emerged: Mental Illness, Criminality, and Not Otherwise Specified. The subtypes varied significantly on their levels of mental illness, substance use, and criminal activity. Findings suggest that reducing fragmentation between law enforcement and mental health service providers might be a crucial goal for suicide intervention and prevention, at least among cases of SbC.

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

PubMed Central

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 Ca2+ 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 Ca2+ 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 Ca2+ 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. PMID:21104039

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

PubMed

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.

Comparison of effects on macrophage cultures of glass fibre, glass powder, and chrysotile asbestos

PubMed Central

Beck, E. G.; Holt, P. F.; Manojlović, N.

1972-01-01

Beck, E. G., Holt, P. F., and Manojlović, N. (1972).Brit. J. industr. Med.,29, 280-286. Comparison of effects on macrophage cultures of glass fibre, glass powder, and chrysotile asbestos. The effects on macrophage cultures of glass fibre, glass powder, and chrysotile asbestos are compared. Glass fibre behaves like chrysotile in producing an increase in cell membrane permeability in cultured macrophages. This is demonstrable by the increase in lactic dehydrogenase activity in the supernatant fluid. The metabolism, measured by lactate production, is not reduced as it is when quartz is phagocytosed. Glass powder behaves like the inert dust corundum, producing little change in the number of cells stained by erythrosin B and a small increase in lactate dehydrogenase activity, both being in the range of the control. There is an increase in lactate production as a result of higher metabolism due to phagocytosis. Dusts may produce two basic effects, namely a toxic effect and change in cell membrane permeability. A non-specific effect on the cell membrane due to the slow and sometimes incomplete process of ingestion of long fibres is probably a function of the morphology, particularly the length of the fibres. A primary specific effect induced by some dusts immediately follows contact with the cell membrane. Images PMID:4339803

Cereal β-glucan quantification with calcofluor-application to cell culture supernatants.

PubMed

Rieder, Anne; Knutsen, Svein H; Ballance, Simon; Grimmer, Stine; Airado-Rodríguez, Diego

2012-11-06

The specific binding of the fluorescent dye calcofluor to cereal β-glucan results in increased fluorescence intensity of the formed complex and is in use for the quantification of β-glucan above a critical molecular weight (MW) by flow injection analysis. In this study, this method was applied in a fast and easy batch mode. In order to emphasize the spectral information of the emission spectra of the calcofluor/β-glucan complexes, derivative signals were calculated. A linear relationship was found between the amplitude of the second derivative signals and the β-glucan concentration between 0.1 and 0.4 μg/mL. The low detection limit of this new method (0.045 μg/mL) enabled its use to study the transport of cereal β-glucans over differentiated Caco-2 cell monolayers. Additionally, the method was applied to quantify β-glucan in arabinoxylan samples, which correlated well with data by an enzyme based method. Copyright © 2012 Elsevier Ltd. All rights reserved.

Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Lytechinus pictus sectioned embryos. M.S. Thesis

NASA Technical Reports Server (NTRS)

Garciaflack, Ana Leticia

1988-01-01

Indirect immunofluorescence was used to localize specific extracellular components in embryos of the sea urchin Lytechinus pictus. Hyalin and S2 (a group of components found in the disaggregation supernatant from Strongylocentrotus purpuratus blastulae) were uniformly present at all stages (unfertilized up to 32 hr) except hyalin could not be detected at the 12 hour early blastula stage. Laminin was found in 16 cell, 32 cell, 6 hour, 18 hour, 24 hour, and 32 hour stages, with especially bright fluorescence at 18 hours. Collagen I was present at all stages (freshly fertilized up to 32 hour) except little was detected at 12 hours. Fibronectin was uniformly present in blastocoelar fibers stained with anto-collagen I and anti-fibronectin. These results were compared with those for S. purpuratus to produce an overview of the localization of specific extracellular matrix components during development of two species of sea urchins. The results set the stage for future studies that will examine the function of these components at the various developmental stages.

Study of the recovery of phosphorus from struvite precipitation in supernatant line from anaerobic digesters of sludge.

PubMed

Xavier, Luciano Dias; Cammarota, Magali Christe; Yokoyama, Lídia; Volschan Junior, Isaac

2014-01-01

The goal of this work was to study the effective recovery of phosphorus from the supernatant of anaerobic digestion of sewage sludge by precipitation as struvite. The formation of struvite is envisioned as a promising process for nutrient removal and subsequent recovery, thus providing a strong incentive for its implementation, since the sewage is a renewable source of phosphorus. Struvite precipitation was obtained by controlled addition of Mg(OH)2 or MgCl2. We evaluated the removal of ammonia and phosphate under equimolar conditions of magnesium and magnesium stoichiometric excess of 100 to 200% relative to the limiting reagent, under a stirring speed of 300 rpm at pH 8, 9 and 10. The best condition was MgCl2 in 1:1 molar ratio to phosphate, considering the stoichiometric ratio [PO4(3-)]:[NH4(+)] of 0.13 (presented by raw sample). The results show the best cost-benefit ratio, removal of phosphate of 90.6% and ammonium removal of 29%, resulting in 23 mg l(-1) PO4(3-) and 265 mg l(-1) NH4(+) concentration in effluent.

Clinical and diagnosis characteristics of breast cancers in women with a history of radiotherapy in the first 30years of life: A French multicentre cohort study.

PubMed

Demoor-Goldschmidt, C; Supiot, S; Oberlin, O; Helfre, S; Vigneron, C; Brillaud-Meflah, V; Bernier, V; Laprie, A; Ducassou, A; Claude, L; Mahé, M A; de Vathaire, F

2017-08-01

Irradiation (>3Gy) to the breast or axillae before 30years of age increases the risk of secondary breast cancer (SBC). The purpose of this article is to describe the clinical characteristics of SBC and the way of diagnosis in young women (before the age of national screening) in France who had received previous radiotherapy for a childhood or a young adulthood cancer. This retrospective, multicentre study reviewed the medical records of women with SBC before the age of the national screening who had received irradiation (≥3Gy) on part or all of the breast before 30years of age, for any type of tumour except BC. A total of 121 SBC were detected in 104 women with previous radiotherapy. Twenty percent of SBC were detected during regular breast screening and 16% of the women had a regular radiological follow-up. Our results points out that the main proportion of childhood cancer survivors did not benefit from the recommended breast cancer screening. This result is comparable to other previously published studies in other countries. A national screening programme is necessary and should take into account the patient's age, family history, personal medical history and previous radiotherapy to reduce the number of SBC diagnosed at an advanced stage. Copyright © 2017. Published by Elsevier B.V.

Sodium bicarbonate induces crystalline wax generation, activates host-resistance, and increases imazalil level in rind wounds of oranges, improving the control of green mold during storage.

PubMed

Dore, Antonio; Molinu, Maria Giovanna; Venditti, Tullio; D'Hallewin, Guy

2010-06-23

Imazalil (IMZ) was quantified in the flavedo and albedo (Citrus fruits outer and inner tissue of the exocarp) of wounded and unwounded Valencia L. Olinda oranges following a 2 min immersion at 25 degrees C in 50, 100, or 250 microg mL(-1) of the fungicide mixture with or without 3% sodium bicarbonate (SBC). The addition of SBC significantly reduced the decay incidence throughout 30 d of storage at 10 degrees C with 95% RH and 6 d of simulated marketing period at 25 degrees C and 75% RH. In unwounded oranges, IMZ uptake was not changed by the coapplication of SBC, and the fungicide was predominantly recovered in the flavedo. To the contrary, in the albedo of wounded fruit, the residue level increased by about 6-fold when the fungicide was applied with SBC. When SBC was coapplied to wounded fruit, the phytoalexin scoparone was induced in the albedo and the accumulation was not affected by IMZ. When fruit was treated with SBC, scanning electron microscopy observations evidenced a production of crystalline wax patches with branched stripes and the magnitude was positively correlated to the salt concentration in the mixture. The generation as fast as 24 h post-treatment, and the different morphology of the new wax suggests a displacement of intracuticular waxes which can affect the fungicide sorption and diffusion coefficient into the rind.

Evaluation of Phytate-Degrading Lactobacillus Culture Administration to Broiler Chickens

PubMed Central

Askelson, Tyler E.; Campasino, Ashley; Lee, Jason T.

2014-01-01

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. PMID:24271165

Evaluation of phytate-degrading Lactobacillus culture administration to broiler chickens.

PubMed

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.

Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis.

PubMed

Golowczyc, M A; Mobili, P; Garrote, G L; Abraham, A G; De Antoni, G L

2007-09-30

Eight Lactobacillus kefir strains isolated from different kefir grains were tested for their ability to antagonize Salmonella enterica serovar Enteritidis (Salmonella enteritidis) interaction with epithelial cells. L. kefir surface properties such as autoaggregation and coaggregation with Salmonella and adhesion to Caco-2/TC-7 cells were evaluated. L. kefir strains showed significantly different adhesion capacities, six strains were able to autoaggregate and four strains coaggregated with Salmonella. Coincubation of Salmonella with coaggregating L. kefir strains significantly decreased its capacity to adhere to and to invade Caco-2/TC-7 cells. This was not observed with non coaggregating L. kefir strains. Spent culture supernatants of L. kefir contain significant amounts of S-layer proteins. Salmonella pretreated with spent culture supernatants (pH 4.5-4.7) from all tested L. kefir strains showed a significant decrease in association and invasion to Caco-2/TC-7 cells. Artificially acidified MRS containing lactic acid to a final concentration and pH equivalent to lactobacilli spent culture supernatants did not show any protective action. Pretreatment of this pathogen with spent culture supernatants reduced microvilli disorganization produced by Salmonella. In addition, Salmonella pretreated with S-layer proteins extracted from coaggregating and non coaggregating L. kefir strains were unable to invade Caco-2/TC-7 cells. After treatment, L. kefir S-layer protein was detected associated with Salmonella, suggesting a protective role of this protein on association and invasion.

Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.

PubMed

Rodrigues, Valérie; Baudier, Jean Baptiste; Chantal, Isabelle

2017-09-01

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R 2  > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

The Reproducibility of 4-km Time Trial (TT) Performance Following Individualised Sodium Bicarbonate Supplementation: a Randomised Controlled Trial in Trained Cyclists.

PubMed

Gough, Lewis Anthony; Deb, Sanjoy Kumar; Sparks, Andy; McNaughton, Lars Robert

2017-09-21

Individual time to peak blood bicarbonate (HCO 3 - ) has demonstrated good to excellent reproducibility following ingestion of both 0.2 g kg -1 body mass (BM) and 0.3 g kg -1 BM sodium bicarbonate (NaHCO 3 ), but the consistency of the time trial (TT) performance response using such an individualised NaHCO 3 ingestion strategy remains unknown. This study therefore evaluated the reproducibility of 4-km TT performance following NaHCO 3 ingestion individualised to time to peak blood bicarbonate. Eleven trained male cyclists completed five randomised treatments with prior ingestion of 0.2 g kg -1 (SBC2) or 0.3 g kg -1 BM (SBC3) NaHCO 3 , on two separate occasions each, or a control trial entailing no supplementation. Participants completed a 4-km cycling TT on a Velotron ergometer where time to complete, power and speed were measured, whilst acid-base blood parameters were also recorded (pH and blood bicarbonate concentration HCO 3 - ) and lactate [La - ]. Alkalosis was achieved prior to exercise in both SBC2 and SBC3, as pH and HCO 3 - were greater compared to baseline (p < 0.001), with no differences between treatments (p > 0.05). The reproducibility of the mean absolute change from baseline to peak in HCO 3 - was good in SBC2 (r = 0.68) and excellent in SBC3 (r = 0.78). The performance responses following both SBC2 and SBC3 displayed excellent reproducibility (r range = 0.97 to 0.99). Results demonstrate excellent reproducibility of exercise performance following individualised NaHCO 3 ingestion, which is due to the high reproducibility of blood acid-base variables with repeat administration of NaHCO 3 . Using a time to peak HCO 3 - strategy seems to cause no dose-dependent effects on performance for exercise of this duration and intensity; therefore, athletes may consider smaller doses of NaHCO 3 to mitigate gastrointestinal (GI) discomfort.

Avenin fails to induce a Th1 response in coeliac tissue following in vitro culture

PubMed Central

Kilmartin, C; Lynch, S; Abuzakouk, M; Wieser, H; Feighery, C

2003-01-01

Background: It is well established that the wheat protein gliadin triggers inflammation in coeliac patients. However, the potential toxicity of avenin, the equivalent protein in oats, is debated. Aim: To investigate the immunogenicity of avenin using the cytokines interferon γ (IFN-γ) and interleukin (IL)-2 as markers of immunological activity. Methods: Duodenal biopsies from coeliac patients were cultured with 5 mg/ml of peptic tryptic (PT) gliadin (n=9) or 5 mg/ml of PT avenin (n=8) for four hours. Biopsies cultured with RPMI 1640 alone served as controls. Non-coeliac biopsies were also cultured with PT gliadin (n=8) and PT avenin (n=8). Total RNA was extracted from the tissue after culture. Cytokine mRNA was quantified by TaqMan polymerase chain reaction. Secreted cytokine protein was measured in the culture supernatant by enzyme linked immunosorbent assay. Results: After culture with PT gliadin, an increase in IFN-γ mRNA was observed in all nine patients with coeliac disease. Increased IFN-γ protein was also found in four of these patients. Smaller increases in IL-2 mRNA were detected in six subjects with increased IL-2 protein found in two patients. In contrast with PT gliadin, there was no significant IFN-γ or IL-2 response when coeliac biopsies were cultured with PT avenin. Similarly, biopsies from normal controls did not respond to PT gliadin or PT avenin stimulation. Conclusions: The findings of this study suggest that the immunogenic sequences in gliadin are not present in avenin. Moreover, they are in keeping with in vivo studies which report that oats are safe for consumption by coeliac patients. PMID:12477758

Characterization of biologic response modifiers in the supernatant of conventional, refrigerated, and cryopreserved platelets.

PubMed

Johnson, Lacey; Tan, Shereen; Jenkins, Emily; Wood, Ben; Marks, Denese C

2018-04-01

Alternatives to room temperature storage of platelets (PLTs) are of interest to support blood banking logistics. The aim of this study was to compare the presence of biologic response modifiers (BRMs) in PLT concentrates stored under conventional room temperature conditions with refrigerated or cryopreserved PLTs. A three-arm pool-and-split study was carried out using buffy coat-derived PLTs stored in 30% plasma/70% SSP+. The three matched treatment arms were as follows: room temperature (20-24°C), cold (2-6°C), and cryopreserved (-80°C with DMSO). Liquid-stored PLTs were tested over a 21-day period, while cryopreserved PLTs were tested immediately after thawing and reconstitution in 30% plasma/70% SSP+ and after storage at room temperature. Coagulation factor activity was comparable between room temperature and cold PLTs, with the exception of protein S, while cryopreserved PLTs had reduced Factor (F)V and FVIII activity. Cold-stored PLTs retained α-granule proteins better than room temperature or cryopreserved PLTs. Cryopreservation resulted in 10-fold higher microparticle generation than cold-stored PLTs, but both groups contained significantly more microparticles than those stored at room temperature. The supernatant from both cold and cryopreserved PLTs initiated faster clot formation and thrombin generation than room temperature PLTs. Cold storage and cryopreservation alter the composition of the soluble fraction of stored PLTs. These differences in coagulation proteins, cytokines, and microparticles likely influence both the hemostatic capacity of the components and the auxiliary functions. © 2017 AABB.

Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures.

PubMed

Laurent, P; Buchon, L; Guespin-Michel, J F; Orange, N

2000-04-01

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

PubMed

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.

77 FR 8803 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-15

...,936. Needs and Uses: This request is for an extension of a current information collection. Under... owner or operator of a vessel for which a permit has been issued under Sec. 635.4 and that uses handline... block letters or Arabic numerals in a color that contrasts with the background color of the float or...

Biocontrol Potential of Streptomyces hydrogenans Strain DH16 toward Alternaria brassicicola to Control Damping Off and Black Leaf Spot of Raphanus sativus

PubMed Central

Manhas, Rajesh K.; Kaur, Talwinder

2016-01-01

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 S. 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 vigor 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. PMID:28018402

1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

PubMed Central

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. PMID:25337708

Sodium bicarbonate improves 4 km time trial cycling performance when individualised to time to peak blood bicarbonate in trained male cyclists.

PubMed

Gough, Lewis A; Deb, Sanjoy K; Sparks, S Andy; McNaughton, Lars R

2018-08-01

The aim of this study was to investigate the effects of sodium bicarbonate (NaHCO 3 ) on 4 km cycling time trial (TT) performance when individualised to a predetermined time to peak blood bicarbonate (HCO 3 - ). Eleven male trained cyclists volunteered for this study (height 1.82 ± 0.80 m, body mass (BM) 86.4 ± 12.9 kg, age 32 ± 9 years, peak power output (PPO) 382 ± 22 W). Two trials were initially conducted to identify time to peak HCO 3 - following both 0.2 g . kg -1 BM (SBC2) and 0.3 g . kg -1 BM (SBC3) NaHCO 3 . Thereafter, on three separate occasions using a randomised, double-blind, crossover design, participants completed a 4 km TT following ingestion of either SBC2, SBC3, or a taste-matched placebo (PLA) containing 0.07 g . kg -1 BM sodium chloride (NaCl) at the predetermined individual time to peak HCO 3 - . Both SBC2 (-8.3 ± 3.5 s; p < 0.001, d = 0.64) and SBC3 (-8.6 ± 5.4 s; p = 0.003, d = 0.66) reduced the time to complete the 4 km TT, with no difference between SBC conditions (mean difference = 0.2 ± 0.2 s; p = 0.87, d = 0.02). These findings suggest trained cyclists may benefit from individualising NaHCO 3 ingestion to time to peak HCO 3 - to enhance 4 km TT performance.

Near infrared spectroscopy combined with multivariate analysis for monitoring the ethanol precipitation process of fraction I + II + III supernatant in human albumin separation

NASA Astrophysics Data System (ADS)

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.

PubMed

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.

Pasteurization of naturally contaminated water with solar energy.

PubMed

Ciochetti, D A; Metcalf, R H

1984-02-01

A solar box cooker (SBC) was constructed with a cooking area deep enough to hold several 3.7-liter jugs of water, and this was used to investigate the potential of using solar energy to pasteurize naturally contaminated water. When river water was heated either in the SBC or on a hot plate, coliform bacteria were inactivated at temperatures of 60 degrees C or greater. Heating water in an SBC to at least 65 degrees C ensures that the water will be above the milk pasteurization temperature of 62.8 degrees C for at least an hour, which appears sufficient to pasteurize contaminated water. On clear or partly cloudy days, with the SBC facing magnetic south in Sacramento, bottom water temperatures of at least 65 degrees C could be obtained in 11.1 liters of water during the 6 weeks on either side of the summer solstice, in 7.4 liters of water from mid-March through mid-September, and in 3.7 liters of water an additional 2 to 3 weeks at the beginning and end of the solar season. Periodic repositioning of the SBC towards the sun, adjusting the back reflective lid, and preheating water in a simple reflective device increased final water temperatures. Simultaneous cooking and heating water to pasteurizing temperatures was possible. Additional uses of the SBC to pasteurize soil and to decontaminate hospital materials before disposal in remote areas are suggested.

Pasteurization of naturally contaminated water with solar energy.

PubMed Central

Ciochetti, D A; Metcalf, R H

1984-01-01

A solar box cooker (SBC) was constructed with a cooking area deep enough to hold several 3.7-liter jugs of water, and this was used to investigate the potential of using solar energy to pasteurize naturally contaminated water. When river water was heated either in the SBC or on a hot plate, coliform bacteria were inactivated at temperatures of 60 degrees C or greater. Heating water in an SBC to at least 65 degrees C ensures that the water will be above the milk pasteurization temperature of 62.8 degrees C for at least an hour, which appears sufficient to pasteurize contaminated water. On clear or partly cloudy days, with the SBC facing magnetic south in Sacramento, bottom water temperatures of at least 65 degrees C could be obtained in 11.1 liters of water during the 6 weeks on either side of the summer solstice, in 7.4 liters of water from mid-March through mid-September, and in 3.7 liters of water an additional 2 to 3 weeks at the beginning and end of the solar season. Periodic repositioning of the SBC towards the sun, adjusting the back reflective lid, and preheating water in a simple reflective device increased final water temperatures. Simultaneous cooking and heating water to pasteurizing temperatures was possible. Additional uses of the SBC to pasteurize soil and to decontaminate hospital materials before disposal in remote areas are suggested. PMID:6712206

Mechanisms of antimony adsorption onto soybean stover-derived biochar in aqueous solutions.

PubMed

Vithanage, Meththika; Rajapaksha, Anushka Upamali; Ahmad, Mahtab; Uchimiya, Minori; Dou, Xiaomin; Alessi, Daniel S; Ok, Yong Sik

2015-03-15

Limited mechanistic knowledge is available on the interaction of biochar with trace elements (Sb and As) that exist predominantly as oxoanions. Soybean stover biochars were produced at 300 °C (SBC300) and 700 °C (SBC700), and characterized by BET, Boehm titration, FT-IR, NMR and Raman spectroscopy. Bound protons were quantified by potentiometric titration, and two acidic sites were used to model biochar by the surface complexation modeling based on Boehm titration and NMR observations. The zero point of charge was observed at pH 7.20 and 7.75 for SBC300 and SBC700, respectively. Neither antimonate (Sb(V)) nor antimonite (Sb(III)) showed ionic strength dependency (0.1, 0.01 and 0.001 M NaNO3), indicating inner sphere complexation. Greater adsorption of Sb(III) and Sb(V) was observed for SBC300 having higher -OH content than SBC700. Sb(III) removal (85%) was greater than Sb(V) removal (68%). Maximum adsorption density for Sb(III) was calculated as 1.88 × 10(-6) mol m(-2). The Triple Layer Model (TLM) successfully described surface complexation of Sb onto soybean stover-derived biochar at pH 4-9, and suggested the formation of monodentate mononuclear and binuclear complexes. Spectroscopic investigations by Raman, FT-IR and XPS further confirmed strong chemisorptive binding of Sb to biochar surfaces. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pasteurization of naturally contaminated water with solar energy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ciochetti, D.A.; Metcalf, R.H.

1984-02-01

A solar box cooker (SBC) was constructed with a cooking area deep enough to hold several 3.7-liter jugs of water, and this was used to investigate the potential of using solar energy to pasteurize naturally contaminated water. When river water was heated either in the SBC or on a hot plate, coliform bacteria were inactivated at temperatures of 60/sup 0/C or greater. Heating water in an SBC to at least 65/sup 0/C ensures that the water will be above the milk pasteurization temperature of 62.8/sup 0/C for at least an hour, which appears sufficient to pasteurize contaminated water. On clearmore » or partly cloudy days, with the SBC facing magnetic south in Sacramento, bottom water temperatures of at least 65/sup 0/C could be obtained in 11.1 liters of water during the 6 weeks on either side of the summer solstice, in 7.4 liters of water from mid-March through mid-September, and in 3.7 liters of water an additional 2 to 3 weeks at the beginning and end of the solar season. Periodic repositioning of the SBC towards the sun, adjusting the back reflective lid, and preheating water in a simple reflective device increased final water temperatures. Simultaneous cooking and heating water to pasteurizing temperatures was possible. Additional uses of the SBC to pasteurize soil and to decontaminate hospital materials before disposal in remote areas are suggested. 14 references.« less

Experimental prestorage filtration removes antibodies and decreases lipids in RBC supernatants mitigating TRALI in vivo

PubMed Central

Kelher, Marguerite R.; Khan, Samina Y.; LaSarre, Monica; West, F. Bernadette; Land, Kevin J.; Mish, Barbara; Ceriano, Linda; Sowemimo-Coker, Samuel

2014-01-01

Transfusion-related acute lung injury (TRALI) remains a significant cause of transfusion-related mortality with red cell transfusion. We hypothesize that prestorage filtration may reduce proinflammatory activity in the red blood cell (RBC) supernatant and prevent TRALI. Filters were manufactured for both small volumes and RBC units. Plasma containing antibodies to human lymphocyte antigen (HLA)-A2 or human neutrophil antigen (HNA)-3a was filtered, and immunoglobulins and specific HNA-3a and HLA-2a neutrophil (PMN) priming activity were measured. Antibodies to OX27 were added to plasma, and filtration was evaluated in a 2-event animal model of TRALI. RBC units from 31 donors known to have antibodies against HLA antigens and from 16 antibody-negative controls were filtered. Furthermore, 4 RBC units were drawn and underwent standard leukoreduction. Immunoglobulins, HLA antibodies, PMN priming activity, and the ability to induce TRALI in an animal model were measured. Small-volume filtration of plasma removed >96% of IgG, antibodies to HLA-A2 and HNA-3a, and their respective priming activity, as well as mitigating antibody-mediated in vivo TRALI. In RBC units, experimental filtration removed antibodies to HLA antigens and inhibited the accumulation of lipid priming activity and lipid-mediated TRALI. We conclude that filtration removes proinflammatory activity and the ability to induce TRALI from RBCs and may represent a TRALI mitigation step. PMID:24747436

270GHz SiGe BiCMOS manufacturing process platform for mmWave applications

NASA Astrophysics Data System (ADS)

Kar-Roy, Arjun; Preisler, Edward J.; Talor, George; Yan, Zhixin; Booth, Roger; Zheng, Jie; Chaudhry, Samir; Howard, David; Racanelli, Marco

2011-11-01

TowerJazz has been offering the high volume commercial SiGe BiCMOS process technology platform, SBC18, for more than a decade. In this paper, we describe the TowerJazz SBC18H3 SiGe BiCMOS process which integrates a production ready 240GHz FT / 270 GHz FMAX SiGe HBT on a 1.8V/3.3V dual gate oxide CMOS process in the SBC18 technology platform. The high-speed NPNs in SBC18H3 process have demonstrated NFMIN of ~2dB at 40GHz, a BVceo of 1.6V and a dc current gain of 1200. This state-of-the-art process also comes with P-I-N diodes with high isolation and low insertion losses, Schottky diodes capable of exceeding cut-off frequencies of 1THz, high density stacked MIM capacitors, MOS and high performance junction varactors characterized up to 50GHz, thick upper metal layers for inductors, and various resistors such as low value and high value unsilicided poly resistors, metal and nwell resistors. Applications of the SBC18H3 platform for millimeter-wave products for automotive radars, phased array radars and Wband imaging are presented.

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

PubMed

Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

2016-02-02

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. Copyright © 2015 Elsevier B.V. All rights reserved.

[ANTIMICROBIAL ACTION OF NOCARDIA VACCINII IMV B-7405 SURFACTANTS].

PubMed

Pirog, T P; Beregova, K A; Savenko, I V; Shevchuk, T A; Iutynska, G O

2015-01-01

To study the effect of Nocardia vaccinii IMV B-7405 surfactants on some bacteria (including pathogens of genera Proteus, Staphylococcus, Enterobacter), yeast of Candida species and fungi (Aspergillus niger R-3, Fusarium culmorum T-7). The antimi- crobial properties of surfactant were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). It is shown that the antimicrobial properties of N. vaccinii IMV B-7405 surfactant depended on the degree of purification (supernatant, solution of surfactant), concentration and exposure. Survival of Escherichia coli IEM-1 and Bacillus subtilis BT-2 (both vegetative cells and spores) after treatment for 1-2 hours with surfactants solution and the supernatant (the surfactant concentration 21 µg/ml) was 3-28%. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants on studied bacteria, yeast and micromycetes were 11.5-85.0; 11.5-22.5 and 165.0-325.0 µ/ml respectively. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants are comparable to those of the known microbial surfactants. The possibility of using the supernatant of culture liquid as an effective antimicrobial agent noticeably simplifies and reduces the cost of the technology of its obtaining.

Rapid evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos.

PubMed

Rebmann, Vera; Switala, Magdalena; Eue, Ines; Schwahn, Eva; Merzenich, Markus; Grosse-Wilde, Hans

2007-04-01

Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p < 0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly

PubMed Central

Liu, Yue; Gibson, Glenn R.; Walton, Gemma E.

2016-01-01

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. PMID:27612304

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly.

PubMed

Liu, Yue; Gibson, Glenn R; Walton, Gemma E

2016-01-01

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.

Soft sensor for monitoring biomass subpopulations in mammalian cell culture processes.

PubMed

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.

Bovine lactotroph cultures for the study of prolactin synthesis functions.

PubMed

Wang, Jianfa; Yang, Zhanqing; Fu, Shoupeng; Liu, Bingrun; Wu, Dianjun; Wang, Wei; Sun, Dongbo; Wu, Rui; Liu, Juxiong

2016-03-01

The aim of this study was to establish a bovine anterior pituitary-derived lactotroph (BAPDL) line that expresses prolactin (PRL) in vitro to study the mechanisms of bovine PRL synthesis and secretion. Immunohistochemistry assay of PRL in the newborn calves' anterior pituitary glands showed that most lactotrophs were located within the superior border of the lateral wings of the anterior pituitary. Tissues of the superior border of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The limiting dilution method was used to establish BAPDL from single cell clone. BAPDL cells constantly expressed mRNAs for PRL and pituitary-specific transcription factor 1 (Pit-1) gene and grew steadily and rapidly in the DMEM supplemented with 10% FBS. PRL immunoreactivity was present in BAPDL at passage 20. The concentration of bovine PRL in BAPDL at passage 20 culture supernatant was decreased to below 35% compared with that in BAPDL at passage 1. The effects of human epidermal growth factor (hEGF) and dopamine (DA) on the expression and secretion of PRL in BAPDL at passage 4 were also investigated. The results are consistent with those of previous studies. Thus, it can be used successfully for studying the mechanisms of stimuli regulating PRL synthesis and release.

Comparison of spectroscopy technologies for improved monitoring of cell culture processes in miniature bioreactors

PubMed Central

van den Berg, Frans; Racher, Andrew J.; Martin, Elaine B.; Jaques, Colin

2017-01-01

Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the throughput of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in large‐scale bioreactors. The small volume of miniature bioreactor cultures (15 mL) is incompatible with the large sample volume (600 µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman, and 2D‐fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design‐of‐experiment approach is described that utilizes previously analyzed cell culture supernatant to assess metabolite concentrations under various conditions while providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D‐fluorescence could more accurately measure ammonium concentration (RMSECV 0.031 g L−1) than Raman and NIR. Raman spectroscopy, however, was more robust at measuring lactate and glucose concentrations (RMSECV 1.11 and 0.92 g L−1, respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D‐fluorescence. The implementation of Raman spectroscopy increases at‐line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:337–346, 2017 PMID:28271638

Comparison of spectroscopy technologies for improved monitoring of cell culture processes in miniature bioreactors.

PubMed

Rowland-Jones, Ruth C; van den Berg, Frans; Racher, Andrew J; Martin, Elaine B; Jaques, Colin

2017-03-01

Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the throughput of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in large-scale bioreactors. The small volume of miniature bioreactor cultures (15 mL) is incompatible with the large sample volume (600 µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman, and 2D-fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design-of-experiment approach is described that utilizes previously analyzed cell culture supernatant to assess metabolite concentrations under various conditions while providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D-fluorescence could more accurately measure ammonium concentration (RMSE CV 0.031 g L -1 ) than Raman and NIR. Raman spectroscopy, however, was more robust at measuring lactate and glucose concentrations (RMSE CV 1.11 and 0.92 g L -1 , respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D-fluorescence. The implementation of Raman spectroscopy increases at-line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:337-346, 2017. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on

Fermentation supernatants of Lactobacillus delbrueckii inhibit growth of human colon cancer cells and induce apoptosis through a caspase 3-dependent pathway.

PubMed

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.

[Effects of non-saccharomyces albicans metabolic products on the proliferation of human umbilical vein endothelial cell ECV304].

PubMed

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 < 0.05). After adding various non-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 < 0.05). 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

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

PubMed

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).

Influence of two barrier membranes on staged guided bone regeneration and osseointegration of titanium implants in dogs: part 1. Augmentation using bone graft substitutes and autogenous bone.

PubMed

Schwarz, Frank; Mihatovic, Ilja; Golubovic, Vladimir; Hegewald, Andrea; Becker, Jürgen

2012-01-01

To assess the influence of two barrier membranes and two bone graft substitutes mixed with autogenous bone (AB) on staged guided bone regeneration and osseointegration of titanium implants in dogs. Four saddle-type defects each were prepared in the upper jaw of six fox hounds and randomly filled with a natural bone mineral (NBM)+AB and a biphasic calcium phosphate (SBC)+AB and allocated to either an in situ gelling polyethylene glycol (PEG) or a collagen membrane (CM). At 8 weeks, modSLA titanium implants were inserted and left to heal in a submerged position. At 8+2 weeks, dissected blocks were processed for histomorphometrical analysis (e.g., treated area [TA], bone-to-implant contact [BIC]). The mean TA values (mm(2) ) and BIC values (%) tended to be higher in the PEG groups(TA: NBM+AB [10.4 ± 2.5]; SBC+AB [10.4 ± 5.8]/BIC: NBM+AB [86.4 ± 20.1]; SBC+AB [80.1 ± 21.5]) when compared with the corresponding CM groups (TA: NBM+AB [9.7 ± 4.8]; SBC+AB [7.8 ± 4.3]/BIC: NBM+AB [71.3 ± 20.8]; SBC+AB [72.4 ± 20.3]). A significant difference was observed for the mean TA values in the SBC+AB groups. It was concluded that all augmentation procedures investigated supported bone regeneration and staged osseointegration of modSLA titanium implants. However, the application of PEG may be associated with increased TA values. © 2011 John Wiley & Sons A/S.

Low-level laser irradiation modifies the effect of hyperglycemia on adhesion molecule levels.

PubMed

Góralczyk, Krzysztof; Szymańska, Justyna; Gryko, Łukasz; Fisz, Jacek; Rość, Danuta

2018-05-03

Endothelium plays a key role in maintaining vascular homeostasis by secreting active factors involved in many biological processes such as hemostasis, angiogenesis, and inflammation. Hyperglycemia in diabetic patients causes dysfunction of endothelial cells. Soluble fractions of adhesion molecules like sE-selectin and vascular cell adhesion molecule (sVCAM) are considered as markers of endothelial damage. The low-level laser therapy (LLLT) effectively supports the conventional treatment of vascular complications in diabetes, for example hard-to-heal wounds in patients with diabetic foot syndrome. The aim of our study was to evaluate the effect of low-energy laser at the wavelength of 635 nm (visible light) and 830 nm (infrared) on the concentration of adhesion molecules: sE-selectin and sVCAM in the supernatant of endothelial cell culture of HUVEC line. Cells were cultured under high-glucose conditions of 30 mM/L. We have found an increase in sE-selectin and sVCAM levels in the supernatant of cells cultured under hyperglycemic conditions. This fact confirms detrimental influence of hyperglycemia on vascular endothelial cell cultures. LLLT can modulate the inflammation process. It leads to a decrease in sE-selectin and sVCAM concentration in the supernatant and an increase in the number of endothelial cells cultured under hyperglycemic conditions. The influence of LLLT is greater at the wavelength of 830 nm.

Interleukin 1 and Tumor Necrosis Factor Inhibit Cardiac Myocyte β -adrenergic Responsiveness

NASA Astrophysics Data System (ADS)

Gulick, Tod; Chung, Mina K.; Pieper, Stephen J.; Lange, Louis G.; Schreiner, George F.

1989-09-01

Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit β -adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contractile response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator. Inhibition occurs in the absence of alteration in β -adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal responsiveness and function of contractile cells. The effects of interleukin 1 and tumor necrosis factor on intracellular cAMP accumulation may be a model for immune modulation of other cellular functions dependent upon cyclic nucleotide metabolism. The uncoupling of agonist-occupied receptors from adenyl cyclase suggests that β -receptor or guanine nucleotide binding protein function is altered by the direct or indirect action of cytokines on cardiac muscle cells.

Exploring the potential environmental functions of viable but non-culturable bacteria.

PubMed

Su, Xiaomei; Chen, Xi; Hu, Jinxing; Shen, Chaofeng; Ding, Linxian

2013-12-01

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in "viable but non-culturable" (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.

Lactic acid bacteria as functional probiotic isolates for inhibiting the growth of Aspergillus flavus, A. parasiticus, A. niger and Penicillium chrysogenum.

PubMed

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 (P<0.05). Antifungal activity evaluation showed that A. flavus was the most inhibited fungus by probiotic bacteria, followed by P. chrysogenum, A. niger 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.

Effect of L-arginine on the growth of Plasmodium falciparum and immune modulation of host cells.

PubMed

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.

Building a discovery partnership with Sarawak Biodiversity Centre: a gateway to access natural products from the rainforests.

PubMed

Yeo, Tiong Chia; Naming, Margarita; Manurung, Rita

2014-03-01

The Sarawak Biodiversity Centre (SBC) is a state government agency which regulates research and promotes the sustainable use of biodiversity. It has a program on documentation of traditional knowledge (TK) and is well-equipped with facilities for natural product research. SBC maintains a Natural Product Library (NPL) consisting of local plant and microbial extracts for bioprospecting. The NPL is a core discovery platform for screening of bioactive compounds by researchers through a formal agreement with clear benefit sharing obligations. SBC aims to develop partnerships with leading institutions and the industries to explore the benefits of biodiversity.

The effect of down regulation of calcineurin Aα by lentiviral vector-mediated RNAi on the biological behavior of small-cell lung cancer and its bone metastasis.

PubMed

Ma, Ning-Qiang; Liu, Li-Li; Min, Jie; Wang, Jun-Wei; Jiang, Wei-Feng; Liu, Yan; Feng, Yan-Guo; Su, Hai-Chuan; Feng, Ying-Ming; Zhang, He-Long

2011-12-01

Bone is the third most common site of cancer metastasis. Over 30 to 40% of lung cancers can develop skeletal metastasis and no effective curative therapy exists in clinic cases. Previously we screened the different expression of proteins between SBC-5 cells and SBC-3 cells by proteomic study methods (MALDI-TOF/TOF-MS) and found that calcineurin (hereafter referred as Cn) overexpresses in SBC-5 which has special priority in metastasis to bone in a multiple-organ metastasis mice model. However the roles of Cn in osteotropism of SCLC remain to be elucidated. At present study, we decrease CnAα expression in SBC-5 by lentiviral vector-mediated RNAi and found that down regulation of CnAα gene expression can decrease the proliferation and colony formation rate, impede the cell cycle progression, reduce the cell migration and invasion, and inhibit cells adhering to bone matrix, but not change the apoptosis rate of SBC-5 in vitro. In vivo down or up regulation of CnAα gene expression can only decrease or increase the bone metastasis rate, but not affect the metastasis rate to the visceral organs. Our research reveals that CnAα is closely related to the osteotropism metastasis of SCLC and a candidate tumor promotor gene for developing bone metastases.

B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

PubMed

Nakaya, K; Mizuno, R; Ohhashi, T

2001-12-01

We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of <1,000 molecular weight (MW), producing complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating or digestion with protease had no significant effect on supernatant-mediated response. These findings suggest that B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

Pyrometric Practice

DTIC Science & Technology

1948-01-01

this type of crucible a primary calibration is made by observing, as described on page 211, .00069 .000-- eI -0 40 60 so 100 12O 140 ISO Aagular ftsMnga... 8583 7.8597 36 8613 8629 8644 8660 8676 8692 8708 8724 8740 8755 37 877L 8787 8803 8818 8834 8850 8865 8881 8897 8912 38 8928 8944 89 898975 8990 9-06

Outbreak of hirame rhabdovirus infection in cultured spotted sea bass Lateolabrax maculatus on the western coast of Korea.

PubMed

Seo, H-G; Do, J W; Jung, S H; Han, H-J

2016-10-01

In this study, we determined the cause of a disease outbreak in spotted sea bass, Lateolabrax maculatus reared in culture cages on the western coast of Korea in 2013. The major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. No external morphological signs of infection were visible, except for a darkening in colour. No parasites or pathological bacteria were isolated from the diseased fish; however, epithelioma papulosum cyprini (EPC) cells inoculated with tissue homogenates from the diseased fish showed cytopathic effects (CPEs). Virus particles in the EPC cells were bullet-shaped, 185-225 nm long and 70-80 nm wide, characteristic of Rhabdoviridae. Polymerase chain reaction analyses of homogenized tissues from the diseased fish and supernatants of cell cultures with CPEs indicated specific, 553-bp-long fragments corresponding to the matrix protein gene of the hirame rhabdovirus (HIRRV). Phylogenetically, the HIRRV phosphoprotein gene of spotted sea bass was more closely related to phosphoproteins from Chinese and Polish HIRRV strains than from other Korean strains. To our knowledge, this is the first report of HIRRV infection in cultured spotted sea bass. © 2016 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.

Mechanisms of antimony adsorption onto soybean stover-derived biochar in aqueous solutions

USDA-ARS?s Scientific Manuscript database

Limited mechanistic knowledge is available to understand how biochar interacts with trace elements that exist predominantly as oxoanions, such as antimony (Sb). Soybean stover biochars were produced at 300 degrees C (SBC300) and 700 degrees C (SBC700), and were characterized by BET, Boehm titration,...

13 CFR 125.8 - What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business...

Code of Federal Regulations, 2012 CFR

2012-01-01

... the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? 125.8 Section 125.8... for the Service-Disabled Veteran-Owned Small Business Concern Program § 125.8 What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? (a) Contracting...

13 CFR 125.8 - What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business...

Code of Federal Regulations, 2013 CFR

2013-01-01

... the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? 125.8 Section 125.8... for the Service-Disabled Veteran-Owned Small Business Concern Program § 125.8 What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? (a) Contracting...

13 CFR 125.8 - What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business...

Code of Federal Regulations, 2014 CFR

2014-01-01

... the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? 125.8 Section 125.8... for the Service-Disabled Veteran-Owned Small Business Concern Program § 125.8 What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? (a) Contracting...

13 CFR 125.8 - What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business...

Code of Federal Regulations, 2011 CFR

2011-01-01

... the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? 125.8 Section 125.8... for the Service-Disabled Veteran-Owned Small Business Concern Program § 125.8 What definitions are important in the Service-Disabled Veteran-Owned (SDVO) Small Business Concern (SBC) Program? (a) Contracting...

Mixed species biofilms of Fusobacterium necrophorum and Porphyromonas levii impair the oxidative response of bovine neutrophils in vitro.

PubMed

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.

Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients.

PubMed

Wang, Peng; Chen, Fei-Xue; Du, Chao; Li, Chang-Qing; Yu, Yan-Bo; Zuo, Xiu-Li; Li, Yan-Qing

2015-05-22

Colonic brain-derived neurotrophic factor (BDNF) plays an essential role in pathogenesis of abdominal pain in diarrhea-predominant irritable bowel syndrome (IBS-D), but regulation on its expression remains unclear. We investigated the role of fecal supernatants (FSN) from IBS-D patients on regulating BDNF expression in colonic epithelial cells of human and mice. Using human Caco-2 cells, we found that IBS-D FSN significantly increased BDNF mRNA and protein levels compared to control FSN, which were remarkably suppressed by the serine protease inhibitor. To further explore the potential mechanisms, we investigated the impact of protease-activated receptor-2 (PAR-2) on BDNF expression. We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation. Knockdown of PAR-2 significantly inhibited IBS-D FSN-induced upregulation of BDNF. Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression. To confirm these results, we intracolonically infused IBS-D or control FSN in mice and found that IBS-D FSN significantly elevated colonic BDNF and visceral hypersensitivity in mice, which were both suppressed by the inhibitor of serine protease or antagonist of PAR-2. Together, our data indicate that activation of PAR-2 signaling by IBS-D FSN promotes expression of colonic BDNF, thereby contributing to IBS-like visceral hypersensitivity.

A post-processing method to simulate the generalized RF sheath boundary condition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myra, James R.; Kohno, Haruhiko

For applications of ICRF power in fusion devices, control of RF sheath interactions is of great importance. A sheath boundary condition (SBC) was previously developed to provide an effective surface impedance for the interaction of the RF sheath with the waves. The SBC enables the surface power flux and rectified potential energy available for sputtering to be calculated. For legacy codes which cannot easily implement the SBC, or to speed convergence in codes which do implement it, we consider here an approximate method to simulate SBCs by post-processing results obtained using other, e.g. conducting wall, boundary conditions. The basic approximationmore » is that the modifications resulting from the generalized SBC are driven by a fixed incoming wave which could be either a fast wave or a slow wave. Finally, the method is illustrated in slab geometry and compared with exact numerical solutions; it is shown to work very well.« less

A post-processing method to simulate the generalized RF sheath boundary condition

DOE PAGES

Myra, James R.; Kohno, Haruhiko

2017-10-23

For applications of ICRF power in fusion devices, control of RF sheath interactions is of great importance. A sheath boundary condition (SBC) was previously developed to provide an effective surface impedance for the interaction of the RF sheath with the waves. The SBC enables the surface power flux and rectified potential energy available for sputtering to be calculated. For legacy codes which cannot easily implement the SBC, or to speed convergence in codes which do implement it, we consider here an approximate method to simulate SBCs by post-processing results obtained using other, e.g. conducting wall, boundary conditions. The basic approximationmore » is that the modifications resulting from the generalized SBC are driven by a fixed incoming wave which could be either a fast wave or a slow wave. Finally, the method is illustrated in slab geometry and compared with exact numerical solutions; it is shown to work very well.« less

Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

PubMed

Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

2013-12-01

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

PubMed

Heenen, M; Giacomoni, P U; Golstein, P

2001-10-01

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an

Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons

PubMed Central

Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C.

2013-01-01

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. PMID:24058626

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

PubMed

Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

2013-01-01

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.

Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus, Neurospora intermedia: controlled synthesis and antibacterial activity.

PubMed

Hamedi, Sepideh; Shojaosadati, Seyed Abbas; Shokrollahzadeh, Soheila; Hashemi-Najafabadi, Sameereh

2014-02-01

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV-visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV-visible spectra of colloidal AgNPs. Comparing the UV-visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag(+) and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.

Immunomodulatory effect of non-viable components of probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus on holoxenic mice.

PubMed

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.

Effect of thalidomide and arsenic trioxide on the release of tumor necrosis factor-α and vascular endothelial growth factor from the KG-1a human acute myelogenous leukemia cell line.

PubMed

Girgis, Erian H; Mahoney, John P; Khalil, Rafaat H; Soliman, Magdi R

2010-07-01

Studies conducted in our lab have indicated that thalidomide cytotoxicity in the KG-1a human acute myelogenous leukemia (AML) cell line was enhanced by combining it with arsenic trioxide. The current investigation was conducted in order to evaluate the effect of thalidomide either alone or in combination with arsenic trioxide on the release of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) from this cell line in an attempt to clarify its possible cytotoxic mechanism(s). Human AML cell line KG-1a was used in this study. The cells were cultured for 48 h in the presence or absence of thalidomide (5 mg/l), and or arsenic trioxide (4 μM). The levels of TNF-α and VEGF in the supernatant were determined by ELISA. Results obtained indicate that the levels of TNF-α in the supernatant of KG-1a cell cultures incubated with thalidomide, arsenic trioxide, or combination were statistically lower than those observed in the supernatant of control cells (2.89, 5.07, 4.15 and 16.88 pg/ml, respectively). However, the levels of VEGF in the supernatant of thalidomide-treated cells were statistically higher than those in the supernatant of control cells (69.61 vs. 11.48 pg/l). Arsenic trioxide, whether alone or in combination with thalidomide, did not produce any statistically significant difference in the levels of VEGF as compared to the control or thalidomide-treated cell supernatant. These findings indicate that thalidomide and the arsenic trioxide inhibition of TNF-α production by KG-1a cells may play an important role in their cytotoxic effect.

Grain dust induces IL-8 production from bronchial epithelial cells: effect on neutrophil recruitment.

PubMed

Park, H S; Suh, J H; Kim, S S; Kwon, O J

2000-06-01

There have been several investigations suggesting an involvement of activated neutrophils in the development of grain dust (GD)-induced occupational asthma. Interleukin-8 in the sputa from GD-induced asthmatic patients increased significantly after the exposure to GD. To confirm IL-8 production from bronchial epithelial cells when exposed to GD, and to evaluate the role of IL-8 on neutrophil recruitment. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four different concentrations ranging from 1 to 200 microg/mL of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production and neutrophil chemotaxis, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant derived from subjects with GD-induced asthma exposed to 10 microg/mL of GD, and then compared with those without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity of the culture supernatant was determined by modified Boyden chamber method. Interleukin-8 production and neutrophil chemotactic activity from bronchial epithelial cells significantly increased with additions of GD in a dose-dependent manner (P < .05, respectively), and were significantly augmented with additions of PBMC supernatant (P < .05, respectively) at each concentration. Close correlation was noted between neutrophil chemotactic activity and IL-8 level (r = 0.87, P < .05). Compared with the untreated sample, pre-treatment of anti-IL-8 antibody induced a significant suppression (up to 67.2%) of neutrophil chemotactic activity in a dose-dependent manner. These results suggest that IL-8 produced from bronchial epithelial cells may be a major cytokine, which induces neutrophil migration into the airways when exposed to GD.

Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells stimulated with Strongylus vulgaris antigen.

PubMed

Dennis, V A; Klei, T R; Chapman, M R

1993-07-01

Supernatants generated by stimulation of peripheral blood mononuclear cells (PBMC) from Strongylus vulgaris sensitized or immunized ponies were assayed in vitro for eosinophil chemotactic activity (ECA) using the filter system in blind well chambers. The supernatants from these cultures were chemotactic for eosinophils, but not for neutrophils. Supernates from cultures of unsensitized PBMC stimulated with S. vulgaris antigen were not chemotactic for eosinophils. ECA was first detected in culture supernatants after 1.5 h of incubation and was dependent on both antigen and PBMC concentrations, but independent of serum concentrations. Both female and male S. vulgaris worm antigens stimulated ECA production from sensitized PBMC. ECA was not induced by in vitro stimulation of sensitized S. vulgaris PBMC by female Strongylus edentatus worm antigen. Partial characterization of the eosinophil chemotactic cytokine showed it to be nondialyzable, greater than 8000 molecular weight (MW), and sensitive to heating (56 and 95 degrees C), trypsin, and sodium metaperiodate treatments, suggesting that the cytokine is a protein containing some essential carbohydrate moieties. The cytokine described in this paper could partially contribute to the in vivo blood and tissue eosinophilia in experimental S. vulgaris infection.

The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis

DTIC Science & Technology

2007-03-23

as determined by sucrose density gradient flotation and immunoprecipitation analysis. However, co-expression of F and G along with M revealed a...total protein detected (total lysate + supernatant). Experiments described in Chapter 4 did not 35 include a flotation step. Rather, following...culture supernatant were prepared as described above except the top 1.4 ml of the flotation gradient was mixed with 3 ml of PBS and centrifuged for an

Metagenomic analysis of microbial consortium from natural crude oil that seeps into the marine ecosystem offshore Southern California

PubMed Central

Hawley, Erik R.; Piao, Hailan; Scott, Nicole M.; Malfatti, Stephanie; Pagani, Ioanna; Huntemann, Marcel; Chen, Amy; Glavina del Rio, Tijana; Foster, Brian; Copeland, Alex; Jansson, Janet; Pati, Amrita; Tringe, Susannah; Gilbert, Jack A.; Lorenson, Thomas D.; Hess, Matthias

2014-01-01

Crude oils can be major contaminants of the marine ecosystem and microorganisms play a significant role in the degradation of its main constituents. To increase our understanding of the microbial hydrocarbon degradation process in the marine ecosystem, we collected crude oil from an active seep area located in the Santa Barbara Channel (SBC) and generated a total of about 52 Gb of raw metagenomic sequence data. The assembled data comprised ~500 Mb, representing ~1.1 million genes derived primarily from chemolithoautotrophic bacteria. Members of Oceanospirillales, a bacterial order belonging to the Deltaproteobacteria, recruited less than 2% of the assembled genes within the SBC metagenome. In contrast, the microbial community associated with the oil plume that developed in the aftermath of the Deepwater Horizon (DWH) blowout in 2010, was dominated by Oceanospirillales, which comprised more than 60% of the metagenomic data generated from the DWH oil plume. This suggests that Oceanospirillales might play a less significant role in the microbially mediated hydrocarbon conversion within the SBC seep oil compared to the DWH plume oil. We hypothesize that this difference results from the SBC oil seep being mostly anaerobic, while the DWH oil plume is aerobic. Within the Archaea, the phylum Euryarchaeota, recruited more than 95% of the assembled archaeal sequences from the SBC oil seep metagenome, with more than 50% of the sequences assigned to members of the orders Methanomicrobiales and Methanosarcinales. These orders contain organisms capable of anaerobic methanogenesis and methane oxidation (AOM) and we hypothesize that these orders – and their metabolic capabilities – may be fundamental to the ecology of the SBC oil seep. PMID:25197496

Metagenomic analysis of microbial consortium from natural crude oil that seeps into the marine ecosystem offshore Southern California

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawley, Erik R.; Piao, Hailan; Scott, Nicole M.

2014-01-02

Crude oils can be major contaminants of the marine ecosystem and microorganisms play a significant role in the degradation of the main constituents of crude oil. To increase our understanding of the microbial hydrocarbon degradation process in the marine ecosystem, we collected crude oil from an active seep area located in the Santa Barbara Channel (SBC) and generated a total of about 52 Gb of raw metagenomic sequence data. The assembled data comprised ~500 Mb, representing ~1.1 million genes derived primarily from chemolithoautotrophic bacteria. Members of Oceanospirillales, a bacterial order belonging to the Deltaproteobacteria, recruited less than 2% of themore » assembled genes within the SBC metagenome. In contrast, the microbial community associated with the oil plume that developed in the aftermath of the Deepwater Horizon (DWH) blowout in 2010, was dominated by Oceanospirillales, which comprised more than 60% of the metagenomic data generated from the DWH oil plume. This suggests that Oceanospirillales might play a less significant role in the microbially mediated hydrocarbon conversion within the SBC seep oil compared to the DWH plume oil. We hypothesize that this difference results from the SBC oil seep being mostly anaerobic, while the DWH oil plume is aerobic. Within the Archaea, the phylum Euryarchaeota, recruited more than 95% of the assembled archaeal sequences from the SBC oil seep metagenome, with more than 50% of the sequences assigned to members of the orders Methanomicrobiales and Methanosarcinales. These orders contain organisms capable of anaerobic methanogenesis and methane oxidation (AOM) and we hypothesize that these orders and their metabolic capabilities may be fundamental to the ecology of the SBC oil seep.« less

Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue.

PubMed

Chabaud, Stéphane; Marcoux, Thomas-Louis; Deschênes-Rompré, Marie-Pier; Rousseau, Alexandre; Morissette, Amélie; Bouhout, Sara; Bernard, Geneviève; Bolduc, Stéphane

2015-11-01

The time needed to produce engineered tissue is critical. A self-assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L-arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis. Copyright © 2013 John Wiley & Sons, Ltd.

Beta-carotene-rich carotenoid-protein preparation and exopolysaccharide production by Rhodotorula rubra GED8 grown with a yogurt starter culture.

PubMed

Frengova, Ginka I; Simova, Emilina D; Beshkova, Dora M

2006-01-01

The underlying method for obtaining a beta-carotene-rich carotenoid-protein preparation and exopolysaccharides is the associated cultivation of the carotenoid-synthesizing lactose-negative yeast strain Rhodotorula rubra GED8 with the yogurt starter culture (Lactobacillus bulgaricus 2-11 + Streptococcus thermophilus 15HA) in whey ultrafiltrate (45 g lactose/l) with a maximum carotenoid yield of 13.37 mg/l culture fluid on the 4.5th day. The chemical composition of the carotenoid-protein preparation has been identified. The respective carotenoid and protein content is 497.4 microg/g dry cells and 50.3% per dry weight, respectively. An important characteristic of the carotenoid composition is the high percentage (51.1%) of beta-carotene (a carotenoid pigment with the highest provitamin A activity) as compared to 12.9% and 33.7%, respectively, for the other two individual pigments--torulene and torularhodin. Exopolysaccharides (12.8 g/l) synthesized by the yeast and lactic acid cultures, identified as acid biopolymers containing 7.2% glucuronic acid, were isolated in the cell-free supernatant. Mannose, produced exclusively by the yeast, predominated in the neutral carbohydrate biopolymer component (76%). The mixed cultivation of R. rubra GED8 with the yogurt starter (L. bulgaricus 2-11 + S. thermophilus 15HA) in ultrafiltrate under conditions of intracellular production of maximum amount of carotenoids and exopolysaccharides synthesis enables combined utilization of the culture fluid from the fermentation process.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

PubMed

Ruiz, Christian; Kustermann, Stefan; Pietilae, Elina; Vlajnic, Tatjana; Baschiera, Betty; Arabi, Leila; Lorber, Thomas; Oeggerli, Martin; Savic, Spasenija; Obermann, Ellen; Singer, Thomas; Rothschild, Sacha I; Zippelius, Alfred; Roth, Adrian B; Bubendorf, Lukas

2016-01-01

The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

48 CFR 1852.219-84 - Limitation of the principal investigator-STTR program.

Code of Federal Regulations, 2010 CFR

2010-10-01

... SBC/Contractor or RI. (b) The PI is considered to be key personnel in the performance of this contract. The SBC/Contractor, whether or not the employer of the PI, shall exercise primary management direction and control over the PI and be overall responsible for the PI's performance under this contract...

Staff Behavior toward Children and Adolescents in a Residential Facility: A Self-Report Questionnaire

ERIC Educational Resources Information Center

Huitink, C.; Embregts, P. J. C. M.; Veerman, J. W.; Verhoeven, L.

2011-01-01

The purpose of the present study was to examine psychometric properties of the Staff Behavior toward Clients questionnaire (SBC), a self-report measure for care staff working with children and adolescents with mild to borderline intellectual disabilities in residential care. Ninety-nine care staff completed the SBC and the Strengths and…

Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

EPA Science Inventory

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

Further analyses of human kidney cell populations separated on the Space Shuttle

NASA Technical Reports Server (NTRS)

Stewart, Robin M.; Todd, Paul; Cole, Kenneth D.; Morrison, Dennis R.

1992-01-01

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantization of plasminogen activators in these samples. These assays of frozen-culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator-producing cells from nonproducing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one other.

The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions

PubMed Central

Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J.; Infanger, Manfred; Grimm, Daniela

2015-01-01

We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: “NanoRacks-CellBox-Thyroid Cancer”. The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell–cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids. PMID:26633361

Influence of two barrier membranes on staged guided bone regeneration and osseointegration of titanium implants in dogs. Part 2: augmentation using bone graft substitutes.

PubMed

Mihatovic, Ilja; Becker, Jürgen; Golubovic, Vladimir; Hegewald, Andrea; Schwarz, Frank

2012-03-01

To assess the influence of two barrier membranes and two bone graft substitutes on staged guided bone regeneration and osseointegration of titanium implants in dogs. Saddle-type defects were prepared in the lower jaws of 6 fox hounds and randomly filled with a natural bone mineral (NBM) and a biphasic calcium phosphate (SBC) and allocated to either an in situ gelling polyethylene glycol (PEG) or a collagen membrane (CM). At 8 weeks, modSLA titanium implants were inserted and left to heal in a submerged position. At 8+2 weeks, respectively, dissected blocks were processed for histomorphometrical analysis (e.g., mineralized tissue [MT], bone-to-implant contact [BIC]). The mean MT values (mm2) and BIC values (%) tended to be higher in the PEG groups (MT: NBM [3.4±1.7]; SBC [4.2±2]/BIC: NBM [67.7±16.9]; SBC [66.9±17.8]) when compared with the corresponding CM groups (MT: NBM [2.5±0.8]; SBC [2.3±1.6]/BIC: NBM [54.1±22.6]; SBC [61±8.7]). These differences, however, did not reach statistical significance. It was concluded that all augmentation procedures investigated supported bone regeneration and staged osseointegration of modSLA titanium implants. © 2011 John Wiley & Sons A/S.

Sequential production of leukaemia inhibitory factor by blood cell culture in patients with ARDS.

PubMed

Gruson, D; Hilbert, G; Juzan, M; Taupin, J L; Coulon, V; Moreau, J F; Gualde, N; Gbikpi-Benissan, G

1998-04-01

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine integrated in cytokine networks and its concentration has been shown to be elevated in bronchoalveolar lavage fluid of patients with the acute respiratory distress syndrome (ARDS). The aim of our study was to evaluate the production of LIF by culturing blood cells from patients with ARDS. 8 patients with ARDS, 8 patients with pneumonia and 5 healthy subjects. The blood samples were taken on day 1 after onset of ARDS. LIF was measured, in the cell-free supernatant, with an enzyme-linked immunosorbent assay after 24 h, 48 h and 72 h of blood cell culture. LIF was detectable in some patients in the ARDS group: at i) at 24 h and 48 h: in 2 patients ii) at 72 h in 4/5 patients (140 +/- 231 pg/ml). Only in the 4 patients in whom LIF was measured at 72 h was ARDS associated with the multiple organ dysfunction syndrome. Furthermore, among the 5 patients with ARDS who subsequently died, 4 had a detectable LIF. We have observed that LIF was produced only in ARDS, but not in all patients. The production of LIF seems to be a good indicator of the severity of ARDS. These preliminary results must be confirmed by a larger study.

48 CFR 1852.219-84 - Limitation of the principal investigator-STTR program.

Code of Federal Regulations, 2011 CFR

2011-10-01

...—STTR Program (OCT 2006) (a) The primary employment of the principal investigator (PI) identified in... in the employ of the SBC/Contractor or RI. (b) The PI is considered to be key personnel in the performance of this contract. The SBC/Contractor, whether or not the employer of the PI, shall exercise...

Security Engineering Project - System Aware Cyber Security for an Autonomous Surveillance System On Board an Unmanned Aerial Vehicle

DTIC Science & Technology

2014-01-31

59 Figure 26. Raspberry Pi SBC... Raspberry Pi single compute board (SBC) (see section 3.3.1.2). These snoopers can intercept the serial data, decode the information, and retransmit the...data. The Raspberry Pi contains two serial ports that allow receiving, altering, and retransmitting of serial data. These monitor points will provide

48 CFR 1852.219-84 - Limitation of the principal investigator-STTR program.

Code of Federal Regulations, 2012 CFR

2012-10-01

...—STTR Program (OCT 2006) (a) The primary employment of the principal investigator (PI) identified in... in the employ of the SBC/Contractor or RI. (b) The PI is considered to be key personnel in the performance of this contract. The SBC/Contractor, whether or not the employer of the PI, shall exercise...

48 CFR 1852.219-84 - Limitation of the principal investigator-STTR program.

Code of Federal Regulations, 2013 CFR

2013-10-01

...—STTR Program (OCT 2006) (a) The primary employment of the principal investigator (PI) identified in... in the employ of the SBC/Contractor or RI. (b) The PI is considered to be key personnel in the performance of this contract. The SBC/Contractor, whether or not the employer of the PI, shall exercise...

48 CFR 1852.219-84 - Limitation of the principal investigator-STTR program.

Code of Federal Regulations, 2014 CFR

2014-10-01

...—STTR Program (OCT 2006) (a) The primary employment of the principal investigator (PI) identified in... in the employ of the SBC/Contractor or RI. (b) The PI is considered to be key personnel in the performance of this contract. The SBC/Contractor, whether or not the employer of the PI, shall exercise...

101. View of transmitter building no. 102, missile warning operation ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

101. View of transmitter building no. 102, missile warning operation center, close up view of DRED (detection radar environmental display) console in operation showing target. Official photograph BMEWS Project by Hansen, 14 March 1963, clear as negative no. A-8803. - Clear Air Force Station, Ballistic Missile Early Warning System Site II, One mile west of mile marker 293.5 on Parks Highway, 5 miles southwest of Anderson, Anderson, Denali Borough, AK

Bioleaching of rare earth elements from waste phosphors and cracking catalysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reed, David W.; Fujita, Yoshiko; Daubaras, Dayna L.

Four microbial cultures were evaluated for organic acid production and their potential utility for leaching of rare earth elements (REE) from retorted phosphor powder (RPP) and spent fluidized cracking catalyst (FCC). Three of the cultures (2 bacterial, 1 fungal) were isolated from environmental and industrial materials known to contain rare earth elements. The other was the well-known and industrially important bacterium Gluconobacter oxydans. Gluconic acid was the predominant identified organic acid produced by all of the cultures; citric and acetic acid were among the other acids detected. There was also maximum REE leaching by cell free culture supernatants obtained withmore » Gluconobacter and the FCC; 49% of total REE was recovered, with preferential recovery of lanthanum over cerium. The phosphor powder was more difficult to leach; only ~2 % total REE was leached from RPP with Gluconobacter. Tests with the RPP indicated that the extent of REE solubilization was similar whether whole cell cultures or cell-free supernatants were used. However, Gluconobacter cell-free culture supernatants with 10-15 mM gluconic acid outperformed abiotically prepared leaching solutions with 30 mM gluconic acid concentrations. Abiotic tests showed that increasing gluconic acid concentrations increased leaching efficiency; for example, total REE leaching from FCC increased from 24 to 36 to 45% when gluconic acid was increased from 10 to 30 to 90 mM. Our research shows that utilizing microorganisms that produce gluconic acid can result in effective leaching of REE from waste materials, and optimizing gluconic acid production will improve recovery.« less

Bioleaching of rare earth elements from waste phosphors and cracking catalysts

DOE PAGES

Reed, David W.; Fujita, Yoshiko; Daubaras, Dayna L.; ...

2016-08-22

Four microbial cultures were evaluated for organic acid production and their potential utility for leaching of rare earth elements (REE) from retorted phosphor powder (RPP) and spent fluidized cracking catalyst (FCC). Three of the cultures (2 bacterial, 1 fungal) were isolated from environmental and industrial materials known to contain rare earth elements. The other was the well-known and industrially important bacterium Gluconobacter oxydans. Gluconic acid was the predominant identified organic acid produced by all of the cultures; citric and acetic acid were among the other acids detected. There was also maximum REE leaching by cell free culture supernatants obtained withmore » Gluconobacter and the FCC; 49% of total REE was recovered, with preferential recovery of lanthanum over cerium. The phosphor powder was more difficult to leach; only ~2 % total REE was leached from RPP with Gluconobacter. Tests with the RPP indicated that the extent of REE solubilization was similar whether whole cell cultures or cell-free supernatants were used. However, Gluconobacter cell-free culture supernatants with 10-15 mM gluconic acid outperformed abiotically prepared leaching solutions with 30 mM gluconic acid concentrations. Abiotic tests showed that increasing gluconic acid concentrations increased leaching efficiency; for example, total REE leaching from FCC increased from 24 to 36 to 45% when gluconic acid was increased from 10 to 30 to 90 mM. Our research shows that utilizing microorganisms that produce gluconic acid can result in effective leaching of REE from waste materials, and optimizing gluconic acid production will improve recovery.« less

Optimal turnaround time for direct identification of microorganisms by mass spectrometry in blood culture.

PubMed

Randazzo, Adrien; Simon, Marc; Goffinet, Pierre; Classen, Jean-François; Hougardy, Nicolas; Pierre, Pascal; Kinzinger, Philippe; Mauel, Etienne; Goffinet, Jean-Sébastien

2016-11-01

During the past few years, several studies describing direct identification of bacteria from blood culture using mass spectrometry have been published. These methods cannot, however, be easily integrated into a common laboratory workflow because of the high hands-on time they require. In this paper, we propose a new method of identification with a short hands-on time and a turnaround time shorter than 15min. Positive blood bottles were homogenised and 600μL of blood were transferred to an Eppendorf tube where 600μL of lysis buffer were added. After homogenisation, a centrifugation step of 4min at 10,500g was performed and the supernatant was discarded. The pellet was then washed and loaded in quadruplicate into wells of a Vitek® MS-DS plate. Each well was covered with a saturated matrix solution and a MALDI-TOF mass spectrometry analysis was performed. Species were identified using the software Myla 3.2.0-2. We analysed 266 positive blood culture bottles. A microorganism grew in 261 cultures, while five bottles remained sterile after 48h of incubation in subculture. Our method reaches a probability of detection at the species level of 77.8% (203/261) with a positive predictive value of 99.5% (202/203). We developed a new method for the identification of microorganisms using mass spectrometry, directly performed from a positive blood culture. This method has short hands-on time and turnaround time and can easily take place in the workflow of a laboratory, with comparable results in performance with other methods reported in the literature. Copyright © 2016 Elsevier B.V. All rights reserved.

Elucidating the Mechanism of Gain of Toxic Function From Mutant C1 Inhibitor Proteins in Hereditary Angioedema

DTIC Science & Technology

2017-10-01

hours after re-seeding, the supernatant was harvested. C1INH protein in the culture supernatant was measured by ELISA . As shown in the table below...0.551 0.527 0.533 H 0.599 0.593 0.586 0.572 0.577 0.577 0.567 0.560 0.554 0.551 0.552 0.597 The mean ± SD C1INH ELISA OD was 0.553 ± 0.021, and

Proliferation of smooth muscle cells stimulated by Porphyromonas gingivalis is inhibited by apple polyphenol.

PubMed

Inaba, Hiroaki; Tagashira, Motoyuki; Kanda, Tomomasa; Amano, Atsuo

2011-11-01

Porphyromonas gingivalis (Pg) is thought to be involved in the progression of occlusive arterial lesions, whereas vascular smooth muscle cell (SMC) proliferation is considered to be involved in occlusive arterial disease. We previously showed that bacteremia caused by Pg infection induced proliferation of mouse aortic SMCs. Furthermore, human SMCs stimulated with human plasma incubated with Pg showed a marked transformation from the contractile to proliferative phenotype. In the present study, we examine the involvement of Pg gingipains and fimbriae in induction of the SMC transformation and proliferation, and effective inhibitors. Pg strains including gingipain- and fimbria-null mutants were incubated in human plasma, after which the bacteria were removed and the supernatants were added to cultured SMCs. To evaluate the effects of inhibitors, Pg organisms were incubated in plasma in the presence of apple polyphenol (AP), epigallocatechin gallate, KYT-1 (Arg-gingipain inhibitor), and KYT-36 (Lys-gingipain inhibitor). Plasma supernatants from wild-type and fimbria-mutant cultures markedly stimulated cellular proliferation, whereas those containing gingipain-null mutants showed negligible effects. SMC proliferation was also induced by plasma treated with trypsin. Furthermore, plasma supernatants cultured in the presence of KYT-1/KYT-36 and AP showed significant inhibitory effects on SMC proliferation, whereas cultures with epigallocatechin gallate did not. Our results suggest that Pg gingipains are involved in the induction of SMC transformation and proliferation, whereas this was inhibited by AP.

Effect of Microcystin-LR on Cultured Rat Endothelial Cells

DTIC Science & Technology

1990-02-01

silymarin (SM) [81, in \\ticrocystin. silymarin . order to determine if thcse agents could prevent changes 4 induced by supernatants derived from MCLR...commercially from the indicated sources: silymarin (SM) such as deformation of ceillsblebbing) rapid risc in intra- (Aldrich, Milwaukee. WVI

Phanerochaete flavido-alba Laccase Induction and Modification of Manganese Peroxidase Isoenzyme Pattern in Decolorized Olive Oil Mill Wastewaters

PubMed Central

Pérez, J.; de la Rubia, T.; Hamman, O. Ben; Martínez, J.

1998-01-01

Lignin-degrading enzymes were partially purified from supernatant solutions obtained from Phanerochaete flavido-alba-decolorized olive oil mill wastewaters (OMW). The dominant enzymes, manganese peroxidases, exhibited different isoform patterns in decolorized OMW-containing cultures than in residue-free samples. Laccase induction was also detected in OMW-containing cultures but not in control cultures. PMID:9647858

Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

PubMed

Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

2011-01-01

SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

T cell-replacing factor for glucocorticosteroid-induced immunoglobulin production. A unique steroid-dependent cytokine

PubMed Central

1983-01-01

Glucocorticosteroids (GCS) added to otherwise unstimulated cultures of human peripheral blood mononuclear cells (PBMC) induce the synthesis and secretion of all classes of immunoglobulin. The magnitude of this response is similar to that seen with other polyclonal B cell activators such as pokeweed mitogen (PWM), and like that of PWM, the steroid effect is dependent on both T cells and monocytes. To determine the cellular target for GCS in these cultures, separated populations of T cells and non-T cells were preincubated with steroids and then recombined. No immunoglobulin was produced in any of these preincubation experiments. As a different approach to this question, supernatants were collected from various cell populations following stimulation with PWM, concanavalin A (Con A), phytohemagglutinin (PHA), alloantigens, or GCS. These supernatants were tested for their effects on GCS-induced Ig production by B cells. Supernatants from 3-d cultures of unstimulated, as well as GCS-treated, PBMC contained a T cell- replacing factor that permitted T-depleted PBMC to produce Ig upon steroid stimulation. This supernatant factor (TRF-S) could be produced in the absence of steroid stimulation, but both the factor and GCS were necessary for the induction of Ig synthesis. Production of the TRF-S required the presence of both T cells and adherent cells in culture and was found in the highest concentrations at 3-4 d of culture. Supernatants from cultures stimulated with PWM, PHA, Con A, and alloantigens did not contain detectable TRF-S activity, and TRF-S was unable to replace helper T cells for PWM-induced Ig production. TRF-S required the presence of adherent cells in the T cell-depleted responder population for its action. Further, it was effective in inducing Ig production along with GCS in the presence of a sufficient concentration of cyclosporin A to block all T cell helper activity for primary responses of PBMC to PWM or GCS. TRF-S was inactivated by trypsin treatment

Characterization and Clinical Implication of Th1/Th2/Th17 Cytokines Produced from Three-Dimensionally Cultured Tumor Tissues Resected from Breast Cancer Patients

PubMed Central

Kiyomi, Anna; Makita, Masujiro; Ozeki, Tomoko; Li, Na; Satomura, Aiko; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Iwase, Takuji; Hirano, Toshihiko

2015-01-01

OBJECTIVES: Several cytokines secreted from breast cancer tissues are suggested to be related to disease prognosis. We examined Th1/Th2/Th17 cytokines produced from three-dimensionally cultured breast cancer tissues and related them with patient clinical profiles. METHODS: 21 tumor tissues and 9 normal tissues surgically resected from breast cancer patients were cultured in thermoreversible gelatin polymer–containing medium. Tissue growth and Th1/Th2/Th17 cytokine concentrations in the culture medium were analyzed and were related with hormone receptor expressions and patient clinical profiles. RESULTS: IL-6 and IL-10 were expressed highly in culture medium of both cancer and normal tissues. However, IFN-γ, TNF-α, IL-2, and IL-17A were not detected in the supernatant of the three-dimensionally cultured normal mammary gland and are seemed to be specific to breast cancer tissues. The growth abilities of hormone receptor–negative cancer tissues were significantly higher than those of receptor-positive tissues (P = 0.0383). Cancer tissues of stage ≥ IIB patients expressed significantly higher TNF-α levels as compared with those of patients with stage < IIB (P = 0.0096). CONCLUSIONS: The tumor tissues resected from breast cancer patients can grow in the three-dimensional thermoreversible gelatin polymer culture system and produce Th1/Th2/Th17 cytokines. Hormone receptor–positive cancer tissues showed less growth ability. TNF-α is suggested to be a biomarker for the cancer stage. PMID:26310378

An Asymmetric Birdcage Coil for Small-animal MR Imaging at 7T

PubMed Central

Kim, Kyoung-Nam; Han, Sang-Doc; Seo, Jeung-Hoon; Heo, Phil; Yoo, Dongkyeom; Im, Geun Ho; Lee, Jung Hee

2017-01-01

The birdcage (BC) coil is currently being utilized for uniform radiofrequency (RF) transmit/receive (Tx/Rx) or Tx-only configuration in many magnetic resonance (MR) imaging applications, but insufficient magnetic flux (|B1|) density and their non-uniform distribution still exists in high-field (HF) environments. We demonstrate that the asymmetric birdcage (ABC) transmit/receive (Tx/Rx) volume coil, which is a modified standard birdcage (SBC) coil with the end ring split into two halves, is suitable for improving the |B1| sensitivity in 7T small-animal MR imaging. Cylindrical SBC and ABC coils with 35 mm diameter were constructed and bench tested for mouse body MR imaging at 300 MHz using a 7T scanner. To assess the ABC coil performance, computational electromagnetic (EM) simulation and 7T MR experiment were performed by using a cylindrical phantom and in vivo mouse body and quantitatively compared with the SBC coil in terms of |B1| distribution, RF transmit (|B1+|) field, and signal-to-noise ratio (SNR). The bench measurements of the two BC coils are similar, yielding a quality value (Q-value) of 74.42 for the SBC coil and 77.06 for the ABC coil. The computational calculation results clearly show that the proposed ABC coil offers superior |B1| field and |B1+| field sensitivity in the central axial slice compared with the SBC coil. There was also high SNR and uniformly distributed flip angle (FA) under the loaded condition of mouse body in the 7T experiment. Although ABC geometry allows a further increase in the |B1| field and |B1+| field sensitivity in only the central axial slice, the geometrical modification of the SBC coil can make a high performance RF coil feasible in the central axial slice and also make target imaging possible in the diagonal direction. PMID:27725573

Psychological Change in Distressed Young People Who Do Not Receive Counselling: Does Improvement Happen Anyway?

ERIC Educational Resources Information Center

Daniunaite, Akvile; Ahmad Ali, Zenib; Cooper, Mick

2012-01-01

The aim of this article is to explore self-healing processes in young people, and to develop an understanding of the effects of school-based counselling (SBC), by analysing changes in young people who did not receive this intervention. Semi-structured interviews were conducted with 14 young people on a waiting list for SBC. Participants…

13 CFR 126.504 - When is a concern removed from the List?

Code of Federal Regulations, 2010 CFR

2010-01-01

... any time that a HUBZone SBC is not qualified, SBA may de-certify the HUBZone SBC, remove the concern... resolution of a protest also may result in de-certification and removal from the List, and the imposition of... eligibility will result in immediate de-certification, removal from the List, and SBA may seek the imposition...

13 CFR 126.504 - When is a concern removed from the List?

Code of Federal Regulations, 2011 CFR

2011-01-01

... any time that a HUBZone SBC is not qualified, SBA may de-certify the HUBZone SBC, remove the concern... resolution of a protest also may result in de-certification and removal from the List, and the imposition of... eligibility will result in immediate de-certification, removal from the List, and SBA may seek the imposition...

Secretome of transmissible Pseudomonas aeruginosa AES-1R grown in a cystic fibrosis lung-like environment.

PubMed

Scott, Nichollas E; Hare, Nathan J; White, Melanie Y; Manos, Jim; Cordwell, Stuart J

2013-12-06

Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Modified sedimentation-dispersion model for solids in a three-phase slurry column

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, D.N.; Ruether, J.A.; Shah, Y.T.

1986-03-01

Solids distribution data for a three-phase, batch-fluidized slurry bubble column (SBC) are presented, using air as the gas phase, pure liquids and solutions as the liquid phase, and glass beads and carborundum catalyst powder as the solid phase. Solids distribution data for the three-phase SBC operated in a continuous mode of operation are also presented, using nitrogen as the gas phase, water as the liquid phase, and glass beads as the solid phase. A new model to provide a reasonable approach to predict solids concentration distributions for systems containing polydispersed solids is presented. The model is a modification of standardmore » sedimentation-dispersion model published earlier. Empirical correlations for prediction of hindered settling velocity and solids dispersion coefficient for systems containing polydispersed solids are presented. A new method of evaluating critical gas velocity (CGV) from concentrations of the sample withdrawn at the same port of the SBC is presented. Also presented is a new mapping for CGV which separates the two regimes in the SBC, namely, incomplete fluidization and complete fluidization.« less

Fabrication of spherical biochar by a two-step thermal process from waste potato peel.

PubMed

Yang, Xiao; Kwon, Eilhann E; Dou, Xiaomin; Zhang, Ming; Kim, Ki-Hyun; Tsang, Daniel C W; Ok, Yong Sik

2018-06-01

The aim of this study was to develop a new approach for the preparation of spherical biochar (SBC) by employing a two-step thermal technology to potato peel waste (PPW). Potato starch (PS), as a carbon-rich material with microscale spherical shape, was separated from PPW as a precursor to synthesizing SBC. The synthesis process comprised (1) pre-oxidization (preheating under air) of PS at 220 °C and (2) subsequent pyrolysis of the pretreated sample at 700 °C. Results showed that the produced SBC successfully retained the original PS morphology and that pre-oxidization was the key for its shape maintenance, as it reduced surface tension and enhanced structural stability. The SBC possessed excellent chemical inertness (high aromaticity) and uniform particle size (10-30 μm). Zero-cost waste material with a facile and easy-to-control process allows the method to be readily scalable for industrialization, while offering a new perspective on the full use of PPW. Copyright © 2018 Elsevier B.V. All rights reserved.

Biomimetic Synthesis of Selenium Nanospheres by Bacterial Strain JS-11 and Its Role as a Biosensor for Nanotoxicity Assessment: A Novel Se-Bioassay

PubMed Central

Dwivedi, Sourabh; AlKhedhairy, Abdulaziz A.; Ahamed, Maqusood; Musarrat, Javed

2013-01-01

Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate Pseudomonas aeruginosa strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO3 2−) up to 100 mM concentration with an EC50 value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO3 2− to insoluble red elemental selenium (Se0) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO3 2− to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO3 2− to elemental red Se0, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO3 2− bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point. PMID:23483909

Biomimetic synthesis of selenium nanospheres by bacterial strain JS-11 and its role as a biosensor for nanotoxicity assessment: a novel se-bioassay.

PubMed

Dwivedi, Sourabh; Alkhedhairy, Abdulaziz A; Ahamed, Maqusood; Musarrat, Javed

2013-01-01

Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate Pseudomonas aeruginosa strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO3(2-)) up to 100 mM concentration with an EC50 value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO3(2-) to insoluble red elemental selenium (Se(0)) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO3(2-) to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO3(2-) to elemental red Se(0), a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO3(2-) bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point.

Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

PubMed Central

Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

2016-01-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns.

PubMed

Szafran, Adam T; Mancini, Maureen G; Nickerson, Jeffrey A; Edwards, Dean P; Mancini, Michael A

2016-03-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytopathogenicity of Naegleria for cultured neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulford, D.E.

1985-01-01

The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile,more » pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.« less

[The influence of mycophenolate mofetil upon the maturation and allostimulatory activity of cultured dendritic cell progenitors and the effects of tolerance induction in allograft recipients].

PubMed

Han, Cong-hui; Li, Huai-fu; Wang, Yu-xin; Zhang, Ming; Wang, Yin; Yin, Ming; Min, Zhi-lian; Zheng, Ke-li

2005-05-25

To investigate the influence of mycophenolate mofetil (MMF) on the maturation and allostimulatory activity of cultured dendritic cell progenitors (DCPs) and to evaluate the efficacy of pretreatment of donor dendritic cells (DCs) with MMF in tolerance induction in allograft recipients and its possible mechanism. DCPs of Balb/c mice were cultured in culture fluid containing recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), and then divided into 4 groups: control group, MMF group, lipopolysaccharide (LPS) group, and MMF + LPS group. Seven days later, flow cytometry was used to analyze the phenotypes of the DCs. ELISA was used to examine the level of IL-12 in the supernatant. T cells from the spleens of C57/BL6 mice were cultured together with inactivated DCs from Balb/c mice, and added with [(3)H]-TdR. Mixed lymphocyte reaction (MLR) was analyzed. The DCs and MMF-DCs cultured for 5 days were co-cultured with T hybridoma cells of the line MF2.2D9 in culture fluid containing ovalbumin (OVA). Twenty-four hours later, the supernatant was collected and ELISA was used to measure the level of interleukin (IL)-12 so as to reflect the antigen-presenting ability of the DCs. OVA immunized C57/BL6 mice for 4 times. 21 days after T cells were collected from the spleens and co-incubated with DCs and MMF-DCs, [(3)H]-TdR was added and the values of counts per minute (cpm) were calculated so as to analyze the antigen-specific proliferation. Twenty-four Balb/c mice were randomly divided into 3 groups: group A (without treatment), group B (treatment with intravenous injection of untreated DCs of Balb/c mice), and group C (treatment with intravenous injection of DCs of Balb/c mice treated with MMF), and then transplanted with the hearts of C57/BL6 mice. The functions of the transplanted hearts were evaluated by touching the arterial pulse and histological examination. ELISA was used to detect the levels of Th1 cytokines, such as IL-12, IL-4, IL-10, IL-2, and

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.

PubMed

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, P<.0001). Sensitivity, specificity and diagnostic accuracy of anti-tTG detected by ICA were 84.5%, 100% and 88.9%, respectively. Using ICA, anti-tTG are detectable in duodenal culture 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.

Recent developments using TowerJazz SiGe BiCMOS platform for mmWave and THz applications

NASA Astrophysics Data System (ADS)

Kar-Roy, Arjun; Howard, David; Preisler, Edward J.; Racanelli, Marco

2013-05-01

In this paper, we report on the highest speed 240GHz/340GHz FT/FMAX NPN which is now available for product designs in the SBC18H4 process variant of TowerJazz's mature 0.18μm SBC18 silicon germanium (SiGe) BiCMOS technology platform. NFMIN of ~2dB at 50GHz has been obtained with these NPNs. We also describe the integration of earlier generation NPNs with FT/FMAX of 240GHz/280GHz into SBC13H3, a 0.13μm SiGe BiCMOS technology platform. Next, we detail the integration of the deep silicon via (DSV), through silicon via (TSV), high-resistivity substrate, sub-field stitching and hybrid-stitching capability into the 0.18μm SBC18 technology platform to enable higher performance and highly integrated product designs. The integration of SBC18H3 into a thick-film SOI substrate, with essentially unchanged FT and FMAX, is also described. We also report on recent circuit demonstrations using the SBC18H3 platform: (1) a 4-element phased-array 70-100GHz broadband transmit and receive chip with flat saturated power greater than 5dBm and conversion gain of 33dB; (2) a fully integrated W-band 9-element phase-controllable array with responsivity of 800MV/W and receiver NETD is 0.45K with 20ms integration time; (3) a 16-element 4x4 phased-array transmitter with scanning in both the E- and H-planes with maximum EIRP of 23-25 dBm at 100-110GHz; (4) a power efficient 200GHz VCO with -7.25dBm output power and tuning range of 3.5%; and (5) a 320GHz 16-element imaging receiver array with responsivity of 18KV/W at 315GHz, a 3dB bandwidth of 25GHz and a low NEP of 34pW/Hz1/2. Wafer-scale large-die implementation of the phased-arrays and mmWave imagers using stitching in TowerJazz SBC18 process are also discussed.

Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment.

PubMed

Wang, San-Lang; Wu, Pei-Chen; Liang, Tzu-Wen

2009-05-26

We have developed a culture system for efficient production of chitosanase by Bacillus sp. TKU004. TKU004 was cultivated by using squid pen powder as the sole carbon/nitrogen source. The effects of autoclave treatments of the medium on the production of chitosanase were investigated. Autoclave treatment of squid pen powder for 45 min remarkably promoted enzyme productivity. When the culture medium containing an initial squid pen powder concentration of 3% was autoclaved for 45 min, the chitosanase activity was optimal and reached 0.14-0.16 U/mL. In addition, extracellular surfactant-stable chitosanase was purified from the TKU004 culture supernatant. The antioxidant activity of TKU004 culture supernatant was determined through the scavenging ability of DPPH, with 70% per mL. With this method, we have shown that marine wastes can be utilized efficiently through prolonged autoclave treatments to generate a high value-added product, and have revealed its hidden potential in the production of functional foods.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Release of lysosomal enzymes in Candida albicans phagocytosis by rat peritoneal macrophages.

PubMed

Fontenla de Petrino, S E; Sirena, A

1984-02-15

The present paper reports the in vitro release of lysosomal enzymes in the supernatant of cultures of rat peritoneal macrophages, with the addition of Candida albicans cells. Macrophages were taken from the rat peritoneal cavity 72 hr after non-specific activation with Brain-Heart-Infusion (B.H.I.) broth containing 10% proteose-peptone No. 3. They were then cultured in Parker medium No. 199 (TC 199). After 24 hr a suspension of Candida albicans cells, in a determined concentration, was added to the peritoneal macrophage cultures. At that time, and during pre-determined periods, the following enzymes in the culture supernatants were studied using colorimetric methods: beta-glucuronidase, beta-galactosidase and acid phosphatase. It is concluded that, under identical conditions, the release of beta-galactosidase and acid phosphatase is higher than for beta-glucuronidase. The release rate of all three enzymes is the highest at a 6 hr incubation period, after which, a gradual decrease leads to the rate down to 50% at 24 hr.

Comprehensive Profiling of Immune Responses in MARV Survivors Demonstrates Robust Th1-Skewing with Short Lived Neutralizing Antibody Responses

DTIC Science & Technology

2017-03-29

Beyond IgG or IgM ELISAs performed for diagnostic purposes, virtually the entirety of the literature available regarding filovirus immune responses in...supernatants for an expanded cytokine analysis by ELISA . A representative set of flow plots for CD4 and CD8 T cell responses from a MARV survivor is shown in...performed a multiplex ELISA assay with the culture supernatants to analyze a broader range of cytokines. We focused on five cytokines that are germane

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

PubMed

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.

A numerical study on shear buckling capacity of Z-section steel purlin with opening

NASA Astrophysics Data System (ADS)

De'nan, Fatimah; Keong, Choong Kok; Hashim, Nor Salwani; Yuting, Ng

2017-10-01

Cold-formed Z-section steel purlin is one of the most commonly available steel purlin worldwide. A numerical study on Z-section steel purlin with opening under shear loading was carried out. Six (6) variables such as opening size, opening shape, section type, opening distance, opening position and opening arrangement were identified and tested to investigate their effect on shear capacity of Z-section steel purlin. Results indicated that the presence of web opening did not improve the shear behaviour of Z-section steel purlin. However, non-negligible improvement in terms of volume reduction was observed. Each 0.1D (where D is the section height) enlargement of opening size caused an approximate 10% drop in shear buckling capacity (SBC) while volume reduction (VR) increased exponentially. Diamond shape performed up to 55% better in SBC compared to circular shape opening but circular shape opening performed up to 24% better in VR. Sections with smaller section height had higher SBC compared to sections with bigger section height. Nevertheless, sections with bigger section height had higher VR. No significant difference in terms of shear buckling capacity was observed when opening distance was manipulated. Opening position had no effect on SBC and VR. Opening arrangement had no significant effect on SBC and VR. An optimal section of Z100-19 with 0.3D to 0.5D diamond shaped opening and an opening distance of 100mm centre to centre depending on design engineer's specification is proposed.

[Response of peripheral blood mononuclear leukocyte to nickel stimulation in patients with systemic and contact allergy to nickel].

PubMed

Czarnobilska, Ewa; Thor, Piotr; Kaszuba-Zwoinska, Jolanta; Słodowska-Hajduk, Zofia; Stobiecki, Marcin; Dyga, Wojciech; Wsołek, Katarzyna; Obtułowicz, Krystyna

2006-01-01

Nickel is knows as the most common cause of allergic contact dermatitis, as well as diffuse eczema, allergic rhinitis and bronchial asthma. The mechanism of contact allergy to nickel is well known. In spite of numerous investigations, the mechanism of systemic allergy to nickel is still not clear. 22 patients with positive patch tests to nickel were analyzed. On basis of clinical symptoms the patients were divided into two groups: 1. with contact allergy dermatitis to nickel--8 patients 2. with systemic allergy to nickel (allergic rhinitis and/or diffuse eczema--14 patients. The control group included non-atopic patients with negative patch test to nickel--6 patients. 10 ml of blood were taken from each patient and peripheral mononuclear blood cells (PMBC) were isolated. In PBMC culture, NiSO4 and PHA were stimulated. The control group was non-stimulated cells. The supernatants were collected after 3 and 6 days of culture and the levels of cytokines IL-5, 4 and IFNgamma were measured (ELISA). The concentration of IFNgamma in supernatants from stimulated as well as non-stimulated cells from patients with contact allergy to nickel was higher in comparison to the control group. The concentration of IL-5 in this group was low. There was an increase in the production of IFNgamma and IL-5 after NiSO4 stimulation in patients with systemic allergy to nickel. The higher concentration of IFNgamma in the same groups of patients investigated was in supernatants from the third day of PBMC culture were compared to the sixth day. After 3 and 6 days of culture, the concentration of IL-4 (ELISA) was below detection level in all supernatants analyzed. IFNgamma plays an essential role in the mechanism of developing of contact allergy to nickel; and IFNgamma as well as IL-5 play a role in the mechanism of developing systemic allergy to nickel. The third day of PBMC culture is more reliable for IFNgamma estimation.

An Information Processing Analysis of the Function of Conceptual Understanding in the Learning of Arithmetic Procedures

DTIC Science & Technology

1988-08-01

J. R. (1986). Knowledge compilation: The general learning mechanism. In R. S . Michalski, J. G. Carbonell, & T. M. Mitchell (Eds.), Machine learning...REPORTApproval for public release, 2b. DECLASSIFICATION I DOWNGRADING SCHEDULE d i s tri bution un limi ted 4. PERFORMING ORGANIZATION REPORT NUMBER( S ) S ...MONITORING ORGANIZATION REPORT NUMBER( S ) UPITT/LRDC/ONR/KUL-88-03 6a. NAME OF PERFORMING ORGANIZATION I6b. OFFICE SYMBOL 7a. NAME OF MONITORING

Table of Interplanar Spacings for Crystal-Structure Determinations by X-ray Diffraction with Molybdenum, Copper, Cobalt, Iron, and Chromium Radiations

DTIC Science & Technology

1945-10-01

8834. .8830 .8827 .8823 .8820 .8/316 .8813 .8810 .,8806 .8803 .8799 . 8796 :$;8; 08786 .8782 .8779 08775 leti307 1.0303 1.0299 1.0295 1.02 1 ?1002 7...Fe Kal Cr Kq . 15400 15~.1 154.2 1540 ?1~~. 1540 1*. 2 1*. 154.z 15409 155.0 155.1 155.2 1550 X55.z 155. 1550z 155. 155.z 155.9 * 8796 .8 993 ,88031

The Establishment of a Management Information Systems Research Center at the Naval Postgraduate School

DTIC Science & Technology

1989-09-01

Project WP 88-03 GDSS Technology in Practice: A Study D. Straub R. Beauclair WP 88-04 Interaction Analysis in GDSS Research: I. Zigurs Description of an...03 GDSS Technology in Practice: A Study (D. Straub and R. Beauclair ) 4. WP 88-04 Interaction Analysis in GDSS Research: Description of an Experience...Implementing an Information Architecture," Data Base, forthcoming. 25 Straub, D. W. and R. A. Beauclair . "A New Dimension to Decision Support

Performance evaluation of FlowCytomix assays to quantify cytokines in patients with rheumatoid arthritis

PubMed Central

Wang, Xuefeng; Dong, Liyang; Liang, Yong; Ni, Hongchang; Tang, Jun; Xu, Chengcheng; Zhou, Yuepeng; Su, Yuting; Wang, Jun; Chen, Deyu; Mao, Chaoming

2015-01-01

Objectives: To compare the cytokine profile in RA patients and healthy control by using two methods-FlowCytomix assay and traditional ELISA. Methods: Cytokine levels were evaluated by FlowCytomix assay and ELISA in serum and supernatants of peripheral blood mononuclear cells (PBMC) cultures with and without stimulation by phytohaemagglutinin (PHA). Results: The levels of IL-6, IL-1β, and TNF-α were significantly higher in sera of RA patients than those of healthy controls. The levels of IL-22, IL-6, IL-1β, TNF-α, and IL-10 were higher in unstimulated PBMC culture supernatant of RA patients than those of healthy controls. PHA stimulation significantly increased the production of proinflammatory cytokines from PBMC with RA patients. Compared with detectable cytokine levels in sera, cytokine concentration in the supernatant of PBMCs was remarkably higher. FlowCytomix and ELISA showed significant correlation in detecting cytokines. However, the FlowCytomix assay detected more cytokines than ELISA. Conclusion: The supernatant of PBMCs provide a fine condition for the study of cytokine production because of the lack of interference factors in sera. The FlowCytomix assay is more sensitive than ELISA in detecting cytokines from RA patients. Multiple cytokine signatures using FlowCytomix assay may represent a more realistic approach in the future of personalized medicine in RA. PMID:26629129

Primary culture system of adrenocortical cells from dogs to evaluate direct effects of chemicals on steroidogenesis.

PubMed

Morishita, K; Okumura, H; Ito, N; Takahashi, N

2001-08-28

The present study was conducted to confirm the usefulness of a primary culture system of adrenocortical cells from dogs for detecting the direct effects of the chemicals on adrenal cortex. Corticosteroid levels in the culture supernatant were measured using high-performance liquid chromatography (HPLC) following 24-h incubation with the chemicals. Ketoconazole, miconazole, metyrapone, aminoglutethimide, and 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (o,p-DDD), which were known to inhibit cortisol production were evaluated in this system. Both viable cells and corticosteroid levels were decreased by o,p-DDD treatment. Other chemicals showed various inhibition patterns of corticosteroid levels as follows without affecting cell viability. Ketoconazole decreased total corticosteroids level by mainly due to the decreases in cortisol and 11-deoxycortisol levels. Miconazole decreased cortisol and 11-deoxycortisol levels, however, slightly increased corticosterone level. Metyrapone decreased cortisol and corticosterone levels as 11-deoxycortisol and 11-deoxycorticosterone levels were increased. Aminoglutethimide decreased total corticosteroids level by mainly decreasing cortisol, corticosterone and 11-deoxycortisol levels. These results suggested that determination of the pattern of corticosteroid levels by HPLC in this system well reflected the mode of their action on steroidogenesis. Thus, we conclude this simple system was useful to determine the direct effects of chemicals on steroidogenesis in the adrenal cortex.

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons.

PubMed

Vaillier, D; Daculsi, R; Gualde, N

1992-01-01

Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells. The association of IL-2 and LPS had an additive effect on induction of cytotoxicity. The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture. Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN gamma by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN alpha/beta, (c) and that indomethacin (1 microM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN). When cultures were treated by an anti-IFN gamma antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN alpha/beta serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h. When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN alpha/beta the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor. Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous production of prostaglandin E2 and INF alpha/beta.

Copper(II)-based metal affinity chromatography for the isolation of the anticancer agent bleomycin from Streptomyces verticillus culture.

PubMed

Gu, Jiesi; Codd, Rachel

2012-10-01

The glycopeptide-based bleomycins are structurally complex natural products produced by Streptomyces verticillus used in combination therapy against testicular and other cancers. Bleomycin has a high affinity towards a range of transition metal ions with the 1:1 Fe(II) complex relevant to its mechanism of action in vivo and the 1:1 Cu(II) complex relevant to its production from culture. The affinity between Cu(II) and bleomycin was the underlying principle for using Cu(II)-based metal affinity chromatography in this work to selectively capture bleomycin from crude S. verticillus culture. A solution of standard bleomycin was retained at a binding capacity of 300 nmol mL(-1) on a 1-mL bed volume of Cu(II)-loaded iminodiacetate (IDA) resin at pH 9 via the formation of the heteroleptic immobilized complex [Cu(IDA)(bleomycin)]. Bleomycin was eluted from the resin at pH 5 as the metal-free ligand under conditions where pK(a) (IDA)culture that was pre-processed using XAD-2 resin to remove endogenously bound Cu(II). The approximate 25-fold purification of bleomycin from complex culture supernatant under aqueous conditions in a single step demonstrates the potential of Cu(II)-based metal affinity chromatography as a green chemistry platform for streamlined access to this high-value therapeutic agent. Copyright © 2012 Elsevier Inc. All rights reserved.

Establishment and Characterization of an Air-Liquid Canine Corneal Organ Culture Model To Study Acute Herpes Keratitis

PubMed Central

Harman, Rebecca M.; Bussche, Leen; Ledbetter, Eric C.

2014-01-01

ABSTRACT Despite the clinical importance of herpes simplex virus (HSV)-induced ocular disease, the underlying pathophysiology of the disease remains poorly understood, in part due to the lack of adequate virus–natural-host models in which to study the cellular and viral factors involved in acute corneal infection. We developed an air-liquid canine corneal organ culture model and evaluated its susceptibility to canine herpesvirus type 1 (CHV-1) in order to study ocular herpes in a physiologically relevant natural host model. Canine corneas were maintained in culture at an air-liquid interface for up to 25 days, and no degenerative changes were observed in the corneal epithelium during cultivation using histology for morphometric analyses, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, and transmission electron microscopy (TEM). Next, canine corneas were inoculated with CHV-1 for 48 h, and at that time point postinfection, viral plaques could be visualized in the corneal epithelium and viral DNA copies were detected in both the infected corneas and culture supernatants. In addition, we found that canine corneas produced proinflammatory cytokines in response to CHV-1 infection similarly to what has been described for HSV-1. This emphasizes the value of our model as a virus–natural-host model to study ocular herpesvirus infections. IMPORTANCE This study is the first to describe the establishment of an air-liquid canine corneal organ culture model as a useful model to study ocular herpesvirus infections. The advantages of this physiologically relevant model include the fact that (i) it provides a system in which ocular herpes can be studied in a virus–natural-host setting and (ii) it reduces the number of experimental animals needed. In addition, this long-term explant culture model may also facilitate research in other fields where noninfectious and infectious ocular diseases of dogs and humans are being studied. PMID

[Separate entrance of the main pancreatic duct and of the common bile duct: is it synonymous with easy retrograde cholangiography using endoscopic approach?

PubMed

Benatta, Mohammed Amine

2018-01-01

This study focused on endoscopic examination of a prominent papilla which showed two separate orifices revealing separate entrance of the main pancreatic duct and of the common bile duct (A). This was the most rare anatomoendoscopic variation, accounting for only 10% of retrograde cholangiopancreatographies using endoscopic approach (ERCP). However, it facilitated selective biliary catheterization (SBC) in our patient, as one might have expected. Papilla had a pseudo polypoid appearance and a biliary orifice (BO) at the right angle toward the duodenal wall; hence this was a more difficult approach than that via the pancreatic orifice (PO) which, on the other hand, required undesired pancreatic catheterization (B). As SBC was the approach we decided to adopt, the special techniques to consider in this particular case were: transpancreatic sphincterotomy (TPS) alone or TPS after implantation of a pancreatic stent (PS). We opted for the second technique (STP + PS) and we noticed that the implantation of a PS resulted in better BO visualization (C). We could perform SBC without recourse to TPS. In this specific case, as in other cases of difficult SBC in our practice, PS has proven effectiveness.

Proteomic characterization of fresh spermatozoa and supernatant after cryopreservation in relation to freezability of carp (Cyprinus carpio L) semen

PubMed Central

Ciereszko, Andrzej

2018-01-01

Our recent studies suggested that the freezability of carp semen is related to seminal plasma protein profiles. Here, we aimed to compare the spermatozoa proteomes of good (GF) and poor (PF) freezability semen of carp. To achieve this, we used two-dimensional difference in gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF or PF based on sperm motility after freeze/thawing. We identified proteins enriched in spermatozoa of GF (22 proteins) and PF (18 proteins) semen. We also identified 12 proteins enriched in the supernatant after cryopreservation of PF semen. Good freezability is related to high concentrations of proteins involved in the maintenance of flagella structure, membrane fluidity, efficient control of Ca2+ and sperm motility, energy production, and antioxidative protection, which likely reflects the full maturation status of spermatozoa of GF semen. On the other hand poor freezability seems to be related to the presence of proteins identified as released in high quantities from cryopreserved sperm of PF. Thus, the identified proteins might be useful bioindicators of freezing resilience and could be used to screen carp males before cryopreservation, thus improve long-term sperm preservation in carp. Data are available via ProteomeXchange with identifier PXD008187. PMID:29565997

A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

PubMed

Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

2005-05-01

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3

Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: Potential presence of novel polyamine metabolism and transport genes.

PubMed

Sugiyama, Yuta; Nara, Misaki; Sakanaka, Mikiyasu; Gotoh, Aina; Kitakata, Aya; Okuda, Shujiro; Kurihara, Shin

2017-12-01

Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of the membrane remnants of transferrin receptor with domain-specific antibodies.

PubMed

Baynes, R D; Shih, Y J; Hudson, B G; Cook, J D

1994-03-01

Tissue culture studies with K562 and HL60 cells have demonstrated the production of a soluble form of transferrin receptor identical to that identified in human serum. The present study was undertaken to search for membrane remnants of the truncated receptor with peptide antibodies specific for the extracellular and cytoplasmic domain of transferrin receptor. In cell membranes, a 105K remnant was identified that is consistent with truncation of one extracellular domain monomer of the transferrin receptor. In the exosomal fraction of the culture supernatant, a smaller 20K remnant consistent with truncation of both extracellular domains was also demonstrated. These findings provide evidence that soluble receptor is the product of proteolytic cleavage of intact membrane-bound transferrin receptor. Prior studies showing that the concentration of the extracellular domain in exosomes remained stable during incubation in culture supernatant suggest that this cleavage possibly occurs intracellularly.

Long-Term Cost-Effectiveness of Transanal Irrigation in Patients with Neurogenic Bowel Dysfunction.

PubMed

Emmanuel, Anton; Kumar, Gayathri; Christensen, Peter; Mealing, Stuart; Størling, Zenia M; Andersen, Frederikke; Kirshblum, Steven

2016-01-01

People suffering from neurogenic bowel dysfunction (NBD) and an ineffective bowel regimen often suffer from fecal incontinence (FI) and related symptoms, which have a huge impact on their quality of life. In these situations, transanal irrigation (TAI) has been shown to reduce these symptoms and improve quality of life. To investigate the long-term cost-effectiveness of initiating TAI in patients with NBD who have failed standard bowel care (SBC). A deterministic Markov decision model was developed to project the lifetime health economic outcomes, including quality-adjusted life years (QALYs), episodes of FI, urinary tract infections (UTIs), and stoma surgery when initiating TAI relative to continuing SBC. A data set consisting of 227 patients with NBD due to spinal cord injury (SCI), multiple sclerosis, spina bifida and cauda equina syndrome was used in the analysis. In the model a 30-year old individual with SCI was used as a base-case. A probabilistic sensitivity analysis was applied to evaluate the robustness of the model. The model predicts that a 30-year old SCI patient with a life expectancy of 37 years initiating TAI will experience a 36% reduction in FI episodes, a 29% reduction in UTIs, a 35% reduction in likelihood of stoma surgery and a 0.4 improvement in QALYs, compared with patients continuing SBC. A lifetime cost-saving of £21,768 per patient was estimated for TAI versus continuing SBC alone. TAI is a cost-saving treatment strategy reducing risk of stoma surgery, UTIs, episodes of FI and improving QALYs for NBD patients who have failed SBC.

Inconsistencies in the Weather Research and Forecasting Model of the Marine Boundary Layer Along the Coast of California

NASA Astrophysics Data System (ADS)

Fisher, Andrew M.

The late spring and summer low-level wind field along the California coast is primarily controlled by the pressure gradient between the Pacific high and the thermal low over the desert southwest. Strong northwesterly winds within the marine boundary layer (MBL) are common and the flow is often described as a two-layer shallow water hydraulic system, capped above by subsidence and bounded laterally by high coastal topography. Hydraulic features such as an expansion fan can occur near major coastal headlands. Numerical simulations using the Weather Research and Forecasting (WRF) modeling system were conducted over a two-month period and compared to observations from several buoy stations and aircraft measurements from the Precision Atmospheric Marine Boundary Layer Experiment (PreAMBLE). Model performance of the atmospheric adjustment near the Point Arguello and Point Conception (PAPC) headlands and into the Santa Barbara Channel (SBC) is assessed. Substantial inconsistencies are revealed, especially in the SBC. The strength of the synoptic forcing impacts model performance upstream of PAPC. The model maintains stronger winds than observed under weak forcing regimes, inadequately representing periods of wind relaxation. The large-scale forcing has minimal impact on the flow in the SBC, where poor modeling of the MBL characteristics exists throughout the entire period. Similar results are found in the coarser North American Mesoscale (NAM) model. In general, WRF overestimates the wind speed around PAPC and the expansion fan extends too far into the SBC. Previous conceptual models were based on similar flawed model results and limited observations. PreAMBLE measurements reveal a more complex lower atmosphere in the SBC than the simulations can represent. Mischaracterization of surface wind stress in the SBC has implications for forcing ocean models with WRF. Understanding model biases of the vertical profile of temperature and humidity are also critical to several

Contrasting effects of biochar on phosphorus dynamics and bioavailability in different soil types.

PubMed

Bornø, Marie Louise; Müller-Stöver, Dorette Sophie; Liu, Fulai

2018-06-15

We investigated how two different biochars (wood biochar - WBC and straw biochar - SBC) affected P dynamics and bioavailability in five different soils differing in pH, C%, texture, Fe, Al, Ca, and Mg giving a range of soils with low (S1 and S2), intermediate (S4), and high (S3 and S5) P sorption capacities. Langmuir and Freundlich equations were fitted to the sorption data of soil and soil/biochar mixtures. P fertilizer applied to all treatments was fractioned into strongly sorbed P (qS), easily available sorbed P (qA) and solution P (c) by determining the anion exchange resin (AER)-extractable P in samples from the sorption experiment. A pot experiment was conducted to measure P uptake by maize grown in S1, S2 and S3 amended with WBC or SBC at two P fertilizer levels (0 or 70mgPkg -1 ). Only WBC could sorb P from solution partly due to a high content of calcite. SBC did not have any effect on P sorption isotherms, whereas WBC increased the P sorption in S1, S2, and S4, yet decreased P sorption in acidic soil S5. qS increased in S1, S2, and S4, and decreased in S5 in WBC treatments, whereas, qS decreased in SBC treatments in soils S2, S4, and S5. Accordingly, there was a significant interaction between soil type and biochar on maize growth and P uptake. Biochar had no effect in an alkaline soil (S3), whereas, WBC and SBC had positive effects on maize growth in slightly acidic soils S1 and S2, depending on the soil P status, however, the P uptake was lower in WBC compared to SBC treatments. Biochar and soil properties and the P status of the soil affect P bioavailability. The study provides useful information for optimizing the use of biochar in agricultural P management. Copyright © 2018 Elsevier B.V. All rights reserved.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis

PubMed Central

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X.C.; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-01-01

Abstract The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection. To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10. The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples. The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%. The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. PMID:29390519

Validity of Antibodies in Lymphocyte Supernatant in Diagnosing Tuberculosis in Severely Malnourished Children Presenting with Pneumonia

PubMed Central

Chisti, Mohammod Jobayer; Salam, Mohammed Abdus; Raqib, Rubhana; Banu, Sayera; Shahid, Abu ASMSB; Shahunja, KM; Sharmin, Lazina; Ashraf, Hasan; Faruque, Abu Syed Golam; Bardhan, Pradip Kumar; Ahmed, Tahmeed

2015-01-01

Background 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. Methods 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”. Results 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. Conclusions and Significance Our data suggest that ALS is not sufficiently accurate to improve the diagnosis of TB in children with severe malnutrition. PMID:26020966

Agarolytic culturable bacteria associated with three antarctic subtidal macroalgae.

PubMed

Sánchez Hinojosa, Verónica; Asenjo, Joel; Leiva, Sergio

2018-05-21

Bacterial communities of Antarctic marine macroalgae remain largely underexplored in terms of diversity and biotechnological applications. In this study, three Antarctic subtidal macroalgae (Himantothallus grandifolius, Pantoneura plocamioides and Plocamium cartilagineum), two of them endemic of Antarctica, were investigated as a source for isolation of agar-degrading bacteria. A total of 21 epiphytic isolates showed agarolytic activity at low temperature on agar plates containing agar as the sole carbon source. 16S rRNA identification showed that the agar-degrading bacteria belonged to the genera Cellulophaga, Colwellia, Lacinutrix, Olleya, Paraglaciecola, Pseudoalteromonas and Winogradskyella. The agarase enzyme from a potential new species of the genus Olleya was selected for further purification. The enzyme was purified from the culture supernatant of Olleya sp. HG G5.3 by ammonium sulfate precipitation and ion-exchange chromatography. Molecular weight of the agarase was estimated to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme exhibited activity at 4 °C, retaining > 50% of its maximum activity at this temperature. This is the first study reporting the phylogeny of agar-degrading bacteria isolated from Antarctic subtidal macroalgae and the results suggest the huge potential of Antarctic algae-associated bacteria as a source of cold-active hydrolytic enzymes of biotechnological interest.

Sodium bicarbonate intake improves high-intensity intermittent exercise performance in trained young men.

PubMed

Krustrup, Peter; Ermidis, Georgios; Mohr, Magni

2015-01-01

Sodium bicarbonate intake has been shown to improve exercise tolerance, but the effects on high-intensity intermittent exercise are less clear. Thus, the aim of the present study was to determine the effect of sodium bicarbonate intake on Yo-Yo intermittent recovery test level 2 performance in trained young men. Thirteen men aged 23 ± 1 year (height: 180 ± 2 cm, weight: 78 ± 3 kg; VO2max: 61.3 ± 3.3 mlO2 · kg(-1) · min(-1); means ± SEM) performed the Yo-Yo intermittent recovery test level 2 (Yo-Yo IR2) on two separate occasions in randomized order with (SBC) and without (CON) prior intake of sodium bicarbonate (0.4 g · kg(-1) body weight). Heart rate and rating of perceived exertion (RPE) were measured during the test and venous blood samples were taken frequently. Yo-Yo IR2 performance was 14 % higher (P = 0.04) in SBC than in CON (735 ± 61 vs 646 ± 46 m, respectively). Blood pH and bicarbonate were similar between trials at baseline, but higher (P = 0.003) immediately prior to the Yo-Yo IR2 test in SBC than in CON (7.44 ± 0.01 vs 7.32 ± 0.01 and 33.7 ± 3.2 vs 27.3 ± 0.6 mmol · l(-1), respectively). Blood lactate was 0.9 ± 0.1 and 0.8 ± 0.1 mmol · l(-1) at baseline and increased to 11.3 ± 1.4 and 9.4 ± 0.8 mmol · l(-1) at exhaustion in SBC and CON, respectively, being higher (P = 0.03) in SBC. Additionally, peak blood lactate was higher (P = 0.02) in SBC than in CON (11.7 ± 1.2 vs 10.2 ± 0.7 mmol · l(-1)). Blood glucose, plasma K(+) and Na(+) were not different between trials. Peak heart rate reached at exhaustion was 197 ± 3 and 195 ± 3 bpm in SBC and CON, respectively, with no difference between conditions. RPE was 7% lower (P = 0.003) in SBC than in CON after 440 m, but similar at exhaustion (19.3 ± 0.2 and 19.5 ± 0.2). In conclusion, high-intensity intermittent exercise performance is improved by prior

ALTERATIONS OF MACROPHAGE FUNCTIONS BY MEDIATORS FROM LYMPHOCYTES

PubMed Central

Nathan, Carl F.; Karnovsky, Manfred L.; David, John R.

1971-01-01

Sensitized lymphocytes were incubated in vitro with the specific antigen Supernatants from these cultures were chromatographed on Sephadex G-100 columns. Supernatant fractions containing MIF, chemotactic factor, and lymphotoxin, but free of antigen and antibody, were incubated with normal peritoneal exudate macrophages. Macrophage adherence, phagocytosis, spreading, motility, and direct hexose monophosphate oxidation were enhanced, while protein synthesis was unaffected. Thus, antigen-stimulated lymphocytes secrete a factor or factors which enhance certain macrophage functions. Implications for models of cellular immunity and cellular hypersensitivity are discussed. PMID:5576335

Metabolomics Investigation of an Association of Induced Features and Corresponding Fungus during the Co-culture of Trametes versicolor and Ganoderma applanatum

PubMed Central

Xu, Xiao-Yan; Shen, Xiao-Ting; Yuan, Xiao-Jie; Zhou, Yuan-Ming; Fan, Huan; Zhu, Li-Ping; Du, Feng-Yu; Sadilek, Martin; Yang, Jie; Qiao, Bin; Yang, Song

2018-01-01

The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current work sets

Metabolomics Investigation of an Association of Induced Features and Corresponding Fungus during the Co-culture of Trametes versicolor and Ganoderma applanatum.

PubMed

Xu, Xiao-Yan; Shen, Xiao-Ting; Yuan, Xiao-Jie; Zhou, Yuan-Ming; Fan, Huan; Zhu, Li-Ping; Du, Feng-Yu; Sadilek, Martin; Yang, Jie; Qiao, Bin; Yang, Song

2017-01-01

The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13 C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13 C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13 C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor , 6 from G. applanatum , and 5 features were synthesized by both T. versicolor and G. applanatum . 13 C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13 C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13 C incorporation in the mono-culture of both T. versicolor and G. applanatum , was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor.

PubMed

Flores-Pliego, Arturo; Espejel-Nuñez, Aurora; Castillo-Castrejon, Marisol; Meraz-Cruz, Noemi; Beltran-Montoya, Jorge; Zaga-Clavellina, Veronica; Nava-Salazar, Sonia; Sanchez-Martinez, Maribel; Vadillo-Ortega, Felipe; Estrada-Gutierrez, Guadalupe

2015-01-01

The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. In this work we confirm that placental leukocytes from human term

Grain dust induces IL-8 production from bronchial epithelial cells: the effect of dexamethasone on IL-8 production.

PubMed

Park, H S; Suh, J H; Kim, H Y; Kwon, O J; Choi, D C

1999-04-01

Recent publications have suggested an active participation of neutrophils to induce bronchoconstriction after inhalation of grain dust (GD). To further understand the role of neutrophils in the pathogenesis of GD-induced asthma, this investigation was designed to determine whether human bronchial epithelial cells could produce IL-8 production and to observe the effect of dexamethasone on IL-8 production. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four concentrations (1 to 200 microg/mL) of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant, which was derived from the culture of PBMC from a GD-induced asthmatic subject under the exposure to 10 microg/mL of GD, and compared with those cultured without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. To evaluate the effect of dexamethasone on IL-8 production, four concentrations (5 to 5000 ng/mL) of dexamethasone were pre-incubated for 24 hours and the same experiments were repeated. There was significant production of IL-8 from bronchial epithelial cells with additions of GD in a dose-dependent manner (P < .05), which was significantly augmented with additions of PBMC supernatant (P < .05) at each concentration. Compared with the untreated sample, pretreatment of dexamethasone could induced a remarkable inhibitions (15% to 55%) of IL-8 production from bronchial epithelial cells in a dose-dependent manner. These results suggest that IL-8 production from bronchial epithelial cells may contribute to neutrophil recruitment occurring in GD-induced airway inflammation. The downregulation of IL-8 production by dexamethasone from bronchial epithelial cells may contribute to the efficacy of this compound in

Cholesterol ester hydrolase inhibitors reduce the production of synaptotoxic amyloid-β oligomers.

PubMed

McHale-Owen, Harriet; Bate, Clive

2018-03-01

The production of amyloid-β (Aβ) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aβ within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aβ were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aβ oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aβ 42 to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aβ 42 oligomers and increased concentrations of Aβ 42 monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aβ 42 oligomers and increased concentrations of Aβ 42 monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aβ 42 oligomers and increased concentrations of Aβ 42 monomers in cell supernatants. The Aβ monomers produced by treated cells protected neurons against Aβ oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aβ monomer:oligomer released and consequently their effects on synapses. Copyright © 2017 Elsevier B.V. All rights reserved.

Stimulation of matrix metalloproteinases by black-pigmented Bacteroides in human pulp and periodontal ligament cell cultures.

PubMed

Chang, Yu-Chao; Lai, Chung-Chih; Yang, Shun-Fa; Chan, You; Hsieh, Yih-Shou

2002-02-01

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes capable of degrading most components of the extracellular matrix. Recently, evidence has shown that MMPs may play a role in tissue degradation in inflamed dental pulp. To date very little is known regarding the mechanism of extracellular matrix destruction at the site of bacterial infection. The purpose of this study was to determine the effects of the supernatants from Porphyromonas endodontalis and Porphyromonas gingivalis on the production and secretion of MMPs by primary human pulp and periodontal ligament (PDL) cell cultures in vitro. The results were evaluated by substrate gel zymography from long-term cultures. The main gelatinase secreted by human pulp and PDL cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period, P. endodontalis and P. gingivalis were found to elevate MMP-2 production both in human pulp and PDL cell cultures. In addition, the stimulation was in a dose- and time-dependent manner. Both human pulp and PDL cells, however, treated with either P. endodontalis or P. gingivalis had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These results indicate that black-pigmented Bacteroides species play an important role in tissue destruction and disintegration of extracellular matrix in pulpal and periapical diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of microbial-induced pulpal and periapical lesion. An understanding of the actions of these black-pigmented Bacteroides species on pulp and PDL cells may result in new therapies to augment current treatment of pulpal and periapical lesions.

[Partial biological characteristics and algicidal activity of an algicidal bacterium].

PubMed

Li, San-Hua; Zhang, Qi-Ya

2013-02-01

An algicidal bacterium was isolated from freshwater (Lake Donghu in Wuhan) and coded as A01. The morphology of the algicidal bacterium was observed using optical microscope and electron microscopes, the results showed that A01 was rod-shaped, approximately 1.5 microm in length and 0.45 microm in width and with no flagella structure. A01 was Gram-negative and belongs to the family Acinetobacter sp. though identification by Gram's staining and 16S rDNA gene analysis. A01 exhibited strong algicidal activity on the bloom-forming cyanobacterium Anabaena eucompacta under laboratory conditions. The removal rate of chlorophyll a after 7-day incubation with the culture supernatant of A01 and thalli were 77% and 61%, respectively. Microscopic observation showed that almost all cyanobacterial cells were destroyed within 3 d of co-incubation with the supernatant of algicidal bacterium, but a mass of the cyanobacterial cell lysis was observed only after 5 d of co-incubation with the thalli of algicidal bacterium. These results indicated that the main algicidal component of A01 was in its culture supernatant. In other words, the strain A01 could secrete algicidal component against Anabaena eucompacta.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

PubMed

Rüger, Beate M; Buchacher, Tanja; Giurea, Alexander; Kubista, Bernd; Fischer, Michael B; Breuss, Johannes M

2018-01-01

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro , and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45 + leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by

IL-1β Enhances Wnt Signal by Inhibiting DKK1.

PubMed

Yoshida, Yusuke; Yamasaki, Satoshi; Oi, Katsuhiro; Kuranobu, Tatsuomi; Nojima, Takaki; Miyaki, Shigeru; Ida, Hiroaki; Sugiyama, Eiji

2018-06-28

Aberrant endochondral bone formation in the physis is a unique bone lesion in neonatal-onset multisystem inflammatory disease (NOMID), also called chronic infantile neurologic cutaneous articular (CINCA), the most severe of the cryopyrin-associated periodic syndrome (CAPS) diseases, which are interleukin-1β (IL-1β)-related monogenic autoinflammatory diseases. The wingless (Wnt) pathway plays an important role in osteoblast differentiation. In this study, we explored the potential role of IL-1β on the expression of WNT genes and the Wnt antagonist Dickkopf-1 (DKK1). The expression of WNT and DKK1 in fibroblast-like synoviocytes (FLS), which are articular resident cells, was quantified by quantitative PCR and enzyme-linked immunosorbent assay. Additionally, we used T cell factor (TCF) reporter assays to evaluate the activity of the canonical Wnt signal pathway in the presence or absence of the supernatant of cultured FLS treated with or without IL-1β and IL-6. Anti-DKK1 antibodies were used to neutralize DKK1. The expression of both canonical and non-canonical WNT genes as well as DKK1 was observed in FLS. The supernatant of cultured FLS suppressed the luciferase activity of the TCF reporter, and this effect was reduced by its pre-treatment with an anti-DKK1 antibody. Both IL-1β and IL-6 significantly reduced DKK1 production. Furthermore, the supernatant of FLS cultured with IL-1β or IL-6 showed a reduced inhibitory effect on Wnt signaling, compared with the supernatant of untreated FLS. These data suggest that IL-1β, like IL-6, dampens DKK1 production, and thereby promotes Wnt signal activation. Therefore, increased levels of IL-1β may contribute to the dysregulation of endochondral ossification in NOMID/CINCA.

Blood transfusions may impair endothelium-dependent vasodilatation during coronary artery bypass surgery.

PubMed

Rungatscher, Alessio; Milani, Elisabetta; Covajes, Cecilia; Hallström, Seth; Gottin, Leonardo; Guidi, Gian Cesare; Luciani, Giovanni Battista; Faggian, Giuseppe

2017-07-01

The hemolytic product free-hemoglobin (fHb) reduces nitric oxide (NO) bioavailability. The present study aims to establish whether administration of different blood transfusions result in increased circulating fHb levels and NO consumption with effects on arterial NO-dependent blood flow in patients undergoing CABG surgery. Ninety-five consecutive patients undergoing elective CABG surgery were prospectively divided in four groups based on blood transfusion requirements during surgery: stored blood cells (SBC, n. 21), intraoperative autologous salvaged blood (ASB, n. 25), SBC and ASB (n.22), no transfusion (control, n. 27). Blood samples were collected before and after intervention to analyse plasma levels of fHb and NO consumption. Endothelium-dependent relaxation was assessed in left internal mammary artery (LIMA) rings harvested before chest closure. Peripheral artery tonometry was assessed after intervention. Transfusions with SBC increased plasma fHb (p<0.05). Transfusions of ASB resulted in higher plasma fHb compared to SBC (p<0.01). fHb concentrations directly correlated with NO consumption (r=0.65, p<0.001). Maximal endothelium-dependent relaxation in LIMA was significantly attenuated in SBC and ASB patients compared to control (15.2±3.1% vs 21.1±2.5% vs 43±5.0% respectively; p<0.01). Significant correlations were identified between the aortic pressure wave velocity, plasma fHb concentration and NO consumption (p<0.01). Intraoperative blood transfusions and particularly autologous salvaged blood impair endothelium-dependent relaxation through NO scavenging by fHb. These findings obtained in vitro and in vivo provide new insights into the adverse relation between blood transfusions and patient outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

Signatures of post-zygotic structural genetic aberrations in the cells of histologically normal breast tissue that can predispose to sporadic breast cancer

PubMed Central

Forsberg, Lars A.; Rasi, Chiara; Pekar, Gyula; Davies, Hanna; Piotrowski, Arkadiusz; Absher, Devin; Razzaghian, Hamid Reza; Ambicka, Aleksandra; Halaszka, Krzysztof; Przewoźnik, Marcin; Kruczak, Anna; Mandava, Geeta; Pasupulati, Saichand; Hacker, Julia; Prakash, K. Reddy; Dasari, Ravi Chandra; Lau, Joey; Penagos-Tafurt, Nelly; Olofsson, Helena M.; Hallberg, Gunilla; Skotnicki, Piotr; Mituś, Jerzy; Skokowski, Jaroslaw; Jankowski, Michal; Śrutek, Ewa; Zegarski, Wojciech; Tiensuu Janson, Eva; Ryś, Janusz; Tot, Tibor; Dumanski, Jan P.

2015-01-01

Sporadic breast cancer (SBC) is a common disease without robust means of early risk prediction in the population. We studied 282 females with SBC, focusing on copy number aberrations in cancer-free breast tissue (uninvolved margin, UM) outside the primary tumor (PT). In total, 1162 UMs (1–14 per breast) were studied. Comparative analysis between UM(s), PT(s), and blood/skin from the same patient as a control is the core of the study design. We identified 108 patients with at least one aberrant UM, representing 38.3% of cases. Gains in gene copy number were the principal type of mutations in microscopically normal breast cells, suggesting that oncogenic activation of genes via increased gene copy number is a predominant mechanism for initiation of SBC pathogenesis. The gain of ERBB2, with overexpression of HER2 protein, was the most common aberration in normal cells. Five additional growth factor receptor genes (EGFR, FGFR1, IGF1R, LIFR, and NGFR) also showed recurrent gains, and these were occasionally present in combination with the gain of ERBB2. All the aberrations found in the normal breast cells were previously described in cancer literature, suggesting their causative, driving role in pathogenesis of SBC. We demonstrate that analysis of normal cells from cancer patients leads to identification of signatures that may increase risk of SBC and our results could influence the choice of surgical intervention to remove all predisposing cells. Early detection of copy number gains suggesting a predisposition toward cancer development, long before detectable tumors are formed, is a key to the anticipated shift into a preventive paradigm of personalized medicine for breast cancer. PMID:26430163

NanR Regulates nanI Sialidase Expression by Clostridium perfringens F4969, a Human Enteropathogenic Strain.

PubMed

Li, Jihong; Evans, Daniel R; Freedman, John C; McClane, Bruce A

2017-09-01

Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N -acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI , are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo . Copyright © 2017 American Society for Microbiology.

Effects of bioactive factors of the pineal gland on thymus function and cell composition of the bone marrow and spleen in mice of different age.

PubMed

Labunets, I F; Butenko, G M; Khavinson, V Kh

2004-05-01

The effects of factors from the pineal gland on the titer of thymic serum factor in the supernatant of 3-h thymus stroma cultures, number of stromal precursor fibroblasts and CD4+ cells in the bone marrow, and CD8+ cells in the spleens of adult and old CBA mice were studied in vitro. Epithalamin, Epithalon, and melatonin appreciably increased the titer of thymic serum factor in the supernatant of thymus stroma cultures from mice of different age and increased the percentage of CD4+ cells in the bone marrow suspension from old animals in vitro. The percentage of CD8+ lymphocytes decreased after incubation of splenic cells from old mice with melatonin. The percentage of bone marrow fibroblast precursor cells from adult and old mice did not appreciably change after incubation with the preparations.

Micro-computed tomography and histomorphometric analysis of the effects of platelet-rich fibrin on bone regeneration in the rabbit calvarium.

PubMed

Acar, Ahmet Hüseyin; Yolcu, Ümit; Gül, Mehmet; Keleş, Ali; Erdem, Necip Fazıl; Altundag Kahraman, Sevil

2015-04-01

The present study aimed to investigate the effectiveness of platelet-rich fibrin (PRF) on bone regeneration when used alone or in combination with hydroxyapatite (HA)/beta-tricalcium phosphate (βTCP). In this study, 20 New Zealand white rabbits were used and four calvarial defects were prepared in each animal. PRF, Straumann(®) Bone Ceramic (SBC), or PRF+SBC was applied to the defects; one defect was left untreated as a control. Ten rabbits were sacrificed at week 4 (T1) and 10 at week 8 (T2). After micro-computed tomography (micro-CT) scanning, the samples were sent for histological and histomorphometric analysis to evaluate and compare the volume and area of regenerated bone. Histomorphometric and micro-CT analysis showed that both PRF and SBC significantly increased bone regeneration at T1 and T2 (P<0.01). When PRF was used in combination with HA/βTCP, a further significant increase in new bone formation was observed at T1 and T2 compared with that when PRF or SBC was used alone (P<0.01). PRF has a positive effect on bone formation when used alone and in combination with HA/βTCP. Copyright © 2014 Elsevier Ltd. All rights reserved.

The critical limiting temperature and selective brain cooling: neuroprotection during exercise?

PubMed

Marino, Frank E

2011-01-01

There is wide consensus that long duration exercise in the heat is impaired compared with cooler conditions. A common observation when examining exercise tolerance in the heat in laboratory studies is the critical limiting core temperature (CLT) and the apparent attenuation in central nervous system (CNS) drive leading to premature fatigue. Selective brain cooling (SBC) purportedly confers neuroprotection during exercise heat stress by attenuating the increase in brain temperature. As the CLT is dependent on heating to invoke a reduction in efferent drive, it is thus not compatible with SBC which supposedly attenuates the rise in brain temperature. Therefore, the CLT and SBC hypotheses cannot be complimentary if the goal is to confer neuroprotection from thermal insult as it is counter-intuitive to selectively cool the brain if the purpose of rising brain temperature is to down-regulate skeletal muscle recruitment. This presents a circular model for which there is no apparent end to the ultimate physiological outcome; a 'hot brain' selectively cooled in order to reduce the CNS drive to skeletal muscle. This review will examine the postulates of the CLT and SBC with their relationship to the avoidance of a 'hot brain' which together argue for a theoretical position against neuroprotection as the key physiological strategy in exercise-induced hyperthermia.

A potential large and persistent black carbon forcing over Northern Pacific inferred from satellite observations.

PubMed

Li, Zhongshu; Liu, Junfeng; Mauzerall, Denise L; Li, Xiaoyuan; Fan, Songmiao; Horowitz, Larry W; He, Cenlin; Yi, Kan; Tao, Shu

2017-03-07

Black carbon (BC) aerosol strongly absorbs solar radiation, which warms climate. However, accurate estimation of BC's climate effect is limited by the uncertainties of its spatiotemporal distribution, especially over remote oceanic areas. The HIAPER Pole-to-Pole Observation (HIPPO) program from 2009 to 2011 intercepted multiple snapshots of BC profiles over Pacific in various seasons, and revealed a 2 to 5 times overestimate of BC by current global models. In this study, we compared the measurements from aircraft campaigns and satellites, and found a robust association between BC concentrations and satellite-retrieved CO, tropospheric NO 2 , and aerosol optical depth (AOD) (R 2  > 0.8). This establishes a basis to construct a satellite-based column BC approximation (sBC*) over remote oceans. The inferred sBC* shows that Asian outflows in spring bring much more BC aerosols to the mid-Pacific than those occurring in other seasons. In addition, inter-annual variability of sBC* is seen over the Northern Pacific, with abundances varying consistently with the springtime Pacific/North American (PNA) index. Our sBC* dataset infers a widespread overestimation of BC loadings and BC Direct Radiative Forcing by current models over North Pacific, which further suggests that large uncertainties exist on aerosol-climate interactions over other remote oceanic areas beyond Pacific.

The Influence of Acidity on Microbial Fuel Cells Containing Shewanella Oneidensis (PREPRINT)

DTIC Science & Technology

2008-09-01

d a fi b i s a h t s p t o m d C H p F 8 ig. 4. Cyclic voltammetry of filter sterilized media after 4 days of growth of S. neidensis MR-1 or S...of autologous mediators in the rowthmedium changeswith pH.We analyzed filter sterilized cul- ure supernatants by cyclic voltammetry (Fig. 4), and HPLC...Marsili et al., 2008). Cyclic voltammetrywas used to detect redox-active compounds n growthmedia supernatants fromMR-1 andDSP10 cultures. Fig. 4 hows

Reductive Dechlorination of Carbon Tetrachloride by Tetrachloroethene and Trichloroethene Respiring Anaerobic Mixed Cultures

NASA Astrophysics Data System (ADS)

Vickstrom, K. E.; Azizian, M.; Semprini, L.

2015-12-01

Carbon tetrachloride (CT) is a toxic and recalcitrant groundwater contaminant with the potential to form a broad range of transformation products. Of the possible biochemical pathways through which CT can be degraded, reductive dehalogenation to less chlorinated compounds and mineralization to carbon dioxide (CO2) appear to be the most frequently utilized pathways by anaerobic organisms. Results will be presented from batch experiments of CT degradation by the Evanite (EV), Victoria Strain (VS) and Point Mugu (PM) anaerobic dechlorinating cultures. The cultures are grown in chemostats and are capable of transforming tetrachloroethene (PCE) or trichloroethene (TCE) to ethene by halorespiration via reductive dehalogenase enzymes. For the batch CT transformation tests, the cells along with supernatant were harvested from chemostats fed PCE or TCE, but never CT. The batch reactors were initially fed 0.0085 mM CT and an excess of formate (EV and VS) or lactate (PM) as electron donor. Transformation of CT was 100% with about 20% converted to chloroform (CF) and undetected products. Multiple additions of CT showed a slowing of pseudo first-order CT transformation rates across all cultures. Batch reactors were then established and fed 0.085 mM CT with an excess of electron donor in order to better quantify the reductive pathway. CT was transformed to CF and dichloromethane (DCM), with trace amounts of chloromethane (CM) detected. Between 60-90% of the mass added to the system was accounted for, showing that the majority of the carbon tetrachloride present is being reductively dehalogenated. Results from batch reactors that were poisoned using sodium azide, and from reactors not provided electron donor will be presented to distinguish between biotic and abiotic reactions. Furthermore, results from reactors prepared with acetylene (a potent, reversible inhibitor of reductive dehalogenases (1)) will be presented as a means of identifying the enzymes involved in the

Stable coexistence of two Caldicellulosiruptor species in a de novo constructed hydrogen-producing co-culture

PubMed Central

2010-01-01

Background Mixed culture enrichments have been used frequently for biohydrogen production from different feedstock. In spite of the several advantages offered by those cultures, they suffer poor H2 yield. Constructing defined co-cultures of known H2 producers may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved. Results The extreme thermophiles Caldicellulosiruptor saccharolyticus DSM 8903 and C. kristjanssonii DSM 12137 were combined in a co-culture for H2 production from glucose and xylose in a continuous-flow stirred tank reactor. The co-culture exhibited a remarkable stability over a period of 70 days under carbon-sufficient conditions, with both strains coexisting in the system at steady states of different dilution rates, as revealed by species-specific quantitative PCR assays. The two strains retained their ability to stably coexist in the reactor even when glucose was used as the sole growth-limiting substrate. Furthermore, H2 yields on glucose exceeded those of either organism alone under the same conditions, alluding to a synergistic effect of the two strains on H2 production. A maximum H2 yield of 3.7 mol (mol glucose)-1 was obtained by the co-culture at a dilution rate of 0.06 h-1; a higher yield than that reported for any mixed culture to date. A reproducible pattern of population dynamics was observed in the co-culture under both carbon and non-carbon limited conditions, with C. kristjanssonii outgrowing C. saccharolyticus during the batch start-up phase and prevailing at higher dilution rates. A basic continuous culture model assuming the ability of C. saccharolyticus to enhance the growth of C. kristjanssonii could mimic the pattern of population dynamics observed experimentally and provide clues to the nature of interaction between the two strains. As a proof, the cell-free growth supernatant of C. saccharolyticus was found

Growth inhibition of Candida species by Wickerhamomyces anomalus mycocin and a lactone compound of Aureobasidium pullulans.

PubMed

Tay, Sun-Tee; Lim, Su-Lin; Tan, Hui-Wee

2014-11-08

The increasing resistance of Candida yeasts towards antifungal compounds and the limited choice of therapeutic drugs have spurred great interest amongst the scientific community to search for alternative anti-Candida compounds. Mycocins and fungal metabolites have been reported to have the potential for treatment of fungal infections. In this study, the growth inhibition of Candida species by a mycocin produced by Wickerhamomyces anomalus and a lactone compound from Aureobasidium pullulans were investigated. Mycocin was purified from the culture supernatant of an environmental isolate of W. anomalus using Sephadex G-75 gel filtration column chromatography. The mycocin preparation was subjected to SDS-PAGE analysis followed by MALDI TOF/TOF mass spectrometry analysis. The thermal and temperature stability of the mycocin were determined. The glucanase activity of the mycocin was investigated by substrate staining of the mycocin with 4-methyl-umbelliferyl-ß-D-glucoside (MUG). Gas chromatography mass spectrometry (GCMS) analysis was used to identify anti-Candida metabolite in the culture supernatant of an environmental isolate of Aureobasidium pullulans. The inhibitory effects of the anti-Candida compound against planktonic and biofilm cultures of various Candida species were determined using broth microdilution and biofilm quantitation methods. A mycocin active against Candida mesorugosa but not C. albicans, C. parapsilosis and C. krusei was isolated from the culture supernatant of W. anomalus in this study. The mycocin, identified as exo-ß-1,3 glucanase by MALDI TOF/TOF mass spectrometry, was stable at pH 3-6 and temperature ranging from 4-37°C. The glucanase activity of the mycocin was confirmed by substrate staining with MUG. 5-hydroxy-2-decenoic acid lactone (HDCL) was identified from the culture supernatant of A. pullulans. Using a commercial source of HDCL, the planktonic and biofilm MICs of HDCL against various Candida species were determined in this study

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

PubMed Central

Tosato, G; Tanner, J; Jones, K D; Revel, M; Pike, S E

1990-01-01

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions. Images PMID:2159561

[Effects of hydrogen sulfide on the secretion of cytokines in macrophages of deep partial-thickness burn wound in rats].

PubMed

Li, Y; Xu, D B; Wang, H J

2016-07-20

To analyze the effects of exogenous hydrogen sulfide on the secretion of growth factors basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGF-β1), as well as inflammatory mediators tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in macrophages of deep partial-thickness burn wound in rats. Seventy-eight SD rats were divided into normal control group (n=6), pure burn group, sodium hydrosulfide group, propargylglycine (PPG) group, and sodium hydrosulfide+ PPG group according to the random number table, with 18 rats in each of the latter four groups. Rats in normal control group did not receive any treatment, while rats in the other four groups were inflicted with 5% total burn surface area deep partial-thickness scald (hereinafter referred to as burn) on the back. Immediately after burn, rats in pure burn group, sodium hydrosulfide group, and group PPG were intraperitoneally injected with saline 2 mL/kg, sodium hydrosulfide 56 μmol/kg, and PPG 45 mg/kg respectively, while those in sodium hydrosulfide+ PPG group were intraperitoneally injected with sodium hydrosulfide 56 μmol/kg and PPG 45 mg/kg, once a day till the day before harvesting specimen. Six rats of normal control group fed for one week, and 6 rats from each of the rest four groups on post injury day (PID) 3, 7, 14 were collected respectively. Normal skin on the back of rats in normal control group and tissue in the base of wound of rats in the other four groups were harvested to isolate macrophages, and then the content of bFGF, TGF-β1, TNF-α, and IL-1β in culture supernatant of macrophages was detected with enzyme-linked immunosorbent assay. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and LSD test. Compared with that of normal control group [(42.6±2.5) and (18±4) pg/mL], the content of bFGF and TGF-β1 in culture supernatant of macrophages of rats in pure burn group was obviously increased at each time point

Age-related differences in cigarette smoke extract-induced H2O2 production by lung endothelial cells.

PubMed

Downs, Charles A; Montgomery, David W; Merkle, Carrie J

2011-11-01

Cigarette smoke causes oxidative stress in the lung resulting in injury and disease. The purpose of this study was to determine if there were age-related differences in cigarette smoke extract (CSE)-induced production of reactive species in single and co-cultures of alveolar epithelial type I (AT I) cells and microvascular endothelial cells harvested from the lungs (MVECLs) of neonatal, young and old male Fischer 344 rats. Cultures of AT I cells and MVECLs grown separately (single culture) and together (co-culture) were exposed to CSE (1, 10, 50, 100%). Cultures were assayed for the production of intracellular reactive oxygen species (ROS), hydroxyl radical (OH), peroxynitrite (ONOO(-)), nitric oxide (NO) and extracellular hydrogen peroxide (H(2)O(2)). Single and co-cultures of AT I cells and MVECLs from all three ages produced minimal intracellular ROS in response to CSE. All ages of MVECLs produced H(2)O(2) in response to CSE, but young MVECLs produced significantly less H(2)O(2) compared to neonatal and old MVECLs. Interestingly, when grown as a co-culture with age-matched AT I cells, neonatal and old MVECLs demonstrated ~50% reduction in H(2)O(2) production in response to CSE. However, H(2)O(2) production in young MVECLs grown as a co-culture with young AT I cells did not change with CSE exposure. To begin investigating for a potential mechanism to explain the reduction in H(2)O(2) production in the co-cultures, we evaluated single and co-cultures for extracellular total antioxidant capacity. We also performed gene expression profiling specific to oxidant and anti-oxidant pathways. The total antioxidant capacity of the AT I cell supernatant was ~5 times greater than that of the MVECLs, and when grown as a co-culture and exposed to CSE (≥ 10%), the total antioxidant capacity of the supernatant was reduced by ~50%. There were no age-related differences in total antioxidant capacity of the cell supernatants. Gene expression profiling found eight genes to be

Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.

PubMed

Hare, Nathan J; Soe, Cho Zin; Rose, Barbara; Harbour, Colin; Codd, Rachel; Manos, Jim; Cordwell, Stuart J

2012-02-03

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

PubMed

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Structure of nitrogen-converting communities induced by hydraulic retention time and COD/N ratio in constantly aerated granular sludge reactors treating digester supernatant.

PubMed

Cydzik-Kwiatkowska, Agnieszka; Rusanowska, Paulina; Zielińska, Magdalena; Bernat, Katarzyna; Wojnowska-Baryła, Irena

2014-02-01

This study investigated how hydraulic retention time (HRT) and COD/N ratio affect nitrogen-converting consortia in constantly aerated granules treating high-ammonium digester supernatant. Three HRTs (10, 13, 19 h) were tested at COD/N ratios of 4.5 and 2.3. Denaturing gradient gel electrophoresis and relative real-time PCR were used to characterize the microbial communities. When changes in HRT and COD/N increased nitrogen loading, the ratio of the relative abundance of aerobic to anaerobic ammonium-oxidizers decreased. The COD/N ratio determined the species composition of the denitrifiers; however, Thiobacillus denitrificans, Pseudomonas denitrificans and Azoarcus sp. showed a high tolerance to the environmental conditions and occurred in the granules from all reactors. Denitrifier genera that support granule formation were identified, such as Pseudomonas, Shinella, and Flavobacterium. In aerated granules, nirK-possessing bacteria were more diverse than nirS-possessing bacteria. At a low COD/N ratio, N2O-reducer diversity increased because of the presence of bacteria known as aerobic denitrifiers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

PubMed

Deharde, Daniela; Schneider, Christin; Hiller, Thomas; Fischer, Nicolas; Kegel, Victoria; Lübberstedt, Marc; Freyer, Nora; Hengstler, Jan G; Andersson, Tommy B; Seehofer, Daniel; Pratschke, Johann; Zeilinger, Katrin; Damm, Georg

2016-10-01

CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis.

PubMed

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X C; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-12-01

The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection.To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10.The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples.The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%.The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

In Search of the E. coli Compounds that Change the Antibiotic Production Pattern of Streptomyces coelicolor During Inter-species Interaction.

PubMed

Mavituna, Ferda; Luti, Khalid Jaber Kadhum; Gu, Lixing

2016-08-01

The aim of this work was to investigate the interaction between E.coli and Streptomyces coelicolor A3 (2) for the increased production of undecylprodigiosin and identify the E. coli actives mediating this inter-species interaction. The antibiotics of interest were the red-pigmented undecylprodigiosin and blue-pigmented actinorhodin. Pure cultures of S. coelicolor in a defined medium produced higher concentrations of actinorhodin compared to those of undecylprodigiosin. The latter however, is more important due to its immunosuppressive and antitumor properties. As a strategy to increase undecylprodigiosin production, we added separately, live cells and heat-killed cells of E. coli C600, and the cell-free supernatant of E. coli culture to S. coelicolor cultures in shake flasks. The interaction with live cells of E. coli altered the antibiotic production pattern and undecylprodigiosin production was enhanced by 3.5-fold compared to the pure cultures of S. coelicolor and actinorhodin decreased by 15-fold. The heat-killed cells of E. coli however, had no effect on antibiotic production. In all cases, growth and glucose consumption of S. coelicolor remained almost the same as those observed in the pure culture indicating that the changes in antibiotic production were not due to nutritional stress. Results with cell-free supernatant of E. coli culture indicated that the interaction between S. coelicolor and E. coli was mediated via diffusible molecule(s). Using a set of extraction procedures and agar-well diffusion bioassays, we isolated and preliminarily identified a class of compounds. For the preliminary verification, we added the compound which was the common chemical structural moiety in this class of compounds to the pure S. coelicolor cultures. We observed similar effects on antibiotic production as with the live E. coli cells and their supernatant indicating that this class of compounds secreted by E. coli indeed could act as actives during interspecies

Design of Low-Cost Impact Reporting System

DTIC Science & Technology

2015-12-01

Single Board Computers (SBC) available. Arduino and Raspberry Pi are very low cost and have huge communities for hardware design. Most of the SBC... Raspberry Pi Model B has a considerably faster processor than the Arduino. Although it provides only approximately 25 General Purpose Input and Output...reporting system must be able to operate on its own power for more than 2 or 3 hours. The Raspberry Pi Model B operates on 5 volts direct current at

Survey and Analysis of Environmental Requirements for Shipboard Electronic Equipment Applications. Appendix A. Volume 2.

DTIC Science & Technology

1991-07-31

INTELLIGENT SCSI DMV-719 MAS MIL CONTROLLER DY-4 SYSTEMS BYTE-WIDE MEMORY CARD DMV-536 MEM MIL DY-4 SYSTEMS POWER SUPPLY UNIT DMV-870 PWR MIL P age No. 5 06/10...FORCE COMPUTERS PROCESSOR CPU-386 SERIES SBC COM FORCE COMPUTERS ADVANCED SYSTEM CONTROL ASCU -1/2 SBC COM UNITI FORCE COMPUTERS GRAPHICS CONTROLLER AGC...RECORD VENDOR: JANZ COMPUTER AG DIVISION: VENDOR ADDRESS: Im Doerener Feld 3 D-4790 Paderborn Germany MARKETING: Johannes Kunz TECHNICAL: Arnulf

Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

PubMed

Morales-Álvarez, Edwin D; Rivera-Hoyos, Claudia M; Poveda-Cuevas, Sergio A; Reyes-Guzmán, Edwin A; Pedroza-Rodríguez, Aura M; Reyes-Montaño, Edgar A; Poutou-Piñales, Raúl A

2018-03-01

Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using Ganoderma lucidum supernatant and 5.0 ± 0.5% with Pleurotus ostreatus supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using G. lucidum and P. ostreatus concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.

Postharvest behaviour of five Sardinian prune varieties as affected by immersion in heated sodium bicarbonate solution.

PubMed

Molinu, M G; Venditti, T; Dore, A; Agabbio, M; Rosas, G; D'Hallewin, G

2010-01-01

Storage behaviour of 'Core', 'Core Columbu', 'Fradis' and 'Meloni' white prunes, and a black one ('Sighera') of Sardinian germplasm were evaluated following immersion for 0 (control), 15, 30, 45 or 60 sec in water at 20, 50, 55 or 60 degrees C with or without 2% (w/v) NaHCO3 (SBC). As international varieties, fruit from one white plum ('Shiro') and one black prune ('Stanly') were subjected to the same treatments. Fruit was harvested at commercial maturity, treated and then stored for 1 month at 5 degrees C and 90% RH followed by a simulated marketing period at 20 degrees C and 80% RH for 6 days. Fruit appearance, external damage, firmness and decay percentage were monitored after storage and SMP. Treatments did not induce rind damage (browning or discoloration) to any variety. SBC at 20, 45, 50 or 55 degrees C for 15 or 30 sec was not effective in controlling decay and compared to controls no improvement was observed. Immersion for 45 or 60 sec with SBC at all temperatures improved decay control with respect to controls and best results were obtained at 50 or 55 degrees C. Immersions at 60 degrees C improved decay control, but differences were not significant compared to the control attained with solutions of SBC heated at 55 degrees C. The overall appearance of 'Core', 'Core Columbu', 'Fradis' and 'Shiro' decreased significantly after the SMP period, especially when treated at 55 or 60 degrees C for 60 sec. Fruit shrivel was the main cause of the low rating. SBC did not affect shrivel indicating that heat treatment may be the probable cause. In general, local varieties were less affected by decay than other varieties and they performed well during storage.

Isolation and characterisation of phosphate solubilising microorganisms from the cold desert habitat of Salix alba Linn. in trans Himalayan region of Himachal Pradesh.

PubMed

Chatli, Anshu S; Beri, Viraj; Sidhu, B S

2008-06-01

Phosphate solubilising microorganisms (PSM) (bacteria and fungi) associated with Salix alba Linn. from Lahaul and Spiti valleys of Himachal Pradesh were isolated on Pikovskaya (PVK), modified Pikovskaya (MPVK) and National Botanical Research Institute agar (NBRIP) media by spread plating. The viable colony count of P-solubilising bacteria (PSB) and fungi (PSF) was higher in rhizosphere than that of non-rhizosphere. The frequency of PSM was highest on MPVK followed by NBRIP and PVK agar. The maximum proportion of PSM out of total bacterial and fungal count was found in upper Keylong while the least in Rong Tong. The PSB frequently were Gram-positive, endosporeforming, motile rods and belonged to Bacillus sp. The PSF mainly belonged to Penicillium sp., Aspergillus fumigatus, A. niger, A. spp. and non-sporulating sterile. Amongst the isolates with high efficiency for tricalcium phosphate (TCP) solubilisation, seven bacterial and seven fungal isolates dissolved higher amount of P from North Carolina rock phosphate (NCRP) than Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). However, the organisms solubilised higher-P in NBRIP broth than PVK broth. SBC5 (Bacillus sp.) and SBC7 (Bacillus sp.) bacterial isolates exhibited maximun P solubilisation (40 and 33 μg ml(-1) respectively) whereas FC28 (Penicillium sp.) isolate (52.3 μg ml(-1)) amongst fungi while solubilising URP. The amount of P solubilised was positively correlated with the decrease in pH of medium. SBC5 (Bacillus sp.), SBC7 (Bacillus sp.) and SBC4 (Micrococcus) decreased the pH of medium from 6.8 to 6.08 while FC28 (Penicillium sp.) and FC39 (Penicillium sp.) isolates of fungi recorded maximum decrease in pH of medium from 6.8 to 5.96 in NBRIP broth.

Secondary elements of blood pH variation can influence the effort effectiveness based on adaptive changes within a group of elite athletes.

PubMed

Martin, Ştefan Adrian; Tomescu, Valeriu; Voidăzan, Septimiu

2016-01-01

pH is the direct indicator of the body reaction following the activities performed. Establishing precise correlations between pH and blood biochemical parameters might support the balancing of values during periods of marked physical activity. We conducted a case study in a group of elite rowers. Twelve athletes were included in the study. Monitoring was carried out by collecting biological samples several times a day: in the morning, 80 minutes pre-workout, 12 hours after the last physical effort performed, at two different times, 10 days apart. Determinations were aimed at adapting the reported biochemical parameters depending on the effort performed. The following parameters were monitored: pH, HCO3, pCO2, pO2, BE, SBE, SBC, Ca++, Mg++, LDH, GPT, T-Pro, and Alb. The mean value of pH found in athletes was 7.41±0.024. The value obtained was significantly correlated to biochemical parameters such as BE (2.32±1.79), SBC (1.67±1.45), SBE (2.70±1.75). However, bicarbonate (HCO3) was statistically significantly related with SBE, SBC, SBE, and pO2, but did not present a strong association with the pH value (p=0.094). However, values such as Alb, Ca++, LDH, BE, SBC are related to pH value as a result of variations in the data submitted. The processed data evidence the fact that blood pH, in this case, is significantly influenced by a number of indices that correlate energy system activity, individual adaptation to effort, and the recovery process. The parameters under investigation (SBE, SBC, SBE, CPK, LDH) are associated with pH changes that could confirm the recovery efficiency of the athlete, along with a possible metabolic acidosis/alkalosis.

Comparing diagnostic yield of a novel pan-enteric video capsule endoscope with ileocolonoscopy in patients with active Crohn's disease: a feasibility study.

PubMed

Leighton, Jonathan A; Helper, Debra J; Gralnek, Ian M; Dotan, Iris; Fernandez-Urien, Ignacio; Lahat, Adi; Malik, Pramod; Mullin, Gerard E; Rosa, Bruno

2017-01-01

Crohn's disease (CD) is typically diagnosed with ileocolonoscopy (IC); however, when inflammation is localized solely in the small bowel, visualization of the entire small-bowel mucosa can be challenging. The aim of this study was to compare the diagnostic yield of a pan-enteric video capsule endoscope (small-bowel colon [SBC] capsule) versus IC in patients with active CD. This was a prospective, multicenter study. Patients with known active CD and proven bowel luminal patency underwent a standardized colon cleansing protocol followed by ingestion of the capsule. After passage of the capsule, IC was performed and recorded. Lesions indicative of active CD were assessed. One hundred fourteen subjects were screened; 66 subjects completed both endoscopic procedures. The per-subject diagnostic yield rate for active CD lesions was 83.3% for SBC and 69.7% for IC (yield difference, 13.6%; 95% confidence interval [CI], 2.6%-24.7%); 65% of subjects had active CD lesions identified by both modalities. Of the 12 subjects who were positive for active CD by SBC only, 5 subjects were found to have active CD lesions in the terminal ileum. Three subjects were positive for active CD by IC only. Three hundred fifty-five classifying bowel segments were analyzed; the per-segment diagnostic yield rate was 40.6% for SBC and 32.7% for IC (yield difference 7.9%; 95% CI, 3.3%-12.4%). This preliminary study shows that the diagnostic yields for SBC might be higher than IC; however, the magnitude of difference between the two is difficult to estimate. Further study is needed to confirm these findings. Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation.

PubMed

Liang, Tzu-Wen; Hsieh, Jia-Lin; Wang, San-Lang

2012-11-15

A protease- and chitosanase-producing strain was isolated and identified as Bacillus cereus TKU022. The protease and chitosanase were both produced using 1.5% (w/v) shrimp head powder (SHP) as the sole carbon/nitrogen source, and these enzymes were purified from the culture supernatant. The molecular masses of the TKU022 protease and chitosanase determined using SDS-PAGE were approximately 45 and 44kDa, respectively. The high stability of the TKU022 protease toward surfactants, an optimal pH of 10 and an optimal temperature of 50-60°C suggest that this high-alkaline protease has potential applications for various industrial processes. Concomitant with the production of the TKU022 chitosanase, N-acetyl chitooligosaccharides were also observed in the culture supernatant, including (GlcNAc)(2), (GlcNAc)(4), (GlcNAc)(5), and (GlcNAc)(6) at concentrations of 201.5, 12.4, 0.5, and 0.3μg/mL, respectively, as determined using an HPLC analysis. The chitin oligosaccharides products were also characterized using a MALDI-TOF mass spectrometer. A combination of the HPLC and MALDI-TOF MS results showed that the chitin oligosaccharides of the TKU022 culture supernatant comprise oligomers with degree of polymerization (DP) from 2 to 6. Using this method, the production of a protease, a chitosanase, and chitin oligosaccharides may be useful for various industrial and biological applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

Degradation of Triphenyltin by a Fluorescent Pseudomonad

PubMed Central

Inoue, Hiroyuki; Takimura, Osamu; Fuse, Hiroyuki; Murakami, Katsuji; Kamimura, Kazuo; Yamaoka, Yukiho

2000-01-01

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3′,4′,7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 μM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 μM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment. PMID:10919812

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor.

PubMed

Ohashi, R; Yamamoto, T; Suzuki, T

1999-01-01

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.

A novel multivariate approach using science-based calibration for direct coating thickness determination in real-time NIR process monitoring.

PubMed

Möltgen, C-V; Herdling, T; Reich, G

2013-11-01

This study demonstrates an approach, using science-based calibration (SBC), for direct coating thickness determination on heart-shaped tablets in real-time. Near-Infrared (NIR) spectra were collected during four full industrial pan coating operations. The tablets were coated with a thin hydroxypropyl methylcellulose (HPMC) film up to a film thickness of 28 μm. The application of SBC permits the calibration of the NIR spectral data without using costly determined reference values. This is due to the fact that SBC combines classical methods to estimate the coating signal and statistical methods for the noise estimation. The approach enabled the use of NIR for the measurement of the film thickness increase from around 8 to 28 μm of four independent batches in real-time. The developed model provided a spectroscopic limit of detection for the coating thickness of 0.64 ± 0.03 μm root-mean square (RMS). In the commonly used statistical methods for calibration, such as Partial Least Squares (PLS), sufficiently varying reference values are needed for calibration. For thin non-functional coatings this is a challenge because the quality of the model depends on the accuracy of the selected calibration standards. The obvious and simple approach of SBC eliminates many of the problems associated with the conventional statistical methods and offers an alternative for multivariate calibration. Copyright © 2013 Elsevier B.V. All rights reserved.

A potential large and persistent black carbon forcing over Northern Pacific inferred from satellite observations

PubMed Central

Li, Zhongshu; Liu, Junfeng; Mauzerall, Denise L.; Li, Xiaoyuan; Fan, Songmiao; Horowitz, Larry W.; He, Cenlin; Yi, Kan; Tao, Shu

2017-01-01

Black carbon (BC) aerosol strongly absorbs solar radiation, which warms climate. However, accurate estimation of BC’s climate effect is limited by the uncertainties of its spatiotemporal distribution, especially over remote oceanic areas. The HIAPER Pole-to-Pole Observation (HIPPO) program from 2009 to 2011 intercepted multiple snapshots of BC profiles over Pacific in various seasons, and revealed a 2 to 5 times overestimate of BC by current global models. In this study, we compared the measurements from aircraft campaigns and satellites, and found a robust association between BC concentrations and satellite-retrieved CO, tropospheric NO2, and aerosol optical depth (AOD) (R2 > 0.8). This establishes a basis to construct a satellite-based column BC approximation (sBC*) over remote oceans. The inferred sBC* shows that Asian outflows in spring bring much more BC aerosols to the mid-Pacific than those occurring in other seasons. In addition, inter-annual variability of sBC* is seen over the Northern Pacific, with abundances varying consistently with the springtime Pacific/North American (PNA) index. Our sBC* dataset infers a widespread overestimation of BC loadings and BC Direct Radiative Forcing by current models over North Pacific, which further suggests that large uncertainties exist on aerosol-climate interactions over other remote oceanic areas beyond Pacific. PMID:28266532

A potential large and persistent black carbon forcing over Northern Pacific inferred from satellite observations

NASA Astrophysics Data System (ADS)

Li, Zhongshu; Liu, Junfeng; Mauzerall, Denise L.; Li, Xiaoyuan; Fan, Songmiao; Horowitz, Larry W.; He, Cenlin; Yi, Kan; Tao, Shu

2017-03-01

Black carbon (BC) aerosol strongly absorbs solar radiation, which warms climate. However, accurate estimation of BC’s climate effect is limited by the uncertainties of its spatiotemporal distribution, especially over remote oceanic areas. The HIAPER Pole-to-Pole Observation (HIPPO) program from 2009 to 2011 intercepted multiple snapshots of BC profiles over Pacific in various seasons, and revealed a 2 to 5 times overestimate of BC by current global models. In this study, we compared the measurements from aircraft campaigns and satellites, and found a robust association between BC concentrations and satellite-retrieved CO, tropospheric NO2, and aerosol optical depth (AOD) (R2 > 0.8). This establishes a basis to construct a satellite-based column BC approximation (sBC*) over remote oceans. The inferred sBC* shows that Asian outflows in spring bring much more BC aerosols to the mid-Pacific than those occurring in other seasons. In addition, inter-annual variability of sBC* is seen over the Northern Pacific, with abundances varying consistently with the springtime Pacific/North American (PNA) index. Our sBC* dataset infers a widespread overestimation of BC loadings and BC Direct Radiative Forcing by current models over North Pacific, which further suggests that large uncertainties exist on aerosol-climate interactions over other remote oceanic areas beyond Pacific.

The effects of persuasive communication and planning on intentions to be more physically active and on physical activity behaviour among low-active adolescents.

PubMed

Tessier, Damien; Sarrazin, Philippe; Nicaise, Virginie; Dupont, Jean-Philippe

2015-01-01

The purpose of the present study was to examine, using the theory of planned behaviour (TPB) combined with a self-regulatory behaviour change approach, whether persuasive communication based on adolescents' salient beliefs (SBCondition) and planning (PCondition) could promote the intention and physical activity (PA) behaviour of low-active adolescents participating in less than 1 h/day of moderate-to-vigorous physical activity. The protocol tested the effectiveness of two strategies used separately (i.e. SBC or PC) or in combination (i.e. CC = NSBC-SBC-PC) compared to a group receiving a message based on non-salient beliefs (NSBCondition). The 116 low-active students from ten 10th and 11th grade classes were assigned, using a cluster randomisation, into one of the four conditions (i.e. NSBC, SBC, PC and CC). Baseline data were collected two weeks before the intervention. The post-test data collection occurred directly after the intervention, and the follow-up took place two weeks later. Results showed that (1) the NSBC was the least effective strategy, (2) the SBC had no significant effect on PA behaviour and the TPB variables, (3) the PC had no significant effect on PA behaviour but increased the intention and perceived behavioural control and (4) the effects of the PC and the CC were not significantly different.

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

Evaluation of antifungal activity of carbonate and bicarbonate salts alone or in combination with biocontrol agents in control of citrus green mold.

PubMed

Zamani, M; Sharifi Tehrani, A; Ali Abadi, A Alizadeh

2007-01-01

The aim of this research was to determine if the attacks of green mold on orange could be reduced by edible salts alone or in combination with biocontrol agent. For this purpose toxicity to Pantoea digitatum and practical use of sodium carbonate (SC), sodium bicarbonate (SBC) and potassium carbonate, and potassium bicarbonate alone or in combination with antagonistic bacteria (Pseudomonas fluorescens isolate PN, Bacillus subtilis isolate VHN, Pantoea agglomerans isolate CA) to control green mold were determined. All were fungistatic. SC and SBC were equal and superior to the other salts for control of green mold on oranges inoculated 6h before treatment and were chosen for subsequent trails under cold storage conditions. The biocontrol agents were found completely tolerant to 3% sodium bicarbonate and sodium carbonate at room temperature; although their culturability was reduced by > 1000-fold after 60 min in 1% other salt solutions. Satisfactory results were also obtained with the combined treatment for control of green mold. A significant increase in biocontrol activity of all isolate was observed when combined with sodium carbonate and sodium bicarbonate. The treatments comprising CA combined with SB was as effective as fungicide treatment. Thus, use of sodium bicarbonate treatment at 3% followed by the antagonist P. agglomerans CA could be an alternative to chemical fungicides for control of green mold on oranges.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4[S

PubMed Central

Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena

2016-01-01

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4. In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4. Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions.

PubMed

Chifiriuc, Mariana Carmen; Bleotu, Coralia; Pîrcălăbioru, Gratiela; Israil, Anca Michaela; Dinu, Sorin; Rută, Simona Maria; Grancea, Camelia; Lazăr, Veronica

2010-01-01

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.

Influence of Malaria Infection on the Elaboration of Soluble Mediators by Adherent Mononuclear Cells

PubMed Central

Wyler, David J.; Oppenheim, Joost J.; Koontz, Louis C.

1979-01-01

Malaria results in two seemingly paradoxical perturbations of the immune response: polyclonal B-cell activation and immunosuppression. To determine what immunoregulatory role mediators secreted by adherent cells might play in these alterations, we cultured adherent cells from uninfected mice and from mice at different times during infection with Plasmodium berghei or P. yoelii. Culture supernatants obtained from these cells were tested for their ability to enhance the in vitro proliferative responses of thymocytes to suboptimal concentrations of concanavalin A or to inhibit the mitogen-stimulated proliferation of normal spleen cells. Supernatants obtained from adherent cells of mice early in infection (days 1 to 3) contained significantly elevated levels of enhancing activity which on Bio-Gel P-100 chromatography resembled lymphocyte-activating factor. Later in infection (days 4 and 5), these supernatants contained inhibitory activity. Normal adherent cells, when cocultivated in vitro with parasitized erythrocytes, ingested parasite debris and were stimulated to produce the enhancing factor. At high parasite/adherent-cell ratios, cells elaborated an inhibitory factor. These findings suggest that during malaria, adherent cells are converted from a nonspecific helper role to a nonspecific suppressor role. This modulation in function may be due to the direct interaction between adherent cells and parasitized erythrocytes. PMID:457269

Inactivation of virginiamycin by Aureobasidium pullulans

USDA-ARS?s Scientific Manuscript database

Objective: To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans. Results: Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three...

Successful propagation of shrimp yellow head virus in immortal mosquito cells.

PubMed

Gangnonngiw, Warachin; Kanthong, Nipaporn; Flegel, Timothy W

2010-05-18

Research on crustacean viruses is hampered by the lack of continuous cell lines susceptible to them. To overcome this problem, we previously challenged immortal mosquito and lepidopteran cell lines with shrimp yellow head virus (YHV), followed by serial, split-passage of whole cells, and showed that this produced cells that persistently expressed YHV antigens. To determine whether such insect cultures positive for YHV antigens could be used to infect shrimp Penaeus monodon with YHV, culture supernatants and whole-cell homogenates were used to challenge shrimp by injection. Shrimp injected with culture supernatants could not be infected. However, shrimp injection-challenged with whole-cell homogenates from Passage 5 (early-passage) of such cultures died with histological and clinical signs typical for yellow head disease (YHD), while homogenates of mock-passaged, YHV-challenged cells did not. By contrast, shrimp challenged with cell homogenates of late-passage cultures became infected with YHV, but survived, suggesting that YHV attenuation had occurred during its long-term serial passage in insect cells. Thus, YHV could be propagated successfully in C6/36 mosquito cells and used at low passage numbers as a source of inoculum to initiate lethal infections in shrimp. This partially solves the problem of lack of continuous shrimp cell lines for cultivation of YHV.

New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures.

PubMed

Elkhtab, Ebrahim; El-Alfy, Mohamed; Shenana, Mohamed; Mohamed, Abdelaty; Yousef, Ahmed E

2017-12-01

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography-mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask.

PubMed

Ohashi, R; Mochizuki, E; Suzuki, T

1999-01-01

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.

Serum amyloid A secretion from monocytic leukaemia cell line THP-1 and cultured human peripheral monocytes.

PubMed

Yamada, T; Wada, A; Itoh, K; Igari, J

2000-07-01

Serum amyloid A (SAA), an acute-phase protein and a precursor of fibrous components in reactive amyloid deposits, is synthesized mainly in the liver under the stimulation of inflammation-related cytokines. In addition, the SAA gene is expressed in monocytes/macrophages, which are believed to play a central role in amyloid fibrillogenesis. Consequently, the pathogenic implication of SAA produced from these cells has been of major concern. Because SAA synthesis at the protein level in such cells has never been analyzed quantitatively, in this study an enzyme-linked immunosorbent assay was generated with a detection level sufficiently high to measure SAA concentrations in the culture supernatants of the human monocytic leukaemia cell line THP-1. SAA secretion by THP-1 with interleukin (IL)-1beta required the presence of dexamethasone as proposed previously. We also found that unidentified components in fetal calf serum (FCS) could induce SAA production by THP-1 in the presence of dexamethasone. These findings are in contrast to the results obtained from hepatoma cell line HepG2, in which IL-1beta alone could induce SAA secretion, while dexamethasone-supplemented FCS could not. The method was able to quantify SAA secreted from cultured human peripheral monocytes. The findings suggest that monocytes produce SAA in almost the same manner as THP-1. Thus, THP-1 cells can be utilized to investigate a distinctive manner of SAA production from monocytes.

Increased expression of metalloproteinase-2 and -9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1, TIMP-2), and EMMPRIN (CD147) in multiple myeloma.

PubMed

Urbaniak-Kujda, Donata; Kapelko-Slowik, Katarzyna; Prajs, Iwona; Dybko, Jarosław; Wolowiec, Dariusz; Biernat, Monika; Slowik, Miroslaw; Kuliczkowski, Kazimierz

2016-01-01

Activity of metalloproteinases (MMP) is controlled both by specific tissue inhibitors (TIMP) and activators (extracellular matrix metalloproteinase inducer, EMMPRIN). There are few data available concerning concentration the bone marrow of MMP-2, MMP-9, TIMP-1, and TIMP-2, or EMMPRIM expression by bone marrow mesenchymal stromal cells (BMSCs) in patients with multiple myeloma (MM). We studied 40 newly diagnosed, untreated patients: 18 males and 22 females with de novo MM and 11 healthy controls. Bone marrow was collected prior to therapy. BMSCs were derived by culturing bone marrow cells on MesenCult. Protein concentrations were determined in bone marrow plasma and culture supernatants by ELISA. EMMPRIN expression by BMSCs was assessed by flow cytometry. The median concentrations of MMP-9, TIMP-1, and TIMP-2 in both marrow plasma and culture supernatants were significantly higher in MM patients than controls. EMMPRIN expression and ratios MMP-9/TIMP-1 and MMP-2/TIMP-2 were higher in MM patients, our results demonstrate that in MM patients MMP-2 and MMP-9 are secreted in higher amounts and are not balanced by inhibitors.

Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes

PubMed Central

Blank, Christian A.; Anderson, David A.; Beard, Michael; Lemon, Stanley M.

2000-01-01

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ± 12.5% of the total virus yield compared with 0.23% ± 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms. PMID:10864660

[Effects of electromagnetic pulse exposure on the morphological change and excretion function of BV-2 cells and possible mechanism].

PubMed

Yang, Long-long; Zhou, Yan; Li, Hai-juan; Guo, Juan; Zhang, Yan-jun; Ding, Gui-rong; Guo, Guo-zhen

2012-03-01

To study the effects of electromagnetic pulse (EMP) exposure on the morphological change and excretion functions of mouse microglia (BV-2) cells and possible mechanism. BV-2 cells were divided into two groups: the group exposed to EMP at 200 kV/m for 200 pulses and sham exposure group. At 1, 6, 12 and 24 hour after exposure the cells and culture supernatant were collected. Cellular morphological change was observed under invert microscope, the levels of TNF-α, IL-1β and IL-10 in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA), nitric oxide (NO) and reactive oxygen species (ROS) were detected by nitrate reductase method and DCFH-DA probe, respectively. The protein and phosphorylation levels of ERK, JNK and p38 were measured by Western Blot method. After the cells pre-treated with the inhibitor of p38 (SB203580) were exposed to EMP, the levels of NO and ROS in culture supernatant were detected. It was found that the large ameboid shape appeared in some microglia cells exposed to EMP for 1, 6 and 12 h. Moreover, the number of microglia cells with ameboid shape increased significantly at 1 h, 6 h and 12 h after EMP exposure compared with sham group (P < 0.05). The levels of cytokines, such as TNF-α, IL-1β and IL-10, in culture supernatant did not change obviously after EMP exposure. The levels of NO and ROS increased significantly at 1h after EMP exposure, reached the peak at 6 h, began to recover at 12 h and recovered to sham group level at 24 h (P < 0.05). Western blot results showed that the protein and protein phosphorylation levels of ERK and JNK did not change significantly after EMP exposure, however, the protein and protein phosphorylation levels of p38 increased obviously at 1 h and 6 h after EMP exposure, compared with sham group (P < 0.05). In addition, the pretreatment of p38 inhibitor (SB203580) significantly decreased NO and ROS production induced by EMP. EMP exposure may activate microglia cells and promote the

Interaction between the Sbcc Gene of Escherichia Coli and the Gam Gene of Phage λ

PubMed Central

Kulkarni, S. K.; Stahl, F. W.

1989-01-01

gam mutants of phage λ carrying long palindromes fail to form plaques on wild-type Escherichia coli but do grow on strains that are mutant in the sbcC gene. gam(+) λ carrying the same palindrome grow on both hosts and on a host deleted for the recB, C and D genes. These results suggest that the Gam protein of λ, known to interact also with E. coli's recBCD protein, can interact with the product of the sbcC gene. PMID:2531105

Small Business Innovation Research and Small Business Technology Transfer Programs

NASA Technical Reports Server (NTRS)

Garrison, Lynn; Jasper, Gwen

2015-01-01

The Small Business Innovation Research (SBIR)/Small Business Technology Transfer (STTR) programs fund the research, development, and demonstration of innovative technologies that fulfill NASA's needs as described in the annual Solicitations and have significant potential for successful commercialization. The only eligible participants are small business concern (SBC) with 500 or fewer employees or a nonprofit research institute such as a university or a research laboratory with ties to an SBC. These programs are potential sources of seed funding for the development of small business innovations.

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-01-01

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-02-28

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

Biofilm formation by designed co-cultures of Caldicellulosiruptor species as a means to improve hydrogen productivity.

PubMed

Pawar, Sudhanshu S; Vongkumpeang, Thitiwut; Grey, Carl; van Niel, Ed Wj

2015-01-01

Caldicellulosiruptor species have gained a reputation as being among the best microorganisms to produce hydrogen (H2) due to possession of a combination of appropriate features. However, due to their low volumetric H2 productivities (Q H2), Caldicellulosiruptor species cannot be considered for any viable biohydrogen production process yet. In this study, we evaluate biofilm forming potential of pure and co-cultures of Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor owensensis in continuously stirred tank reactors (CSTR) and up-flow anaerobic (UA) reactors. We also evaluate biofilms as a means to retain biomass in the reactor and its influence on Q H2. Moreover, we explore the factors influencing the formation of biofilm. Co-cultures of C. saccharolyticus and C. owensensis form substantially more biofilm than formed by C. owensensis alone. Biofilms improved substrate conversion in both of the reactor systems, but improved the Q H2 only in the UA reactor. When grown in the presence of each other's culture supernatant, both C. saccharolyticus and C. owensensis were positively influenced on their individual growth and H2 production. Unlike the CSTR, UA reactors allowed retention of C. saccharolyticus and C. owensensis when subjected to very high substrate loading rates. In the UA reactor, maximum Q H2 (approximately 20 mmol · L(-1)  · h(-1)) was obtained only with granular sludge as the carrier material. In the CSTR, stirring negatively affected biofilm formation. Whereas, a clear correlation was observed between elevated (>40 μM) intracellular levels of the secondary messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and biofilm formation. In co-cultures C. saccharolyticus fortified the trade of biofilm formation by C. owensensis, which was mediated by elevated levels of c-di-GMP in C. owensensis. These biofilms were effective in retaining biomass of both species in the reactor and improving Q H2 in a UA reactor using

The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

PubMed

Hashim, Puziah

2014-03-01

Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

PubMed Central

Rotman, Ella; Kouzminova, Elena; Plunkett, Guy

2012-01-01

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion. PMID:23042999

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

PubMed

Tárnok, Attila; Valet, Günther K; Emmrich, Frank

2006-01-01

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

PubMed

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Juritz, Stephanie; Birk, Richard; Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Schultz, Johannes; Hörmann, Karl; Kinscherf, Ralf; Faber, Anne

2014-01-01

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM

New phospholipase A1-producing bacteria from a marine fish.

PubMed

Nishihara, Masaaki; Kamata, Masazumi; Koyama, Tomoyuki; Yazawa, Kazunaga

2008-01-01

Phospholipase A1 is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of glycerophospholipids to form 2-acyl lysophospholipids. Lysophospholipids are used in foods, cosmetics, and pharmaceuticals as surfactants. Novel forms of phospholipase A1 that function at low temperatures are desirable for use in lipophilic systems in food processing. However, there is currently little variety in the available sources of phospholipase A1. Given this situation, we screened the intestinal contents of marine animals for phospholipase A1-producing bacteria. Colonies that formed a halo on K28CP screening medium and that grew in K28 medium were cultured in liquid K28 medium, and the supernatant was retrieved for analysis. Phosphatidylcholine was added to the culture supernatant, and the product of the reaction was analyzed by using TLC. For culture supernatants that were able to generate lysophosphatidylcholine, synthetic phosphatidylcholines were added, and the site of the reaction was determined by analyzing the fatty acid compositions of the lysophosphatidylcholines generated by GLC. A bacterial isolate from a flatfish, which we named HFKI0020, was found to have phospholipase A1 activity at low temperatures. We determined that the isolate HFKI0020 is closely related to Pseudomonas by using 16S rDNA sequence analysis and by characterizing the isolate with respect to its physiologic and biochemical properties. From the intestinal contents of a marine fish, we successfully isolated a bacterium that secretes phospholipase A1 that is active at low temperatures.

Probiotic lactobacilli interfere with Streptococcus mutans biofilm formation in vitro.

PubMed

Söderling, Eva M; Marttinen, Aino M; Haukioja, Anna L

2011-02-01

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.

Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone.

PubMed

Fonseca, Cristina T; Pacífico, Lucila G G; Barsante, Michele M; Rassi, Tatiana; Cassali, Geovanni D; Oliveira, Sérgio C

2006-08-01

Schistosomiasis is an endemic disease that affects 200 million people worldwide. DNA-based vaccine is a promising strategy to induce protective immunity against schistosomiasis, since both humoral and cellular immune responses are involved in parasite elimination. In this study, we evaluated the ability of Sm14 cDNA alone or in association with a plasmid expressing murine interleukin (IL)-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, Th1 type of immune response with high levels of interferon (IFN)-gamma and low levels of IL-4 in splenocyte culture supernatants and in bronchoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-gamma and tumor necrosis factor-alpha.

A rapid and simple procedure to detect the presence of MVM in conditioned cell fluids or culture media.

PubMed

Chang, A; Havas, S; Borellini, F; Ostrove, J M; Bird, R E

1997-12-01

During the manufacture of biopharmaceuticals, numerous adventitious agents have been detected in Master Cell Banks, end-of-production cells as well as bulk harvest fluid. Recently, a number of large-scale production bioreactors have become infected with Minute Virus of Mice (MVM) during cGMP (current good manufacturing practices) operations, and this has resulted in both the loss of product and the need for major cleaning validation procedures to be put in place. We have developed a simple DNA extraction/PCR assay to detect the presence of MVM in cell culture supernatant (conditioned cell fluids). This highly specific assay can detect 10 or fewer genome equivalents (copies) of MVM following PCR and gel electrophoresis visualization. For routine high-throughput detection, 300-100 copies could be consistently detected. The extraction procedure was shown to reliably detect MVM at a concentration of 1 TCID50/ml. The combination of the extraction/PCR procedure establishes a powerful, sensitive, specific assay that can detect the presence of MVM sequences with a 1-day turnaround time.

The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

PubMed

Rahiman, F; Pool, E J

2014-01-01

This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P < 0.001). Exposure of blood cells to sucralose-containing sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat.

Examining the interactive effects of ocenaographic and anthropogenic influences with the SST anomaly, or Warm Blob on the bloom response of the toxigenic HAB genus Pseudo-nitzschia in the Santa Barbara Channel.

NASA Astrophysics Data System (ADS)

Amiri, S.

2016-12-01

Harmful algal blooms (HAB's) include a large subset of toxigenic phytoplankton and microbial species responsible for shutting down major fisheries, impairing water quality and threatening public health. Oceanographic and anthropogenic effects on HAB's in concert with climactic stressors may have interactive effects influencing HAB blooms to persist longer than historically documented. This 3 year time-series explores the interactive effects of the SST anomaly known as the Warm Blob across the coastal Pacific on the bloom progression and persistence of the toxigenic Pseudo-nitzschia bloom across the West Coast, ranging from the gulf of Alaska to the Santa Barbara Channel (SBC). This study also explores direct links of the Warm Blob event on nutrient and oxygen concentrations spatially across the Santa Barbara Channel with the highest levels of domoic acid concentrations recorded from the coast wide mega bloom. MODIS and SeaWIFS Satellite imagery of chlorophyll and SST monthly averaged values of the SBC were identified to better understand the regional distribution of the Warm Blob on phytoplankton community structure. These images were ground truthed with monthly samples from 7 transects across the SBC with the Plumes and Blooms time-series, LTER sites and local pier sites across the Santa Barbara County. Preliminary data suggest an interesting correlation with Pseudo-nitzschia species outcompeting other phytoplankton species within the SBC during the 3 degree averaged increase of SST conditions with the Warm Blob event. *Data is still being processed and results should be analyzed before October 2016.

Slow Cholinergic Modulation of Spike Probability in Ultra-Fast Time-Coding Sensory Neurons

PubMed Central

Goyer, David; Kurth, Stefanie; Rübsamen, Rudolf

2016-01-01

Abstract Sensory processing in the lower auditory pathway is generally considered to be rigid and thus less subject to modulation than central processing. However, in addition to the powerful bottom-up excitation by auditory nerve fibers, the ventral cochlear nucleus also receives efferent cholinergic innervation from both auditory and nonauditory top–down sources. We thus tested the influence of cholinergic modulation on highly precise time-coding neurons in the cochlear nucleus of the Mongolian gerbil. By combining electrophysiological recordings with pharmacological application in vitro and in vivo, we found 55–72% of spherical bushy cells (SBCs) to be depolarized by carbachol on two time scales, ranging from hundreds of milliseconds to minutes. These effects were mediated by nicotinic and muscarinic acetylcholine receptors, respectively. Pharmacological block of muscarinic receptors hyperpolarized the resting membrane potential, suggesting a novel mechanism of setting the resting membrane potential for SBC. The cholinergic depolarization led to an increase of spike probability in SBCs without compromising the temporal precision of the SBC output in vitro. In vivo, iontophoretic application of carbachol resulted in an increase in spontaneous SBC activity. The inclusion of cholinergic modulation in an SBC model predicted an expansion of the dynamic range of sound responses and increased temporal acuity. Our results thus suggest of a top–down modulatory system mediated by acetylcholine which influences temporally precise information processing in the lower auditory pathway. PMID:27699207

Lactobacillus plantarum isolated from kefir protects vero cells from cytotoxicity by type-II shiga toxin from Escherichia coli O157:H7.

PubMed

Kakisu, Emiliano; Abraham, Analía G; Farinati, Carla Tironi; Ibarra, Cristina; De Antoni, Graciela L

2013-02-01

Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide-cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils.

PubMed

Dent, Gordon; Loweth, Sam C; Hasan, Anwar Matar; Leslie, Fiona M

2014-10-01

The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines - CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) - were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P<0.005) and CXCL5 levels (P<0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P<0.05) and CXCL8 (P<0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel. Copyright © 2014 Elsevier GmbH. All rights reserved.

Tocotrienols Stimulate Insulin Secretion of Rat Pancreatic Isolated Islets in a Dynamic Culture.

PubMed

Chia, Ling L; Jantan, Ibrahim; Chua, Kien H

2017-01-01

Tocotrienols (T3) are the naturally occurring vitamin E derivatives that possess antioxidant properties and therapeutic potential in diabetic complications. The bioactivities of the derivatives are determined by the number and arrangement of methyl substitution on the structure. The objective of this study was to determine the effects of T3 derivatives, σ-T3, γ-T3 and α-T3 on insulin secretion of rat pancreatic islets in a dynamic culture. Pancreatic islets isolated from male Wistar rats were treated with T3 for 1 h at 37°C in a microfluidic system with continuous operation that provided a stable cell culture environment. Glucose (2.8 mM and 16.7 mM, as basal and stimulant, respectively) and potassium chloride (KCl) (30 mM) were added to the treatment in calcium free medium. The supernatant was collected for insulin measurements. Short-term exposure (1 h) of σ-T3 to β cells in the stimulant glucose condition significantly potentiated insulin secretion in a dose-dependent manner. γ-T3 and α-T3 also displayed dosedependent effect but were less effective in the activation of insulin secretion. Essentially, KCl, a pancreatic β cell membrane depolarizing agent, added into the treatment further enhanced the insulin secretion of σ-T3, γ-T3 and α-T3 with ED50 values of 504, 511 and 588 µM, respectively. The findings suggest the potential of σ-T3 in regulating glucose-stimulated insulin secretion (GSIS) in response to the intracellular calcium especially in the presence of KCl. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Genome sequences of nine vesicular stomatitis virus isolates from South America

USDA-ARS?s Scientific Manuscript database

We report nine full-genome sequences of vesicular stomatitis virus obtrained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species, clade III, while two...

An Ex Vivo Model for Studying Hepatic Schistosomiasis and the Effect of Released Protein from Dying Eggs

PubMed Central

Gobert, Geoffrey N.; Nawaratna, Sujeevi K.; Harvie, Marina; Ramm, Grant A.; McManus, Donald P.

2015-01-01

Background We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis. Methods and Findings Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5. Conclusions This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs. PMID:25965781

Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP-1 and TIMP-2.

PubMed

Sato, Y; Kishi, J; Suzuki, K; Nakamura, H; Hayakawa, T

2009-12-01

To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria (Porphyromonas gingivalis, P. endodontalis and F. nucleatum) on the activation of matrix metalloproteinase (MMP-2) and the inactivation of tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2). Each SBE was added to cultures of human periodontal ligament (PL) cells or HT1080 cells and their supernatants were analysed by zymography for MMP-2. Each SBE was added to PL cell cultures, and the amount of TIMP-1 was determined by ELISA. P. gingivalis SBE was incubated with HT1080 cell culture supernatants, and the amounts of TIMP-1 and TIMP-2 were determined by ELISA. Statistical analysis was performed with the paired Student's t-test. In extracts of PL cells that had been incubated in the presence of P. gingivalis SBE, one representing pro-MMP-2 (72 kDa) and a band corresponding to the active MMP-2 (66 kDa) were observed; but in the other extracts it was not detected. When HT1080 cells were treated with P. gingivalis SBE, the pro-MMPs was processed into 86- and 66-kDa fragments, but in the other extracts, the processing did not occur when the other SBEs were used. When PL cells were incubated with the same SBEs, the amount of TIMP-1 was markedly decreased (P < 0.01), but in the other extracts, it was not. The amounts of both TIMP-1 and TIMP-2 were decreased in a dose-dependent manner when HT1080 cell culture supernatant was incubated with P. gingivalis SBE. These findings suggest that P. gingivalis SBE may cause connective tissue to be destroyed, contributing to the process of periapical disease, by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2.

Standardized ileal digestibility of amino acids in European soya bean and rapeseed products fed to growing pigs.

PubMed

Kaewtapee, C; Mosenthin, R; Nenning, S; Wiltafsky, M; Schäffler, M; Eklund, M; Rosenfelder-Kuon, P

2018-04-01

This study was conducted to determine the chemical composition and standardized ileal digestibility coefficients (SID) of crude protein (CP) and amino acids (AA) of European soya bean and rapeseed products in pigs. Six soya bean and two rapeseed products were used as the sole dietary source of CP and AA, including raw (FFSB) and roasted full-fat soya beans (FFSB R oasted ), soya bean (SBC) and rapeseed cake (RSC), and rapeseed meal (RSM) from Bavaria (Germany), soya bean meal (SBM) from the Danube region (Austria; SBM A ustria ), a commercially available standard SBM (SBM S td ) and an imported genetically modified organism-free SBM (SBM GMO -free ). Eight ileal- cannulated pigs with an initial body weight of 32 ± 2 kg were allotted to a row-column design with eight diets and six periods of seven days each. Trypsin inhibitor activity (TIA) ranged from 1.8 in SBM S td to 24.5 mg/g DM in FFSB. The SID of CP and all AA in FFSB R oasted were greater than in FFSB, but lower when compared to SBC and SBM A ustria (p < .05). The SID of CP and all AA (except glutamic acid) were not different between SBC and SBM A ustria , but the SID of CP and all AA (except methionine) were greater (p < .05) in SBC than in SBM GMO -free . Furthermore, the SID of CP and most AA showed a quadratic response with decreasing TIA, and there exists a quadratic response in SID of CP and all AA with increasing lysine to CP ratio and neutral detergent insoluble nitrogen (p < .05). In conclusion, variation in chemical composition and SID of CP and AA was observed in different European soya bean and rapeseed products as influenced by differences in processing conditions. European SBC and SBM A ustria can be used as alternative to imported SBM GMO -free and SBM S td in diets for growing pigs. © 2017 Blackwell Verlag GmbH.

[Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene].

PubMed

Zhu, Guo-qiang; Wu, Zhi-fen; Li, Yuan-fei; Hu, De-hua; Wang, Qin-tao

2006-12-01

To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene. pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT. IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation. The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.

Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

PubMed

Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

2016-02-01

Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Fungal secretomes enhance sugar beet pulp hydrolysis.

PubMed

Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

2014-04-01

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nematode-trapping fungi and fungus-associated bacteria interactions: the role of bacterial diketopiperazines and biofilms on Arthrobotrys oligospora surface in hyphal morphogenesis.

PubMed

Li, Lei; Yang, Min; Luo, Jun; Qu, Qing; Chen, Ying; Liang, Lianming; Zhang, Keqin

2016-11-01

In soil, nematode-trapping fungi and bacteria often share microhabitats and interact with each other, but effects of fungus-associated bacteria on its trap formation are underestimated. We have ascertained the presence of Stenotrophomonas and Rhizobium genera associated with A. oligospora GJ-1. After A. oligospora GJ-1 without associated bacteria (cured Arthrobotrys) was co-cultivated with Stenotrophomonas and its supernatant extract, microscopic study of hyphae from co-cultivation indicated that bacterial biofilm formation on hyphae was related to trap formation in fungi and Stenotrophomonas supernatant extract. Four diketopiperazines (DKPs) were purified from Stenotrophomonas supernatant extract that could not induce traps in the cured Arthrobotrys. When cured Arthrobotrys was cultured with Stenotrophomonas and one of DKPs, polar attachment, bacterial biofilms on hyphae and trap formation in fungi were observed. After cured Arthrobotrys with bacterial biofilms was consecutively transferred several times on nutrient poor medium, trap formation disappeared with the disappearance of bacterial biofilms on hyphae. DKPs could facilitate chemotaxis of Stenotrophomonas towards fungal extract which was suggested to contribute to bacterial biofilms on hyphae. Furthermore, when cured Arthrobotrys was cultured with Stenotrophomonas and DKPs in soil, trap formation in fungi and bacterial biofilms on hyphae were also observed, and the fungal activity against nematode was enhanced. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Affinity purification of antibodies

USDA-ARS?s Scientific Manuscript database

Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

NASA Technical Reports Server (NTRS)

Green, Lora M.; Patel, Zarana; Murray, Deborah K.; Rightnar, Steven; Burell, Cheryl G.; Gridley, Daila S.; Nelson, Gregory A.

2002-01-01

Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

Biocorrosion and uptake of titanium by human osteoclasts.

PubMed

Cadosch, Dieter; Al-Mushaiqri, Mohamed S; Gautschi, Oliver P; Meagher, James; Simmen, Hans-Peter; Filgueira, Luis

2010-12-15

All metals in contact with a biological system undergo corrosion through an electrochemical redox reaction. This study investigated whether human osteoclasts (OC) are able to grow on titanium and aluminum, and directly corrode the metals leading to the release of corresponding metal ions, which are believed to cause inflammatory reactions and activate osteoclastic differentiation. Scanning electron microscopy analysis demonstrated long-term viable OC cultures on the surface of titanium and aluminum foils. Atomic emission spectrometry investigations showed significantly increased levels of aluminum in the supernatant of OC cultured on aluminum; however, all measurements in the supernatants of cell cultures on titanium were below detection limits. Despite this, confocal microscopy analysis with Newport Green DCF diacetate ester staining depicted intense fluorescence throughout the cytoplasm and nucleolus of OC cultured on titanium foils. Comparable fluorescence intensities were not observed in monocytes and control cells cultured on glass. The present study demonstrated that human osteoclast precursors are able to grow and differentiate toward mature OC on titanium and aluminum. Furthermore, it established that the mature cells are able to directly corrode the metal surface and take up corresponding metal ions, which subsequently may be released and thereby induce the formation of osteolytic lesions in the periprosthetic bone, contributing to the loosening of the implant. Copyright © 2010 Wiley Periodicals, Inc.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

PubMed

Naskou, Maria C; Norton, Natalie A; Copland, Ian B; Galipeau, Jacques; Peroni, John F

2018-01-01

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1β (IL-1β) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1β. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1β production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1β compared to the control groups. This is the first study to demonstrate that ePL suppresses

Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen.

PubMed

Nidialkova, N A; Varbanets, L D; Chernyshenko, V O

2016-01-01

Peptidase of Bacillus thuringiensis var. israelensis IМV В-7465 was isolated from culture supernatant using consecutive fractionations by an ammonium sulphate (60% saturation), ion-exchange chromatography and gel-filtration on the TSK-gels Toyoperl HW-55 and DEAE 650(M). Specific elastase (442 U∙mg of protein-1) and collagenase (212.7 U∙mg of protein-1) activities of the purified enzyme preparation were 8.0- and 6.1-fold, respectively higher than ones of the culture supernatant. Peptidase yields were 33.5% for elastase activity and 30.1% for collagenase activity. It was established that the enzyme is serine metal-dependent alkaline peptidase with Mr about 37 kDa. Maximal hydrolysis of elastin and collagen occurs at the optimum pH 8.0 and t° – 40 and 50 °С, respectively. The purified preparation has high stability at pH in the range of 7.0 to 10.0 and 40-50 °С.

Paracrine action of HO-1-modified mesenchymal stem cells mediates cardiac protection and functional improvement.

PubMed

Zeng, Bin; Ren, Xiaofeng; Lin, Guosheng; Zhu, Chengang; Chen, Honglei; Yin, Jiechao; Jiang, Hong; Yang, Bo; Ding, Danhua

2008-10-01

The aim has been to determine whether the supernatants of mesenchymal stem cells (MSCs) transfected with adenovirus carrying human heme oxygenase-1 (hHO-1) gene protect cardiomyocytes from ischemic injury. We have found that hHO-1 infected MSCs (hHO-1-MSCs) increased expression of hHO-1 protein. Apoptosis of cultured hHO-1-MSCs exposed to hypoxia was suppressed. Several cytokines, including HGF, bFGF, TGF-beta, VEGF and IL-1beta, were produced by hHO-1-MSCs, some being significantly enhanced under hypoxia stimulation. Meanwhile, those cytokines reduced caspase-3 level and activity in cultured adult rat ventricular cardiomyocytes (ARVCs) exposed to hypoxia. Supernatants obtained from hHO-1-MSCs improved left ventricular function, limited myocardial infarct size, increased microvessel density, and inhibited apoptosis of cardiomyocytes in rat myocardial infarction. It can be concluded hHO-1-modified MSCs prevent myocardial cell injury via secretion of paracrine-acting mediators.

Inhibiting effect of bioactive metabolites produced by mushroom cultivation on bacterial quorum sensing-regulated behaviors.

PubMed

Zhu, Hu; Wang, Shou-Xian; Zhang, Shuai-Shuai; Cao, Chun-Xu

2011-01-01

This study aimed to search for novel quorum sensing (QS) inhibitors from mushroom and to analyze their inhibitory activity, with a view to their possible use in controlling detrimental infections. The bioactive metabolites produced by mushroom cultivation were tested for their abilities to inhibit QS-regulated behavior. All mushroom strains were cultivated in potato-dextrose medium by large-scale submerged fermentation. The culture supernatant was condensed into 0.2 vol by freeze-drying. The condensed supernatant was sterilized by filtration through a 0.22-μm membrane filter and added to Chromobacterium violaceum CV026 cultures, which were used to monitor QS inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. The results have revealed that, of 102 mushroom strains, the bioactive metabolites produced by 14 basidiomycetes were found to inhibit violacein production, a QS-regulated behavior in C. violaceum. Higher fungi can produce QS-inhibitory compounds. Copyright © 2011 S. Karger AG, Basel.

Enhancement of a Novel Isolate of Serratia plymuthica as Potential Candidate for an Antianthracnose.

PubMed