Role of intracellular freezing in the death of cells cooled at supraoptimal rates. [Preservation of erythrocytes, bone marrow cells, and yeasts by freezing

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

Mazur, P.

1976-01-01

Cooling velocity is one of the major factors that determines whether viable cells can be frozen to temperatures that permit indefinite storage. Cooling either too slowly or too rapidly tends to be damaging. Optimum cooling rates are reported for mouse marrow stem cells, yeast, and human red cells.

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Bernardini, Jame N.; Stam, Christina N.

2010-01-01

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.

Deterministic separation of cancer cells from blood at 10 mL/min

NASA Astrophysics Data System (ADS)

Loutherback, Kevin; D'Silva, Joseph; Liu, Liyu; Wu, Amy; Austin, Robert H.; Sturm, James C.

2012-12-01

Circulating tumor cells (CTCs) and circulating clusters of cancer and stromal cells have been identified in the blood of patients with malignant cancer and can be used as a diagnostic for disease severity, assess the efficacy of different treatment strategies and possibly determine the eventual location of metastatic invasions for possible treatment. There is thus a critical need to isolate, propagate and characterize viable CTCs and clusters of cancer cells with their associated stroma cells. Here, we present a microfluidic device for mL/min flow rate, continuous-flow capture of viable CTCs from blood using deterministic lateral displacement (DLD) arrays. We show here that a DLD array device can isolate CTCs from blood with capture efficiency greater than 85% CTCs at volumetric flow rates of up to 10 mL/min with no effect on cell viability.

Heat resistance of viable but non-culturable Escherichia coli cells determined by differential scanning calorimetry.

PubMed

Castro-Rosas, Javier; Gómez-Aldapa, Carlos Alberto; Villagómez Ibarra, José Roberto; Santos-López, Eva María; Rangel-Vargas, Esmeralda

2017-10-16

Several reports have suggested that the viable but non-culturable (VBNC) state is a resistant form of bacterial cells that allows them to remain in a dormant form in the environment. Nevertheless, studies on the resistance of VBNC bacterial cells to ecological factors are limited, mainly because techniques that allow this type of evaluation are lacking. Differential scanning calorimetry (DSC) has been used to study the thermal resistance of culturable bacteria but has never been used to study VBNC cells. In this work, the heat resistance of Escherichia coli cells in the VBNC state was studied using the DSC technique. The VBNC state was induced in E. coli ATCC 25922 by suspending bacterial cells in artificial sea water, followed by storage at 3 ± 2°C for 110 days. Periodically, the behaviour of E. coli cells was monitored by plate counts, direct viable counts and DSC. The entire bacterial population entered the VBNC state after 110 days of storage. The results obtained with DSC suggest that the VBNC state does not confer thermal resistance to E. coli cells in the temperature range analysed here. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tumour cell dispersion by the ultrasonic aspirator during brain tumour resection.

PubMed

Preston, J K; Masciopinto, J; Salamat, M S; Badie, B

1999-10-01

Ultrasonic aspirators are commonly used to resect brain tumours because they allow safe, rapid and accurate removal of diseased tissue. Since ultrasonic aspirators generate a spray of aerosolized irrigating fluid around the instrument tip, we questioned whether this spray might contain viable tumours cells that could contribute to intraoperative spread of tumour fragments. To test this hypothesis, we collected the spray produced during the resection of nine brain tumours with an ultrasonic aspirator and semi-quantitatively analysed it for tumour presence. The aerosolized irrigation fluid was found to contain intact tumour cells or clumps of tumour cells in all nine instances, and there was a trend of increasing tumour cell dispersion with increasing ultrasonic aspiration times. Further examination is required to determine if this intraoperative dispersion of apparently viable tumour fragments contributes to local neoplasm recurrence.

Intestinal distribution of Vibrio cholerae in orally infected infant mice: kinetics of recovery of radiolabel and viable cells.

PubMed Central

Baselski, V S; Parker, C D

1978-01-01

Kinetics of distribution of Vibrio cholerae in the gastrointestinal tract of orally challenged infant mice were examined by determining recovery of input dose from the whole gut and from individual segments of stomach, upper bowel, and lower bowel. The strains studied were 569B, CA401, and VB12 (a rough CA401). Recovery was determined as a percentage of either input radiolabel using 35S-labeled cells or input colony-forming units. We found clearance of radiolabel and viable cells from the stomach into the intestines by 2 h. Early whole-gut clearance of label was greater for 569B and heat-killed CA401 than for CA401, VB12, or Formalinized CA401. At early times postchallenge, significant differences occurred between strains in the upper bowel, with greater recovery of label and viable cells for CA401 than for 569B or VB12. Beginning at 8 h postchallenge, radiolabel accumulated in the lower bowel with all experimental groups except CA401-challenged mice, where diarrhea was noted and label disappeared from the intestines. In vitro evaluation of mucosal association of these strains with bowel sections was also done. CA401 and VB12 associated to a greater extent than 569B or heat-killed or Formalin-killed CA401. PMID:689734

Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave.

PubMed

Kilimann, K V; Kitsubun, P; Delgado, A; Gänzle, M G; Chapleau, N; Le Bail, A; Hartmann, C

2006-07-05

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment. (c) 2006 Wiley Periodicals, Inc.

Rapid cell separation with minimal manipulation for autologous cell therapies

NASA Astrophysics Data System (ADS)

Smith, Alban J.; O'Rorke, Richard D.; Kale, Akshay; Rimsa, Roberts; Tomlinson, Matthew J.; Kirkham, Jennifer; Davies, A. Giles; Wälti, Christoph; Wood, Christopher D.

2017-02-01

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

Comparison of epifluorescent viable bacterial count methods

NASA Technical Reports Server (NTRS)

Rodgers, E. B.; Huff, T. L.

1992-01-01

Two methods, the 2-(4-Iodophenyl) 3-(4-nitrophenyl) 5-phenyltetrazolium chloride (INT) method and the direct viable count (DVC), were tested and compared for their efficiency for the determination of the viability of bacterial populations. Use of the INT method results in the formation of a dark spot within each respiring cell. The DVC method results in elongation or swelling of growing cells that are rendered incapable of cell division. Although both methods are subjective and can result in false positive results, the DVC method is best suited to analysis of waters in which the number of different types of organisms present in the same sample is assumed to be small, such as processed waters. The advantages and disadvantages of each method are discussed.

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli.

PubMed

van Frankenhuyzen, Jessica K; Trevors, Jack T; Flemming, Cecily A; Lee, Hung; Habash, Marc B

2013-11-01

Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by realtime polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5-1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.

Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers

PubMed Central

Fujimoto, Junji

2013-01-01

We developed a PCR-based method to detect and quantify viable Bifidobacterium bifidum BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 1010 to 106 cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4′,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 105.3 to 1010.3 cells/g feces (wet weight) (r > 0.99, P < 0.001). After 12 healthy subjects ingested 1010.3 to 1011.0 CFU of BF-1 in a fermented milk product daily for 28 days, 104.5 ± 1.5 (mean ± standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 106.2 ± 0.4 viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 107.6 ± 0.7 BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P < 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces. PMID:23354719

Quantitative assessment of viable cells of Lactobacillus plantarum strains in single, dual and multi-strain biofilms.

PubMed

Fernández Ramírez, Mónica D; Kostopoulos, Ioannis; Smid, Eddy J; Nierop Groot, Masja N; Abee, Tjakko

2017-03-06

Biofilms of Lactobacillus plantarum are a potential source for contamination and recontamination of food products. Although biofilms have been mostly studied using single species or even single strains, it is conceivable that in a range of environmental settings including food processing areas, biofilms are composed of multiple species with each species represented by multiple strains. In this study six spoilage related L. plantarum strains FBR1-FBR6 and the model strain L. plantarum WCFS1 were characterised in single, dual and multiple strain competition models. A quantitative PCR approach was used with added propidium monoazide (PMA) enabling quantification of intact cells in the biofilm, representing the viable cell fraction that determines the food spoilage risk. Our results show that the performance of individual strains in multi-strain cultures generally correlates with their performance in pure culture, and relative strain abundance in multi-strain biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The total biofilm quantified by CV staining of the dual and multi-strain biofilms formed was mainly correlated to CV values of the dominant strain obtained in single strain studies. However, the combination of strain FBR5 and strain WCFS1 showed significantly higher CV values compared to the individual performances of both strains indicating that total biofilm formation was higher in this specific condition. Notably, L. plantarum FBR5 was able to outgrow all other strains and showed the highest relative abundance in dual and multi-strain biofilms. All the dual and multi-strain biofilms contained a considerable number of viable cells, representing a potential source of contamination. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacillus subtilis Mutants with Knockouts of the Genes Encoding Ribonucleases RNase Y and RNase J1 Are Viable, with Major Defects in Cell Morphology, Sporulation, and Competence

PubMed Central

Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin

2013-01-01

The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed. PMID:23504012

Stem cells: science, policy, and ethics

PubMed Central

Fischbach, Gerald D.; Fischbach, Ruth L.

2004-01-01

Human embryonic stem cells offer the promise of a new regenerative medicine in which damaged adult cells can be replaced with new cells. Research is needed to determine the most viable stem cell lines and reliable ways to promote the differentiation of pluripotent stem cells into specific cell types (neurons, muscle cells, etc.). To create new cell lines, it is necessary to destroy preimplantation blastocysts. This has led to an intense debate that threatens to limit embryonic stem cell research. The profound ethical issues raised call for informed, dispassionate debate. PMID:15545983

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus.

PubMed

García-Hernández, J; Moreno, Y; Amorocho, C M; Hernández, M

2012-03-01

We have developed a direct viable count (DVC)-FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC-FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. This technique was successfully applied to detect viable cells in inoculated faeces. Results showed that this DVC-FISH procedure is a quick and culture-independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements.

PubMed

Trumpaitė-Vanagienė, Rita; Čebatariūnienė, Alina; Tunaitis, Virginijus; Pūrienė, Alina; Pivoriūnas, Augustas

2018-02-01

To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann's Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed. The extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway. The apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by alternative methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Viability and Virulence of Experimentally Stressed Nonculturable Salmonella typhimurium

PubMed Central

Caro, Audrey; Got, Patrice; Lesne, Jean; Binard, Sylvie; Baleux, Bernard

1999-01-01

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities. PMID:10388726

Optimization of low energy sonication treatment for granular activated carbon colonizing biomass assessment.

PubMed

Saccani, G; Bernasconi, M; Antonelli, M

2014-01-01

This study is aimed at optimizing a low energy sonication (LES) treatment for granular activated carbon (GAC)-colonizing biomass detachment and determination, evaluating detachment efficiency and the effects of ultrasound exposure on bacterial cell viability. GAC samples were collected from two filters fed with groundwater. Conventional heterotrophic plate count (HPC) and fluorescence microscopy with a double staining method were used to evaluate cell viability, comparing two LES procedures, without and with periodical bulk substitution. A 20 min LES treatment, with bulk substitution after cycles of 5 min as maximum treatment time, allowed to recover 87%/100% of attached biomass, protecting detached bacteria from ultrasound damaging effects. Observed viable cell inactivation rate was 6.5/7.9% cell/min, with membrane-compromised cell damage appearing to be even higher (11.5%/13.1% cell/min). Assessing bacterial detachment and damaging ultrasound effects, fluorescence microscopy turned out to be more sensitive compared to conventional HPC. The optimized method revealed a GAC-colonizing biomass of 9.9 x 10(7) cell/gGAC for plant 1 and 8.8 x 10(7) cell/gGAC for plant 2, 2 log lower than reported in literature. The difference between the two GAC-colonizing biomasses is higher in terms of viable cells (46.3% of total cells in plant 1 GAC-colonizing biomass compared to the 33.3% in plant 2). Studying influent water contamination through multivariate statistical analyses, apossible combined toxic and genotoxic effect of chromium VI and trichloroethylene was suggested as a reason for the lower viable cell fraction observed in plant 2 GAC-colonizing population.

ATP as a biomarker of viable microorganisms in clean-room facilities

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

2003-01-01

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

ATP as a biomarker of viable microorganisms in clean-room facilities.

PubMed

Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T; Kern, Roger

2003-03-01

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

Differential electrophoretic separation of cells and its effect on cell viability

NASA Technical Reports Server (NTRS)

Leise, E. M.; Lesane, F.

1974-01-01

An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.

Non-viable antagonist cells are associated with reduced biocontrol performance by viable cells of the yeast Papiliotrema flavescens against Fusarium head blight of wheat.

USDA-ARS?s Scientific Manuscript database

Microbially-based plant disease control products have achieved commercial market success, but the efficacy of such biocontrol products is sometimes deemed inconsistent. Improper processing of harvested microbial biomass or long-term storage can reduce the proportion of viable cells and necessitate t...

PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

2012-01-01

The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

Dynamic light scattering: A fast and reliable method to analyze bacterial growth during the lag phase.

PubMed

Vargas, Susana; Millán-Chiu, Blanca E; Arvizu-Medrano, Sofía M; Loske, Achim M; Rodríguez, Rogelio

2017-06-01

A comparison between plate counting (PC) and dynamic light scattering (DLS) is reported. PC is the standard technique to determine bacterial population as a function of time; however, this method has drawbacks, such as the cumbersome preparation and handling of samples, as well as the long time required to obtain results. Alternative methods based on optical density are faster, but do not distinguish viable from non-viable cells. These inconveniences are overcome by using DLS. Two different bacteria strains were considered: Escherichia coli and Staphylococcus aureus. DLS was performed at two different illuminating conditions: continuous and intermittent. By the increment of particle size as a function of time, it was possible to observe cell division and the formation of aggregates containing very few bacteria. The scattered intensity profiles showed the lag phase and the transition to the exponential phase of growth, providing a quantity proportional to viable bacteria concentration. The results revealed a clear and linear correlation in both lag and exponential phase, between the Log 10 (colony-forming units/mL) from PC and the Log 10 of the scattered intensity I s from DLS. These correlations provide a good support to use DLS as an alternative technique to determine bacterial population. Copyright © 2017 Elsevier B.V. All rights reserved.

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

The impact of cellular debris on Pseudomonas aeruginosa adherence to silicone hydrogel contact lenses and contact lens storage cases.

PubMed

Burnham, Geoffrey W; Cavanagh, H Dwight; Robertson, Danielle M

2012-01-01

To evaluate neutrophil-enhanced Pseudomonas aeruginosa (PA) biofilm formation on silicone hydrogel contact lenses and to determine the effect of epithelial biodebris on PA adherence in contact lens storage cases. A fully invasive PA corneal isolate stably conjugated to green fluorescent protein was used. Unworn lotrafilcon A contact lenses were incubated at various ratios of PA to polymorphonuclear neutrophil (PMN) for 24 hours at 37°C. Lens-associated PA was evaluated using laser scanning confocal microscopy and nonviable PA were visualized using propidium iodide. Viable bacteria were enumerated by colony-forming unit (CFU) analysis. For acute epithelial cell studies, PA viability was determined after coincubation with freeze-thaw epithelial cell lysates in 96-well polystyrene plates. Levels of residual cellular debris and bacterial viability were further assessed in used contact lens storage cases. Laser scanning confocal microscopy demonstrated that cotreatment with PMA-stimulated neutrophils increased PA adherence over 24 hours to lens surfaces with a striking alteration of PA architecture. Propidium iodide staining showed that the adherent bacteria consisted of a mixture of viable and nonviable PA; a PMN-associated increase in viable PA was confirmed by CFU (PA:PMN 0.1:1, P = 0.025; PA:PMN 1:1, P = 0.005). Acute epithelial cell debris studies revealed a significant increase in viable PA in 96-well plates in the presence of epithelial freeze-thaw lysates (PA:debris 1:1, P = 0.002; PA:debris 100:1, P = 0.002). Crystal violet staining of used lens storage cases revealed residual cellular debris at all time points, which was independent of microbial contamination; all lens cases used for periods of 9 months or more were uniformly associated with high levels of viable microorganisms. These results demonstrate that prolonged corneal inflammation with the presence of PMNs when confronted with simultaneous PA challenge in extended contact lens wear has the potential to stimulate biofilm formation on silicone hydrogel contact lenses. These findings further suggest that a persistent buildup of extracellular debris in lens storage cases may contribute to the heavy biofilms reported on these surfaces.

Structural parameters of collagen nerve grafts influence peripheral nerve regeneration.

PubMed

Stang, Felix; Fansa, Hisham; Wolf, Gerald; Reppin, Michael; Keilhoff, Gerburg

2005-06-01

Large nerve defects require nerve grafts to allow regeneration. To avoid donor nerve problems the concept of tissue engineering was introduced into nerve surgery. However, non-neuronal grafts support axonal regeneration only to a certain extent. They lack viable Schwann cells which provide neurotrophic and neurotopic factors and guide the sprouting nerve. This experimental study used the rat sciatic nerve to bridge 2 cm nerve gaps with collagen (type I/III) tubes. The tubes were different in their physical structure (hollow versus inner collagen skeleton, different inner diameters). To improve regeneration Schwann cells were implanted. After 8 weeks the regeneration process was monitored clinically, histologically and morphometrically. Autologous nerve grafts and collagen tubes without Schwann cells served as control. In all parameters autologous nerve grafts showed best regeneration. Nerve regeneration in a noteworthy quality was also seen with hollow collagen tubes and tubes with reduced lumen, both filled with Schwann cells. The inner skeleton, however, impaired nerve regeneration independent of whether Schwann cells were added or not. This indicates that not only viable Schwann cells are an imperative prerequisite but also structural parameters determine peripheral nerve regeneration.

Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells.

PubMed

Jung, Heejun; Kim, Namyoung; Yoon, Minjung

2016-10-01

The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of six substances on the growth and freeze-drying of Lactobacillus delbrueckii subsp. bulgaricus.

PubMed

Chen, He; Huang, Jie; Shi, Xiaoyu; Li, Yichao; Liu, Yu

2017-01-01

The efficacy of Lactobacillus delbrueckii subsp. bulgaricus as starter cultures for the dairy industry depends largely on the number of viable and active cells. Freeze-drying is the most convenient and successful method to preserve the bacterial cells. However, not all strains survived during freeze-drying. The effects of six substances including NaCl, sorbitol, mannitol, mannose, sodium glutamate, betaine added to the MRS medium on the growth and freeze-drying survival rate and viable counts of Lb. delbrueckii subsp. bulgaricus were studied through a single-factor test and Plackett-Burman design. Subsequently, the optimum freeze-drying conditions of Lb. delbrueckii subsp. bulgaricus were determined. Lb. delbrueckii subsp. bulgaricus survival rates were up to the maximum of 42.7%, 45.4%, 23.6%, while the concentrations of NaCl, sorbitol, sodium glutamate were 0.6%, 0.15%, 0.09%, respectively. In the optimum concentration, the viable counts in broth is 6.1, 6.9, 5.13 (×108 CFU/mL), respectively; the viable counts in freeze-drying power are 3.09, 5.2, 2.7 (×1010 CFU/g), respectively. Three antifreeze factors including NaCl, sorbitol, sodium glutamate have a positive effect on the growth and freeze-drying of Lb. delbrueckii subsp. bulgaricus. The results are beneficial for developing Lb. delbrueckii subsp. bulgaricus.

Studying the rapid bioconversion of lignocellulosic sugars into ethanol using high cell density fermentations with cell recycle

PubMed Central

2014-01-01

Background The Rapid Bioconversion with Integrated recycle Technology (RaBIT) process reduces capital costs, processing times, and biocatalyst cost for biochemical conversion of cellulosic biomass to biofuels by reducing total bioprocessing time (enzymatic hydrolysis plus fermentation) to 48 h, increasing biofuel productivity (g/L/h) twofold, and recycling biocatalysts (enzymes and microbes) to the next cycle. To achieve these results, RaBIT utilizes 24-h high cell density fermentations along with cell recycling to solve the slow/incomplete xylose fermentation issue, which is critical for lignocellulosic biofuel fermentations. Previous studies utilizing similar fermentation conditions showed a decrease in xylose consumption when recycling cells into the next fermentation cycle. Eliminating this decrease is critical for RaBIT process effectiveness for high cycle counts. Results Nine different engineered microbial strains (including Saccharomyces cerevisiae strains, Scheffersomyces (Pichia) stipitis strains, Zymomonas mobilis 8b, and Escherichia coli KO11) were tested under RaBIT platform fermentations to determine their suitability for this platform. Fermentation conditions were then optimized for S. cerevisiae GLBRCY128. Three different nutrient sources (corn steep liquor, yeast extract, and wheat germ) were evaluated to improve xylose consumption by recycled cells. Capacitance readings were used to accurately measure viable cell mass profiles over five cycles. Conclusion The results showed that not all strains are capable of effectively performing the RaBIT process. Acceptable performance is largely correlated to the specific xylose consumption rate. Corn steep liquor was found to reduce the deleterious impacts of cell recycle and improve specific xylose consumption rates. The viable cell mass profiles indicated that reduction in specific xylose consumption rate, not a drop in viable cell mass, was the main cause for decreasing xylose consumption. PMID:24847379

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

1995-11-14

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

1995-01-01

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

Influence of copper ions on the viability and cytotoxicity of Pseudomonas aeruginosa under conditions relevant to drinking water environments.

PubMed

Dwidjosiswojo, Zenyta; Richard, Jessica; Moritz, Miriam M; Dopp, Elke; Flemming, Hans-Curt; Wingender, Jost

2011-11-01

Copper plumbing materials can be the source of copper ions in drinking water supplies. The aim of the current study was to investigate the influence of copper ions on the viability and cytotoxicity of the potential pathogen Pseudomonas aeruginosa that presents a health hazard when occurring in building plumbing systems. In batch experiments, exposure of P. aeruginosa (10(6)cells/mL) for 24h at 20°C to copper-containing drinking water from domestic plumbing systems resulted in a loss of culturability, while total cell numbers determined microscopically did not decrease. Addition of the chelator diethyldithiocarbamate (DDTC) to copper-containing water prevented the loss of culturability. When suspended in deionized water with added copper sulfate (10 μM), the culturability of P. aeruginosa decreased by more than 6 log units, while total cell counts, the concentration of cells with intact cytoplasmic membranes, determined with the LIVE/DEAD BacLight kit, and the number of cells with intact 16S ribosomal RNA, determined by fluorescent in situ hybridization, remained unchanged. When the chelator DDTC was added to copper-stressed bacteria, complete restoration of culturability was observed to occur within 14 d. Copper-stressed bacteria were not cytotoxic towards Chinese hamster ovary (CHO-9) cells, while untreated and resuscitated bacteria caused an almost complete decrease of the concentration of viable CHO-9 cells within 24 h. Thus, copper ions in concentrations relevant to drinking water in plumbing systems seem to induce a viable but non-culturable (VBNC) state in P. aeruginosa accompanied by a loss of culturability and cytotoxicity, and VBNC cells can regain both culturability and cytotoxicity, when copper stress is abolished. Copyright © 2011 Elsevier GmbH. All rights reserved.

Power limits for microbial life.

PubMed

LaRowe, Douglas E; Amend, Jan P

2015-01-01

To better understand the origin, evolution, and extent of life, we seek to determine the minimum flux of energy needed for organisms to remain viable. Despite the difficulties associated with direct measurement of the power limits for life, it is possible to use existing data and models to constrain the minimum flux of energy required to sustain microorganisms. Here, a we apply a bioenergetic model to a well characterized marine sedimentary environment in order to quantify the amount of power organisms use in an ultralow-energy setting. In particular, we show a direct link between power consumption in this environment and the amount of biomass (cells cm(-3)) found in it. The power supply resulting from the aerobic degradation of particular organic carbon (POC) at IODP Site U1370 in the South Pacific Gyre is between ∼10(-12) and 10(-16) W cm(-3). The rates of POC degradation are calculated using a continuum model while Gibbs energies have been computed using geochemical data describing the sediment as a function of depth. Although laboratory-determined values of maintenance power do a poor job of representing the amount of biomass in U1370 sediments, the number of cells per cm(-3) can be well-captured using a maintenance power, 190 zW cell(-1), two orders of magnitude lower than the lowest value reported in the literature. In addition, we have combined cell counts and calculated power supplies to determine that, on average, the microorganisms at Site U1370 require 50-3500 zW cell(-1), with most values under ∼300 zW cell(-1). Furthermore, we carried out an analysis of the absolute minimum power requirement for a single cell to remain viable to be on the order of 1 zW cell(-1).

The importance of the viable but non-culturable state in human bacterial pathogens

PubMed Central

Li, Laam; Mendis, Nilmini; Trigui, Hana; Oliver, James D.; Faucher, Sebastien P.

2014-01-01

Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed. PMID:24917854

Bactericidal effects of various concentrations of enrofloxacin, florfenicol, tilmicosin phosphate, and tulathromycin on clinical isolates of Mannheimia haemolytica.

PubMed

Blondeau, Joseph M; Shebelski, Shantelle D; Hesje, Christine K

2015-10-01

To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. 4 unique isolates of M haemolytica recovered from clinically infected cattle. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

NASA Technical Reports Server (NTRS)

Yu, F. P.; Pyle, B. H.; McFeters, G. A.

1993-01-01

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

Assessments of Total and Viable Escherichia coli O157:H7 on Field and Laboratory Grown Lettuce

PubMed Central

Moyne, Anne-Laure; Harris, Linda J.; Marco, Maria L.

2013-01-01

Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 106 CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 104 greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable. PMID:23936235

Grazing of particle-associated bacteria-an elimination of the non-viable fraction.

PubMed

Gonsalves, Maria-Judith; Fernandes, Sheryl Oliveira; Priya, Madasamy Lakshmi; LokaBharathi, Ponnapakkam Adikesavan

Quantification of bacteria being grazed by microzooplankton is gaining importance since they serve as energy subsidies for higher trophic levels which consequently influence fish production. Hence, grazing pressure on viable and non-viable fraction of free and particle-associated bacteria in a tropical estuary controlled mainly by protist grazers was estimated using the seawater dilution technique. In vitro incubations over a period of 42h showed that at the end of 24h, growth coefficient (k) of particle-associated bacteria was 9 times higher at 0.546 than that of free forms. Further, 'k' value of viable cells on particles was double that of free forms at 0.016 and 0.007, respectively. While bacteria associated with particles were grazed (coefficient of removal (g)=0.564), the free forms were relatively less grazed indicating that particle-associated bacteria were exposed to grazers in these waters. Among the viable and non-viable forms, 'g' of non-viable fraction (particle-associated bacteria=0.615, Free=0.0086) was much greater than the viable fraction (particle-associated bacteria=0.056, Free=0.068). Thus, grazing on viable cells was relatively low in both the free and attached states. These observations suggest that non-viable forms of particle-associated bacteria were more prone to grazing and were weeded out leaving the viable cells to replenish the bacterial standing stock. Particle colonization could thus be a temporary refuge for the "persistent variants" where the viable fraction multiply and release their progeny. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Legionella in water samples: how can you interpret the results obtained by quantitative PCR?

PubMed

Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Arauco Rivera, Susan R; Zotti, Carla M

2015-02-01

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples. In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU). Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Seasonal and spatial distribution of bacterial biomass and the percentage of viable cells in a reservoir of Alabama

USGS Publications Warehouse

Tietjen, T.E.; Wetzel, R.G.

2003-01-01

Spatial community dynamics of bacterioplankton were evaluated along the length of the former stream channel of Elledge Lake, a small reservoir in western Alabama. The reservoir was strongly stratified from April to October with up to a 10??C temperature difference across the 1 m deep metalimnion. Bacterial biomass was highest during late summer, with a general pattern of increasing abundance from the inflowing river (???10 ??g C l-1) to the dam (???20-30 ??g C l-1). Bacterial numbers also increased following a >10-fold increase in turbidity associated with a major precipitation event, although only ???10% of these cells were viable. The percentage of viable cells generally increased through the stratified period with 50-70% viable cells in late summer. Overall, an average of 38% of bacterial cells were viable, with a range from <20 to 70%. Although these values were similar to those found by others, additional patterns were identified that have not been previously observed: a marked decline in viable cells was found following turbid storm inflows and increases in the percentage of viable cells occurred during spring warming and following autumnal mixing events. Although a modest increase in abundance occurred along the gradient from inflow down-reservoir to the dam, bacterial abundance did not increase near the dam in a pattern coincident with the commonly observed increased algal biomass in the lacustrine portion of reservoir ecosystems. The increases observed in bacterial viability moving from the inflowing rivers towards the dam and later in stratified periods stress the importance of differences in environmental conditions in time and space in regulating bacterial biomass and development, as well as of shifts that would be anticipated accompanying altered hydrological regimes under climatic change.

Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family Enterobacteriaceae in Freshwater and Drinking Water

PubMed Central

Baudart, Julia; Coallier, Josée; Laurent, Patrick; Prévost, Michèle

2002-01-01

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 108 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed. PMID:12324357

Properties of genes essential for mouse development

PubMed Central

Kabir, Mitra; Barradas, Ana; Tzotzos, George T.; Hentges, Kathryn E.

2017-01-01

Essential genes are those that are critical for life. In the specific case of the mouse, they are the set of genes whose deletion means that a mouse is unable to survive after birth. As such, they are the key minimal set of genes needed for all the steps of development to produce an organism capable of life ex utero. We explored a wide range of sequence and functional features to characterise essential (lethal) and non-essential (viable) genes in mice. Experimental data curated manually identified 1301 essential genes and 3451 viable genes. Very many sequence features show highly significant differences between essential and viable mouse genes. Essential genes generally encode complex proteins, with multiple domains and many introns. These genes tend to be: long, highly expressed, old and evolutionarily conserved. These genes tend to encode ligases, transferases, phosphorylated proteins, intracellular proteins, nuclear proteins, and hubs in protein-protein interaction networks. They are involved with regulating protein-protein interactions, gene expression and metabolic processes, cell morphogenesis, cell division, cell proliferation, DNA replication, cell differentiation, DNA repair and transcription, cell differentiation and embryonic development. Viable genes tend to encode: membrane proteins or secreted proteins, and are associated with functions such as cellular communication, apoptosis, behaviour and immune response, as well as housekeeping and tissue specific functions. Viable genes are linked to transport, ion channels, signal transduction, calcium binding and lipid binding, consistent with their location in membranes and involvement with cell-cell communication. From the analysis of the composite features of essential and viable genes, we conclude that essential genes tend to be required for intracellular functions, and viable genes tend to be involved with extracellular functions and cell-cell communication. Knowledge of the features that are over-represented in essential genes allows for a deeper understanding of the functions and processes implemented during mammalian development. PMID:28562614

Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry ▿†

PubMed Central

Parthuisot, N.; Binet, M.; Touron-Bodilis, A.; Pougnard, C.; Lebaron, P.; Baudart, J.

2011-01-01

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter−1, and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 103 viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples. PMID:21742913

Fluorine 18 fluorodeoxyglucose PET/CT volume-based indices in locally advanced non-small cell lung cancer: prediction of residual viable tumor after induction chemotherapy.

PubMed

Soussan, Michael; Cyrta, Joanna; Pouliquen, Christelle; Chouahnia, Kader; Orlhac, Fanny; Martinod, Emmanuel; Eder, Véronique; Morère, Jean-François; Buvat, Irène

2014-09-01

To study whether volume-based indices of fluorine 18 fluorodeoxyglucose positron emission tomographic (PET)/computed tomographic (CT) imaging is an accurate tool to predict the amount of residual viable tumor after induction chemotherapy in patients with locally advanced non-small cell lung cancer (NSCLC). This study was approved by institutional review board with waivers of informed consent. Twenty-two patients with locally advanced NSCLC underwent surgery after induction chemotherapy. All had pre- and posttreatment FDG PET/CT scans. CT largest diameter, CT volume, maximum standardized uptake value (SUVmax), mean SUV (SUVmean), metabolic tumor volume (TV), and total lesion glycolysis of primary tumor were calculated. Changes in tumor measurements were determined by dividing follow-up by baseline measurement (ratio index). Amounts of residual viable tumor, necrosis, fibrous tissue, inflammatory infiltrate, and Ki-67 proliferative index were estimated on resected tumor. Correlations between imaging indices and histologic parameters were estimated by using Spearman correlation coefficients or Mann-Whitney tests. No baseline or posttreatment indices correlated with percentage of residual viable tumor. TV ratio was the only index that correlated with percentage of residual viable tumor (r = 0.61 [95% confidence interval: 0.24, 0.81]; P = .003). Conversely, SUVmax and SUVmean ratios were only indices correlated with Ki-67 (r = 0.62 [95% confidence interval: 0.24, 0.82]; P = .003; and r = 0.60 [95% confidence interval: 0.21, 0.81]; P = .004, respectively). Total lesion glycolysis ratio was moderately correlated with residual viable tumor (r = 0.53 [95% confidence interval: 0.13, 0.78]; P = .01) and with Ki-67 (r = 0.57 [95% confidence interval: 0.18, 0.80]; P = .006). No ratios were correlated with presence of inflammatory infiltrate or foamy macrophages. TV and total lesion glycolysis ratios were the only indices correlated with residual viable tumor after induction chemotherapy in locally advanced NSCLC.

Removal of viable bioaerosol particles with a low-efficiency HVAC filter enhanced by continuous emission of unipolar air ions.

PubMed

Huang, R; Agranovski, I; Pyankov, O; Grinshpun, S

2008-04-01

Continuous emission of unipolar ions has been shown to improve the performance of respirators and stationary filters challenged with non-biological particles. In this study, we investigated the ion-induced enhancement effect while challenging a low-efficiency heating, ventilation and air-conditioning (HVAC) filter with viable bacterial cells, bacterial and fungal spores, and viruses. The aerosol concentration was measured in real time. Samples were also collected with a bioaerosol sampler for viable microbial analysis. The removal efficiency of the filter was determined, respectively, with and without an ion emitter. The ionization was found to significantly enhance the filter efficiency in removing viable biological particles from the airflow. For example, when challenged with viable bacteria, the filter efficiency increased as much as four- to fivefold. For viable fungal spores, the ion-induced enhancement improved the efficiency by a factor of approximately 2. When testing with virus-carrying liquid droplets, the original removal efficiency provided by the filter was rather low: 9.09 +/- 4.84%. While the ion emission increased collection about fourfold, the efficiency did not reach 75-100% observed with bacteria and fungi. These findings, together with our previously published results for non-biological particles, demonstrate the feasibility of a new approach for reducing aerosol particles in HVAC systems used for indoor air quality control. Recirculated air in HVAC systems used for indoor air quality control in buildings often contains considerable number of viable bioaerosol particles because of limited efficiency of the filters installed in these systems. In the present study, we investigated - using aerosolized bacterial cells, bacterial and fungal spores, and virus-carrying particles - a novel idea of enhancing the performance of a low-efficiency HVAC filter utilizing continuous emission of unipolar ions in the filter vicinity. The findings described in this paper, together with our previously published results for non-biological particles, demonstrate the feasibility of the newly developed approach.

Distribution of Cryptococcus neoformans in a natural site.

PubMed Central

Ruiz, A; Fromtling, R A; Bulmer, G S

1981-01-01

Pigeon droppings in a vacant tower were assayed for the number and size of viable cells of Cryptococcus neoformans. The dry, thinly scattered floor debris contained 2.6 x 10(6) viable cells per g--300 times more cells than were cultured from a large, compact pile of pigeon droppings (7.4 x 10(3) cells per g). Aerosols generated from floor debris containing pigeon droppings had an average of 360 viable cells in 31 liters of air; 27 of these cells (7.5%) were 1.1 to 3.3 micrometers in diameter and, therefore, capable of human lung deposition. Environmental factors which may influence the distribution, survival, and proliferation of C. neoformans in nature are discussed. PMID:7012011

[Studies on minimum antibiotic concentration of cephapirin against clinically isolated strain SMK-101 of Klebsiella pneumoniae].

PubMed

Takahashi, M; Usui, Y; Ichiman, Y; Yoshida, K; Yonaha, T

1985-01-01

Using strain SMK-101 of K. pneumoniae its nephelometric absorbencies, viable cell numbers and morphological changes were studied during the time course cultured in a broth medium containing cephapirin (CEPR), and following results were obtained. After 1 to 3 hours culture in the presence of varying concentration of the antibiotic, the absorbency increased in spite of without change in the viable cell number. Morphologically, elongation and swelling of central portion of the cells were observed though differences of the degree of these findings varied depending upon the concentration of the antibiotic. At the concentration higher than 1/4 MIC, indistinct structure was shown in cytoplasm. After 6 hours culture, 3 directions of absorbence curves, ascending, descending and no change, and 2 directions of viable cell numbers, decreasing and increasing were shown. As the morphological changes of the cells, filamentation, leaking of intracellular components were shown in rather upper concentration of the antibiotic. Fission was demonstrated around the end of cells cultured in rather lower concentration of the antibiotic. After 9 hours culture, absorbency and viable cell number were parallel. In this period, structural findings of cytoplasm became clear and fission was also demonstrated by light microscope except for the cells cultured in more than 1 MIC of the antibiotic. After 24 hours culture, both absorbency and viable cell number increased again and fission was observed in the cell which showed filamentation in 1 MIC of the antibiotic.

A Novel Method for Determining the Level of Viable Disseminated Prostate Cancer Cells

DTIC Science & Technology

2012-10-01

Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for...need for multiple replicates of plates in time course studies. Recently a naturally secreted luciferase was identified and cloned from the marine ...well solid white flat bottom polystyrene microplates (Corning, Cat#3917, Lowell, MA). After 24 hours, conditioned media was harvested and remaining

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

PubMed

Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2015-06-01

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

The growth of Staphylococcus aureus and Escherichia coli in low-direct current electric fields.

PubMed

Zituni, Dunya; Schütt-Gerowitt, Heidi; Kopp, Marion; Krönke, Martin; Addicks, Klaus; Hoffmann, Christian; Hellmich, Martin; Faber, Franz; Niedermeier, Wilhelm

2014-03-01

Electrical potentials up to 800 mV can be observed between different metallic dental restorations. These potentials produce fields in the mouth that may interfere with microbial communities. The present study focuses on the impact of different electric field strengths (EFS) on the growth of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) in vitro. Cultures of S. aureus and E. coli in fluid and gel medium were exposed to different EFS. Effects were determined by calculation of viable counts and measurement of inhibition zones. In gel medium, anodic inhibition zones for S. aureus were larger than those for E. coli at all field strength levels. In fluid medium, the maximum decrease in the viable count of S. aureus cells was at 10 V⋅m(-1). Field-treated S. aureus cells presented ruptured cell walls and disintegrated cytoplasm. Conclusively, S. aureus is more sensitive to increasing electric field strength than E. coli.

Extracellular delivery induced by ultrasound and microbubbles in cells

NASA Astrophysics Data System (ADS)

Hussein, Farah; Antonescu, Costin; Karshafian, Raffi

2017-03-01

Ultrasound and microbubble treatment (USMB) can enhance the intracellular uptake of molecules, which otherwise would be excluded from the cell, through USMB-mediated transient membrane disruption and through enhanced endocytosis. However, the effect of USMB on the outward movement of molecules from cells is not well understood. This study investigates the effects of USMB on the release of molecules from various cellular compartments including cytoplasm, lysosomes, and recycling endosomes. In vitro ARPE-19 (RPE henceforth) cells were loaded with Alexa fluor-labeled transferrin as a marker for recycling endosomes, LAMP-1 antibody was used to detect the fusion of lysosomes with the plasma membrane, GFP-transfected RPE cells were used to examine the release of GFP from the cytoplasm, and 7-AAD was used to assess cell viability. Subsequently, cells were exposed to USMB (106 cells/mL, 300 kPa peak negative pressure, 1 min treatment duration, and 20 µL/mL Definity microbubbles). Following USMB, the release of the fluorescent markers was examined at 1.5, 11.5, and 21.5 minutes from the start of USMB. The mean fluorescent intensity (MFI) of untreated and USMB treated samples were measured using flow cytometry. USMB increased the extracellular delivery of GFP molecules from the cytoplasm; the MFI in USMB treated GFP-transfected RPE cells decreased by 17% in viable cells and this MFI decreased by 70% in non-viable cells. This could be due to diffusion of GFP through the membrane disruptions induced by USMB. Additionally, the MFI of viable cells stained with LAMP-1 antibody increased by 50% and this increase was 15 folds in the non-viable cells indicating lysosome exocytosis as a mechanism for membrane repair. Furthermore, the MFI of cells loaded with fluorescent transferrin decreased by 22% after USMB treatment in viable cells, indicating a significant increase in transferrin recycling to the cell membrane. However, the increased recycling was not statistically significant in the non-viable cells. This indicates that the increase in transferrin recycling was through an active mechanism that was triggered or enhanced by USMB. It was concluded from this study that USMB enhances the release of molecules from the cytoplasm, lysosomes, and recycling endosomes.

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

NASA Astrophysics Data System (ADS)

Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

2014-12-01

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Formulation and evaluation of Bacillus coagulans-loaded hypromellose mucoadhesive microspheres.

PubMed

Alli, Sk Md Athar

2011-01-01

Development of a novel delivery system has been attempted to deliver viable probiotic cells into the gut for a prolonged period of time while maintaining high numbers of viable cells within the formulation throughout the shelf-life of the product and during the gastrointestinal transit. Core mucoadhesive microspheres of Bacillus coagulans were developed employing several grades of hypromellose, a mucoadhesive polymer, following coacervation and phase separation technique and were subsequently enteric-coated with hypromellose phthalate. Microspheres were evaluated for percent yield; entrapment efficiency; in vitro swelling; surface morphology; particle size, size distribution, and zeta potential; flow property, mucoadhesion property by the ex vivo mucoadhesive strength test and the in vitro wash off test; in vitro release profile and release kinetic; in vivo probiotic activity; and stability. The values for the kinetic constant and regression coefficient of model-dependent approaches and the difference factor (f(1)), the similarity factor (f(2)), and the Rescigno index (ξ(1) and ξ(2)) of model independent approaches were determined for comparing in vitro dissolution profiles. Freeze dried B. coagulans cells were successfully formulated as enteric-coated mucoadhesive microspheres with satisfactory physical structure and yield. The viability of B. coagulans was maintained in the simulated gastric conditions and during processing; in simulated intestinal conditions exhibiting mucoadhesion, and controlling and extending the viable cell release following zero-order; and was satisfactorily stable at room temperature. Test results depict statistically significant effects of the hypromellose grade and their concentration on the performance and release profile of formulations.

Formulation and evaluation of Bacillus coagulans-loaded hypromellose mucoadhesive microspheres

PubMed Central

Alli, Sk Md Athar

2011-01-01

Development of a novel delivery system has been attempted to deliver viable probiotic cells into the gut for a prolonged period of time while maintaining high numbers of viable cells within the formulation throughout the shelf-life of the product and during the gastrointestinal transit. Core mucoadhesive microspheres of Bacillus coagulans were developed employing several grades of hypromellose, a mucoadhesive polymer, following coacervation and phase separation technique and were subsequently enteric-coated with hypromellose phthalate. Microspheres were evaluated for percent yield; entrapment efficiency; in vitro swelling; surface morphology; particle size, size distribution, and zeta potential; flow property, mucoadhesion property by the ex vivo mucoadhesive strength test and the in vitro wash off test; in vitro release profile and release kinetic; in vivo probiotic activity; and stability. The values for the kinetic constant and regression coefficient of model-dependent approaches and the difference factor (f1), the similarity factor (f2), and the Rescigno index (ξ1 and ξ2) of model independent approaches were determined for comparing in vitro dissolution profiles. Freeze dried B. coagulans cells were successfully formulated as enteric-coated mucoadhesive microspheres with satisfactory physical structure and yield. The viability of B. coagulans was maintained in the simulated gastric conditions and during processing; in simulated intestinal conditions exhibiting mucoadhesion, and controlling and extending the viable cell release following zero-order; and was satisfactorily stable at room temperature. Test results depict statistically significant effects of the hypromellose grade and their concentration on the performance and release profile of formulations. PMID:21674019

Cell death induced by hydroxyapatite on L929 fibroblast cells.

PubMed

Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

2004-05-01

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

Impact of Selection of Cord Blood Units from the United States and Swiss Registries on the Cost of Banking Operations

PubMed Central

Bart, Thomas; Boo, Michael; Balabanova, Snejana; Fischer, Yvonne; Nicoloso, Grazia; Foeken, Lydia; Oudshoorn, Machteld; Passweg, Jakob; Tichelli, Andre; Kindler, Vincent; Kurtzberg, Joanne; Price, Thomas; Regan, Donna; Shpall, Elizabeth J.; Schwabe, Rudolf

2013-01-01

Background Over the last 2 decades, cord blood (CB) has become an important source of blood stem cells. Clinical experience has shown that CB is a viable source for blood stem cells in the field of unrelated hematopoietic blood stem cell transplantation. Methods Studies of CB units (CBUs) stored and ordered from the US (National Marrow Donor Program (NMDP) and Swiss (Swiss Blood Stem Cells (SBSQ)) CB registries were conducted to assess whether these CBUs met the needs of transplantation patients, as evidenced by units being selected for transplantation. These data were compared to international banking and selection data (Bone Marrow Donors Worldwide (BMDW), World Marrow Donor Association (WMDA)). Further analysis was conducted on whether current CB banking practices were economically viable given the units being selected from the registries for transplant. It should be mentioned that our analysis focused on usage, deliberately omitting any information about clinical outcomes of CB transplantation. Results A disproportionate number of units with high total nucleated cell (TNC) counts are selected, compared to the distribution of units by TNC available. Therefore, the decision to use a low threshold for banking purposes cannot be supported by economic analysis and may limit the economic viability of future public CB banking. Conclusions We suggest significantly raising the TNC level used to determine a bankable unit. A level of 125 × 107 TNCs, maybe even 150 × 107 TNCs, might be a viable banking threshold. This would improve the return on inventory investments while meeting transplantation needs based on current selection criteria. PMID:23637645

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

PubMed

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

2016-05-06

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

Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

PubMed

de Aguiar Cordeiro, Rossana; Serpa, Rosana; Flávia Uchoa Alexandre, Camila; de Farias Marques, Francisca Jakelyne; Vladia Silva de Melo, Charlline; da Silva Franco, Jônatas; José de Jesus Evangelista, Antonio; Pires de Camargo, Zoilo; Samia Nogueira Brilhante, Raimunda; Fabio Gadelha Rocha, Marcos; Luciano Bezerra Moreira, José; de Jesus Pinheiro Gomes Bandeira, Tereza; Júlio Costa Sidrim, José

2015-11-01

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

Dye Sensitized Solar Cells for Economically Viable Photovoltaic Systems.

PubMed

Jung, Hyun Suk; Lee, Jung-Kun

2013-05-16

TiO2 nanoparticle-based dye sensitized solar cells (DSSCs) have attracted a significant level of scientific and technological interest for their potential as economically viable photovoltaic devices. While DSSCs have multiple benefits such as material abundance, a short energy payback period, constant power output, and compatibility with flexible applications, there are still several challenges that hold back large scale commercialization. Critical factors determining the future of DSSCs involve energy conversion efficiency, long-term stability, and production cost. Continuous advancement of their long-term stability suggests that state-of-the-art DSSCs will operate for over 20 years without a significant decrease in performance. Nevertheless, key questions remain in regards to energy conversion efficiency improvements and material cost reduction. In this Perspective, the present state of the field and the ongoing efforts to address the requirements of DSSCs are summarized with views on the future of DSSCs.

Comparison of the effect of three autogenous bone harvesting methods on cell viability in rabbits

PubMed Central

Moradi Haghgoo, Janet; Arabi, Seyed Reza; Hosseinipanah, Seyyed Mohammad; Solgi, Ghasem; Rastegarfard, Neda; Farhadian, Maryam

2017-01-01

Background. This study was designed to compare the viability of autogenous bone grafts, harvested using different methods, in order to determine the best harvesting technique with respect to more viable cells. Methods. In this animal experimental study, three harvesting methods, including manual instrument (chisel), rotary device and piezosurgery, were used for harvesting bone grafts from the lateral body of the mandible on the left and right sides of 10 rabbits. In each group, 20 bone samples were collected and their viability was assessed using MTS kit. Statistical analyses, including ANOVA and post hoc Tukey tests, were used for evaluating significant differences between the groups. Results. One-way ANOVA showed significant differences between all the groups (P=0.000). Data analysis using post hoc Tukey tests indicated that manual instrument and piezosurgery had no significant differences with regard to cell viability (P=0.749) and the cell viability in both groups was higher than that with the use of a rotary instrument (P=0.000). Conclusion. Autogenous bone grafts harvested with a manual instrument and piezosurgery had more viable cells in comparison to the bone chips harvested with a rotary device. PMID:28748046

Effects of surface chemistry on the optical properties and cellular interaction of lanthanide-based nanoparticles

NASA Astrophysics Data System (ADS)

Pedraza, Francisco J.; Avalos, Julio C.; Mimun, Lawrence C.; Yust, Brian G.; Tsin, Andrew; Sardar, Dhiraj K.

2015-03-01

Fluorescent nanoparticles (NPs) such as KYb2F7:Tm3+ potential in biomedical applications due to their ability to absorb and emit within the biological window, where near infrared light is less attenuated by soft tissue. This results in less tissue damage and deeper tissue penetration making it a viable candidate in biological imaging. Another big factor in determining their ability to perform in a biological setting is the surface chemistry. Biocompatible coatings, including polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), pluronic and folic acid are commonly used because they pose several advantages such as ease of functionalization, better dispersion, and higher cellular uptake. To study the effects of the NP surface chemistry, KYb2F7:Tm3+ a solvothermal method using PEG, PVP, pluronic acid, and folic acid as a capping agent, followed by thorough optical characterizations. Optical changes were thoroughly studied and compared using absorption, emission, and quantum yield data. Cell viability was obtained by treating Rhesus Monkey Retinal Endothelial cells (RhREC) with KYb2F7:Tm3+ and counting viable cells following a 24 hour uptake period. The work presented will compare the optical properties and toxicity dependency on the surface chemistry on KYb2F7:Tm3+. The results will also indicate that KYb2F7:Tm3+ nanoparticles are viable candidates for various biomedical applications.

Microbial Burden Approach : New Monitoring Approach for Measuring Microbial Burden

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Vaishampayan, Parag; Barmatz, Martin

2013-01-01

Advantages of new approach for differentiating live cells/ spores from dead cells/spores. Four examples of Salmonella outbreaks leading to costly destruction of dairy products. List of possible collaboration activities between JPL and other industries (for future discussion). Limitations of traditional microbial monitoring approaches. Introduction to new approach for rapid measurement of viable (live) bacterial cells/spores and its areas of application. Detailed example for determining live spores using new approach (similar procedure for determining live cells). JPL has developed a patented approach for measuring amount of live and dead cells/spores. This novel "molecular" method takes less than 5 to 7 hrs. compared to the seven days required using conventional techniques. Conventional "molecular" techniques can not discriminate live cells/spores among dead cells/spores. The JPL-developed novel method eliminates false positive results obtained from conventional "molecular" techniques that lead to unnecessary delay in the processing and to unnecessary destruction of food products.

Generation of Viable Mice from Induced Pluripotent Stem Cells (iPSCs) Through Tetraploid Complementation.

PubMed

Kang, Lan; Gao, Shaorong

2015-01-01

Tetraploid complementation assay is the most rigorous criteria for pluripotency characterization of pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells could complement the developmental deficiency of tetraploid embryos and thus support the full-term mice development. Here we describe the protocol for tetraploid complementation using iPSCs to produce viable all-iPSC mice.

Stability, antimicrobial activity, and effect of nisin on the physico-chemical properties of fruit juices.

PubMed

de Oliveira Junior, Adelson Alves; de Araújo Couto, Hyrla Grazielle Silva; Barbosa, Ana Andréa Teixeira; Carnelossi, Marcelo Augusto Guitierrez; de Moura, Tatiana Rodrigues

2015-10-15

Heat processing is the most commonly used hurdle for inactivating microorganisms in fruit juices. However, this preservation method could interfere with the organoleptic characteristics of the product. Alternative methods have been proposed and bacteriocins such as nisin are potential candidates. However, the approval of bacteriocins as food additives is limited, especially in foods from vegetal origin. We aimed to verify the stability, the effect on physico-chemical properties, and the antimicrobial activity of nisin in different fruit juices. Nisin remained stable in fruit juices (cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu) for at least 30 days at room or refrigerated temperature and did not cause any significant alterations in the physico-chemical characteristics of the juices. Besides, nisin favored the preservation of vitamin C content in juices. The antimicrobial activity of nisin was tested against Alicyclobacillus acidoterrestris, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in cashew, soursop, peach, and mango juices. Nisin caused a 4-log reduction in viable cells of A. acidoterrestris in soursop, peach, and mango juices after 8h of incubation, and no viable cells were detected in cashew juices. After 24h of incubation in the presence of nisin, no viable cells were detected, independently of the juices. To S. aureus, at 24h of incubation in the presence of nisin, viable cells were only detected in mango juices, representing a 4-log decrease as compared with the control treatment. The number of viable cells of B. cereus at 24h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment. When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24h of incubation. Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively. The most sensitive microorganism to nisin was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes. These results indicate that the use of nisin could be an alternative in fruit juice processing. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Lactic Acid Bacteria as Poultry Probiotic Candidates with Aflatoxin B1 Binding Activities

NASA Astrophysics Data System (ADS)

Damayanti, E.; Istiqomah, L.; Saragih, J. E.; Purwoko, T.; Sardjono

2017-12-01

Our previous studies have selected lactic acid bacteria (LAB) with antifungal activities from traditional fermented foods made from cassava (G7) and silage feed palm leaf (PDS5 and PDS3). In this study we evaluated their ability to bind aflatoxin B1 (AFB1) and probiotic characteristic. The probiotic characteristic assays of LAB consisted of resistance to acidic conditions (pH 3), gastric juice and bile salts 0.3%. We also carried out an in vitro evaluation of LAB aflatoxin binding ability in viable and non-viable cell for 24 and 48 hours of incubation. The measurement of aflatoxin content was performed by ELISA method using AgraQuant Total Aflatoxin Assay kit. The results showed that all isolates were potential as probiotics and the G7 isolate had the highest viability among other isolates in pH 3 (92.61 %) and the bile salts assay (97.71 %). The percentage of aflatoxin reduction between viable and non-viable cell from each LAB isolate were different. The highest aflatoxin reduction in viable cell assay was performed by G7 isolate (69.11 %) whereas in non-viable cell assay was performed by PDS3 isolate (73.75 %) during incubation time 48 hours. In this study, G7 isolate performed the best probiotic characteristics with the highest viability in acid pH assay, bile salt 0.3% assay and percentage of aflatoxin B1 reduction in viable cell condition. Molecular identification using 16S rRNA sequence analysis showed that G7 isolate had homology with Lactobacillus plantarum (99.9%). It was concluded that Lactobacillus plantarum G7 was potential as probiotic with aflatoxin binding activities.

Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKα1 signaling

PubMed Central

Xue, Jianjing; Nelin, Leif D.

2017-01-01

Pulmonary artery smooth muscle cell (PASMC) proliferation is one of the hallmark features of hypoxia-induced pulmonary hypertension. With only supportive treatment options available for this life-threatening disease, treating and preventing the proliferation of PASMCs is a viable therapeutic option. A key promoter of hypoxia-induced increases in the number of viable human PASMCs is arginase II, with attenuation of viable cell numbers following pharmacologic inhibition or siRNA knockdown of the enzyme. Additionally, increased levels of arginase have been demonstrated in the pulmonary vasculature of patients with pulmonary hypertension. The signaling pathways responsible for the hypoxic induction of arginase II in PASMCs, however, remain unknown. Hypoxia is a recognized activator of AMPK, which is known to be expressed in human PASMCs (hPASMCs). Activation of AMPK by hypoxia has been shown to promote cell survival in PASMCs. In addition, pharmacologic agents targeting AMPK have been shown to attenuate chronic hypoxia-induced pulmonary hypertension in animal models. The present studies tested the hypothesis that hypoxia-induced arginase II expression in hPASMCs is mediated through AMPK signaling. We found that pharmacologic inhibitors of AMPK, as well as siRNA knockdown of AMPKα1, prevented hypoxia-induced arginase II. The hypoxia-induced increase in viable hPASMC numbers was also prevented following both pharmacologic inhibition and siRNA knockdown of AMPK. Furthermore, we demonstrate that overexpression of AMPK induced arginase II protein expression and viable cells numbers in hPASMCs. PMID:28213467

A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells.

PubMed

Hvichia, G E; Parveen, Z; Wagner, C; Janning, M; Quidde, J; Stein, A; Müller, V; Loges, S; Neves, R P L; Stoecklein, N H; Wikman, H; Riethdorf, S; Pantel, K; Gorges, T M

2016-06-15

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability-based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array-based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch(®) system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM-negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as "liquid biopsies." © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Development of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges.

PubMed

Soto-Muñoz, Lourdes; Teixidó, Neus; Usall, Josep; Viñas, Inmaculada; Crespo-Sempere, Ana; Torres, Rosario

2014-06-16

Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

Targeting tumor cell motility to prevent metastasis

PubMed Central

Palmer, Trenis D.; Ashby, William J.; Lewis, John D.; Zijlstra, Andries

2011-01-01

Mortality and morbidity in patients with solid tumors invariably results from the disruption of normal biological function caused by disseminating tumor cells. Tumor cell migration is under intense investigation as the underlying cause of cancer metastasis. The need for tumor cell motility in the progression of metastasis has been established experimentally and is supported empirically by basic and clinical research implicating a large collection of migration-related genes. However, there are few clinical interventions designed to specifically target the motility of tumor cells and adjuvant therapy to specifically prevent cancer cell dissemination is severely limited. In an attempt to define motility targets suitable for treating metastasis, we have parsed the molecular determinants of tumor cell motility into five underlying principles including cell autonomous ability, soluble communication, cell-cell adhesion, cell-matrix adhesion, and integrating these determinants of migration on molecular scaffolds. The current challenge is to implement meaningful and sustainable inhibition of metastasis by developing clinically viable disruption of molecular targets that control these fundamental capabilities. PMID:21664937

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

PubMed

Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

2018-01-11

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.

PubMed

Crowston, Jonathan G; Wang, Xiao Y; Khaw, Peng T; Zoellner, Hans; Healey, Paul R

2006-03-01

To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

2011-01-01

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Modeling to Optimize Terminal Stem Cell Differentiation

PubMed Central

Gallicano, G. Ian

2013-01-01

Embryonic stem cell (ESC), iPCs, and adult stem cells (ASCs) all are among the most promising potential treatments for heart failure, spinal cord injury, neurodegenerative diseases, and diabetes. However, considerable uncertainty in the production of ESC-derived terminally differentiated cell types has limited the efficiency of their development. To address this uncertainty, we and other investigators have begun to employ a comprehensive statistical model of ESC differentiation for determining the role of intracellular pathways (e.g., STAT3) in ESC differentiation and determination of germ layer fate. The approach discussed here applies the Baysian statistical model to cell/developmental biology combining traditional flow cytometry methodology and specific morphological observations with advanced statistical and probabilistic modeling and experimental design. The final result of this study is a unique tool and model that enhances the understanding of how and when specific cell fates are determined during differentiation. This model provides a guideline for increasing the production efficiency of therapeutically viable ESCs/iPSCs/ASC derived neurons or any other cell type and will eventually lead to advances in stem cell therapy. PMID:24278782

Burn Eschar Stimulates Fibroblast and Adipose Mesenchymal Stromal Cell Proliferation and Migration but Inhibits Endothelial Cell Sprouting

PubMed Central

Monsuur, Hanneke N.; van den Broek, Lenie J.; Jhingoerie, Renushka L.; Vloemans, Adrianus F. P. M.

2017-01-01

The majority of full-thickness burn wounds heal with hypertrophic scar formation. Burn eschar most probably influences early burn wound healing, since granulation tissue only forms after escharotomy. In order to investigate the effect of burn eschar on delayed granulation tissue formation, burn wound extract (BWE) was isolated from the interface between non-viable eschar and viable tissue. The influence of BWE on the activity of endothelial cells derived from dermis and adipose tissue, dermal fibroblasts and adipose tissue-derived mesenchymal stromal cells (ASC) was determined. It was found that BWE stimulated endothelial cell inflammatory cytokine (CXCL8, IL-6 and CCL2) secretion and migration. However, BWE had no effect on endothelial cell proliferation or angiogenic sprouting. Indeed, BWE inhibited basic Fibroblast Growth Factor (bFGF) induced endothelial cell proliferation and sprouting. In contrast, BWE stimulated fibroblast and ASC proliferation and migration. No difference was observed between cells isolated from dermis or adipose tissue. The inhibitory effect of BWE on bFGF-induced endothelial proliferation and sprouting would explain why excessive granulation tissue formation is prevented in full-thickness burn wounds as long as the eschar is still present. Identifying the eschar factors responsible for this might give indications for therapeutic targets aimed at reducing hypertrophic scar formation which is initiated by excessive granulation tissue formation once eschar is removed. PMID:28820426

Viability of human fibroblasts in coconut water as a storage medium.

PubMed

Moreira-Neto, J J S; Gondim, J O; Raddi, M S G; Pansani, C A

2009-09-01

To evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts. Cell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 microL of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%). Milk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05). Coconut water was worse than milk in maintaining human fibroblast cell viability.

Equilibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells.

PubMed

Dedov, Vadim N; Dedova, Irina V; Nicholson, Garth A

2004-04-01

Starvation arrests cultured mammalian cells in the G(1) restriction point of the cell cycle, whereas cancer cells generally lose the regulatory control of the cell cycle. Human lymphocytes, infected with Epstein-Barr virus (EBV), also lose their cell cycle control and produce immortal lymphoblastoid cell lines. We show that during starvation, EBV-lymphoblasts override the cell cycle arrest in the G(1) restriction point and continue cell division. Simultaneously, starvation activates apoptosis in an approximately half of the daughter cells in each cell generation. Continuos cell division and partial removal of cells by apoptosis results in stabilization of viable cell numbers, where a majority of viable cells are in the G(1) phase of the cell cycle. In contrast to starvation, anticancer drug etoposide activates apoptosis indiscriminately in all EBV-lymphoblasts and convertes all the viable cells into apoptotic. We conclude that the removal of surplus cells by apoptosis may represent a survival mechanism of transformed (i.e., cancer) cell population in nutrient restricted conditions, whereas nontransformed mammalian cells are arrested in the G(1) restriction point of the cell cycle.

Research on fluorescence detection method of Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Wang, Xiao-xiong

2017-07-01

The paper studied the viability determination of Microcystis aeruginosa by FDA and PI staining. The staining results were measured by fluorescence microscopy. The results indicated that viable and dead cells were stained as bright green and red fluorescent respectively by FDA and PI. Through PI-FDA dual color fluorescence staining, the color of green and red distinct obviously by fluorescence microscope. The staining rate has relation with the cell density. If the cell density of M. aeruginosa was 1.0×107-1.0×109 cell·mL-1, the staining rate would be 100.0% or 98.0% by PI and of FDA respectively.

The effect of riboflavin-UV-A treatment on corneal limbal epithelial cells--a study on human cadaver eyes.

PubMed

Vimalin, Jeyalatha; Gupta, Nidhi; Jambulingam, Malathi; Padmanabhan, Prema; Madhavan, Hajib N

2012-09-01

To determine the effect of riboflavin-UV-A treatment on the corneal limbal epithelial cells during a corneal collagen cross-linking (CXL) procedure. Thirty freshly enucleated human cadaveric eyeballs were subjected to a CXL procedure, mimicking the clinical protocol. During the UV-A exposure, one half of the limbus (sector A) was left unprotected, whereas the other half (sector B) was covered by a metal shield. Limbal biopsies from both sectors before and after the procedure were analyzed. Each strip of tissue was divided into 3 segments, for cell count of viable cells, for cultivation on human amniotic membrane (HAM), and for stem cell and differentiated corneal epithelial cell marker studies using reverse transcriptase-polymerase chain reaction. Compared with the cell count before CXL, there was a statistically significant drop in the mean number of viable cells after CXL in sector A but not in sector B. Biopsies from both sectors before CXL and from sector B after CXL showed good growth on HAM. Biopsies from sector A after CXL showed no growth on HAM. The putative stem cell marker ABCG2 was absent in all samples and p63 was absent in 3 of 10 samples taken from sector A after CXL. All markers were present in all samples from sector B after CXL. Riboflavin-UV-A treatment can result in damage to limbal epithelial cells, particularly the stem cells. Covering the limbal region with a metal shield effectively prevents this damage.

Challenge of N95 Filtering Facepiece Respirators with Viable H1N1 Influenza Aerosols

PubMed Central

Harnish, Delbert A.; Heimbuch, Brian K.; Husband, Michael; Lumley, April E.; Kinney, Kimberly; Shaffer, Ronald E.; Wander, Joseph D.

2015-01-01

OBJECTIVE Specification of appropriate personal protective equipment for respiratory protection against influenza is somewhat controversial. In a clinical environment, N95 filtering facepiece respirators (FFRs) are often recommended for respiratory protection against infectious aerosols. This study evaluates the ability of N95 FFRs to capture viable H1N1 influenza aerosols. METHODS Five N95 FFR models were challenged with aerosolized viable H1N1 influenza and inert polystyrene latex particles at continuous flow rates of 85 and 170 liters per minute. Virus was assayed using Madin-Darby canine kidney cells to determine the median tissue culture infective dose (TCID50). Aerosols were generated using a Collison nebulizer containing H1N1 influenza virus at 1 × 108 TCID50/mL. To determine filtration efficiency, viable sampling was performed upstream and downstream of the FFR. RESULTS N95 FFRs filtered 0.8-µm particles of both H1N1 influenza and inert origins with more than 95% efficiency. With the exception of 1 model, no statistically significant difference in filtration performance was observed between influenza and inert particles of similar size. Although statistically significant differences were observed for 2 models when comparing the 2 flow rates, the differences have no significance to protection. CONCLUSIONS This study empirically demonstrates that a National Institute for Occupational Safety and Health–approved N95 FFR captures viable H1N1 influenza aerosols as well as or better than its N95 rating, suggesting that a properly fitted FFR reduces inhalation exposure to airborne influenza virus. This study also provides evidence that filtration efficiency is based primarily on particle size rather than the nature of the particle’s origin. PMID:23571366

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a robust, versatile, and scalable inoculum train for the production of a DNA vaccine.

PubMed

Okonkowski, J; Kizer-Bentley, L; Listner, K; Robinson, D; Chartrain, M

2005-01-01

For many microbial fermentation processes, the inoculum train can have a substantial impact on process performance in terms of productivity, profitability, and process control. In general, it is understood that a well-characterized and flexible inoculum train is essential for future scale-up and implementation of the process in a pilot plant or manufacturing setting. A fermentation process utilizing E. coli DH5 for the production of plasmid DNA carrying the HIV gag gene for use as a vaccine is currently under development in our laboratory. As part of the development effort, we evaluated inoculum train schemes that incorporate one, two, or three stages. In addition, we investigated the effect of inoculum viable-cell concentrations, either thawed or actively growing, over a wide range (from 2.5 x 10(4) to 1.0 x 10(8) viable cells/mL or approximately 0.001% to 4% of final working volume). The various inoculum trains were evaluated in terms of final plasmid yield, process time, reproducibility, robustness, and feasibility at large scale. The results of these studies show that final plasmid yield remained in the desired range, despite the number of stages or inoculation viable-cell concentrations comprising the inoculum train. On the basis of these observations and because it established a large database, the first part of these investigations supports an exceptional flexibility in the design of scalable inoculum trains for this DNA vaccine process. This work also highlighted that a slightly higher level of process reproducibility, as measured by the time for the culture to reach mid-exponential growth, was observed when using actively growing versus frozen cells. It also demonstrated the existence of a viable-cell concentration threshold for the one-stage process, since we observed that inoculation of the production stage with very low amounts of viable cells from a frozen source could lead to increased process sensitivity to external factors such as variation in the quality of the raw materials used in the medium formulation. However, our analysis indicates that, despite this slight disadvantage, a one-stage inoculum train was a viable option in many situations, especially if the inoculation viable-cell concentration was kept above 4.8 x 10(6) viable cells/mL. Because it leads to a reduction in process steps and eliminates some capital investments (i.e., inoculum fermenter), when feasible a one-stage process configuration will positively impact process economics.

Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

NASA Technical Reports Server (NTRS)

Yu, F. P.; McFeters, G. A.

1994-01-01

Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

Detection of chromosomal abnormalities by fluorescent in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test.

PubMed

Zeyneloglu, H B; Baltaci, V; Ege, S; Haberal, A; Batioglu, S

2000-04-01

If randomly selected immotile spermatozoa are used for intracytoplasmic sperm injection (ICSI), pregnancy rates are significantly decreased. The hypo-osmotic swelling test (HOST) is the only method available to detect the viable, but immotile spermatozoa for ICSI. However, evidence is still lacking for the chromosomal abnormalities for the normal-looking, but immotile spermatozoa positive for HOST. Sperm samples from 20 infertile men with normal chromosomal constitution were obtained. After Percoll separation, morphologically normal but immotile spermatozoa were transported individually into HOST solution for 1 min using micropipettes. Cells that showed tail curling with swelling in HOST were then transferred back into human tubal fluid solution to allow reversal of swelling. These sperm cells were fixed and processed for the multi-colour fluorescence in-situ hybridization (FISH) for chromosomes X, Y and 18. The same FISH procedure was applied for the motile spermatozoa from the same cohort, which formed the control group. The average aneuploidy rates were 1.70 and 1.54% in 1000 HOST positive immotile and motile spermatozoa respectively detected by FISH for each patient. Our results indicate that morphologically normal, immotile but viable spermatozoa have an aneuploidy rate similar to that of normal motile spermatozoa.

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water

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

Crist, D.K.; Wyza, R.E.; Mills, K.K.

1984-05-01

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly nodulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10/sup 3/ to 10/sup 5/ cells ml/sup -1/, the bacteria multiplied until the viable cell count reached levels of between 10/sub 6/ and 10/sup 7/ cells ml/sup -1/. The viable cell count subsequently remained fairly constant. When themore » rhizobia were diluted to 10/sup 7/ cells ml/sup -1/, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10/sup 9/ cells ml/sup -1/, viability slowly declined to 10/sup 7/ cells ml/sup -1/ during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation. 25 references, 7 figures, 2 tables.« less

In vitro inhibition of Eimeria tenella sporozoite invasion into host cells by probiotics.

PubMed

Hessenberger, S; Schatzmayr, G; Teichmann, K

2016-10-15

The aim was to study the effects of probiotics isolated from the intestinal tract of livestock animals on Eimeria tenella invasion into Madin-Darby bovine kidney (MDBK) cells in vitro. E. tenella sporozoites were purified and labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester before seeding on cell cultures, and invasion was evaluated by fluorescence microscopy. Two protocols (A and B) were used. In protocol A, Enterococcus faecium # 589 or Lactobacillus salivarius subsp. salivarius # 505 were added together with sporozoites to MDBK cell cultures and invasion was evaluated after incubation for approximately 20h. Viable, dead, or spent culture supernatants of probiotics were tested. In protocol B, viable probiotics were incubated with MDBK cells for one hour before sporozoites were added and invasion was evaluated after two more hours of incubation. Parasite invasion of viable, dead, or spent culture supernatant of E. faecium # 589 was assessed. Using protocol A, it was shown that parasite invasion was inhibited by viable (80%) or dead (75%) E. faecium # 589. While inhibition by viable L. salivarius subsp. salivarius # 505 was not valid at the highest concentration and not significant at the other test concentrations, dead cells inhibited parasite invasion up to 45%. Spent culture supernatants of both probiotics had no influence on parasite invasion. Using protocol B, it was shown that viable Bifidobacterium animalis subsp. animalis # 503, E. faecium # 497, E. faecium # 589, L. reuteri # 514, L. salivarius subsp. salivarius # 505, and Bacillus subtilis # 588 inhibited parasite invasion into MDBK cells up to 80%. Anticoccidial activity was strain-specific for E. faecium strains, and the strongest effect was shown by E. faecium # 589. Anticoccidial effects of some of the tested probiotics have already been shown in vivo, which makes them candidates to prevent coccidiosis. These findings have now been confirmed in vitro. The used parasite invasion assay is a fast and inexpensive tool to screen probiotics for prevention of coccidiosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular integration of synthetic nanostructures with viable cells for controlled biochemical manipulation

NASA Astrophysics Data System (ADS)

McKnight, Timothy E.; Melechko, Anatoli V.; Griffin, Guy D.; Guillorn, Michael A.; Merkulov, Vladimir I.; Serna, Francisco; Hensley, Dale K.; Doktycz, Mitchel J.; Lowndes, Douglas H.; Simpson, Michael L.

2003-05-01

We demonstrate the integration of vertically aligned carbon nanofibre (VACNF) elements with the intracellular domains of viable cells for controlled biochemical manipulation. Deterministically synthesized VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and were subsequently inserted into cells. Post insertion viability of the cells was demonstrated by continued proliferation of the interfaced cells and long-term (> 22 day) expression of the introduced plasmid. Adsorbed plasmids were typically desorbed in the intracellular domain and segregated to progeny cells. Covalently bound plasmids remained tethered to nanofibres and were expressed in interfaced cells but were not partitioned into progeny, and gene expression ceased when the nanofibre was no longer retained. This provides a method for achieving a genetic modification that is non-inheritable and whose extent in time can be directly and precisely controlled. These results demonstrate the potential of VACNF arrays as an intracellular interface for monitoring and controlling subcellular and molecular phenomena within viable cells for applications including biosensors, in vivo diagnostics, and in vivo logic devices.

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

Martin, Olga A.; Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC

Purpose: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. Methods and Materials: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. Results: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and inmore » 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. Conclusions: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies.« less

Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries

PubMed Central

Hallow, Daniel M.; Mahajan, Anuj D.; Prausnitz, Mark R.

2007-01-01

This study tested the hypothesis that ultrasound can target intracellular uptake of drugs into vascular endothelial cells (ECs) at low to intermediate energy and into smooth muscle cells (SMCs) at high energy. Ultrasound-enhanced delivery has been shown to enhance and target intracellular drug and gene delivery in the vasculature to treat cardiovascular disease, but quantitative studies of the delivery process are lacking. Viable ex vivo porcine carotid arteries were placed in a solution containing a model drug, TO-PRO®-1, and Optison® microbubbles. Arteries were exposed to ultrasound at 1.1 MHz and acoustic energies of 5.0, 66, or 630 J/cm2. Using confocal microscopy and fluorescent labeling of cells, the artery endothelium and media were imaged to determine the localization and to quantify intracellular uptake and cell death. At low to intermediate ultrasound energy, ultrasound was shown to target intracellular delivery into viable cells that represented 9 – 24% of exposed ECs. These conditions also typically caused 7 – 25% EC death. At high energy, intracellular delivery was targeted to SMCs, which was associated with denuding or death of proximal ECs. This work represents the first known in-depth study to evaluate intracellular uptake into cells in tissue. We conclude that significant intracellular uptake of molecules can be targeted into ECs and SMCs by ultrasound-enhanced delivery suggesting possible applications for treatment of cardivascular diseases and dysfunctions. PMID:17291619

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7

NASA Technical Reports Server (NTRS)

Lisle, J. T.; Pyle, B. H.; McFeters, G. A.

1999-01-01

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.

Granular Formation during Apoptosis in Blastocystis sp. Exposed to Metronidazole (MTZ)

PubMed Central

Suresh, Kumar; Tan, Tian Chye

2016-01-01

The role and function of the granular life cycle stage in Blastocystis sp, remains uncertain despite suggestions being made that the granules are metabolic, reproductive and lipid in nature. This present study aims to understand granular formation by triggering apoptosis in Blastocystis sp. by treating them with metronidazole (MTZ). Blastocystis sp.cultures of 4 sub-types namely 1, 2, 3 and 5 when treated with 0.01 and 0.0001 mg/ml of metronidazole (MTZ) respectively showed many of the parasites to be both viable and apoptotic (VA). Treated subtype 3 isolates exhibited the highest number of granular forms i.e. 88% (p<0.001) (0.0001 mg/ml) and 69% (p<0.01) (0.01 mg/ml) respectively at the 72 h in in vitro culture compared to other subtypes. These VA forms showed distinct granules using acridine orange (AO) and 4’,6-diamino-2-phenylindole (DAPI) staining with a mean per cell ranging from 5 in ST 5 to as high as 16 in ST 3. These forms showed intact mitochondria in both viable apoptotic (VA) and viable non-apoptotic (VNA) cells with a pattern of accumulation of lipid droplets corresponding to viable cells. Granular VA forms looked ultra-structurally different with prominent presence of mitochondria-like organelle (MLO) and a changed mitochondrial trans-membrane potential with thicker membrane and a highly convoluted inner membrane than the less dense non-viable apoptotic (NVA) cells. This suggests that granular formation during apoptosis is a self-regulatory mechanism to produce higher number of viable cells in response to treatment. This study directs the need to search novel chemotherapeutic approaches by incorporating these findings when developing drugs against the emerging Blastocystis sp. infections. PMID:27471855

Separable Bilayer Microfiltration Device for Label-Free Enrichment of Viable Circulating Tumor Cells.

PubMed

Hao, Sijie; Nisic, Merisa; He, Hongzhang; Tai, Yu-Chong; Zheng, Si-Yang

2017-01-01

Analysis of rare circulating tumor cells enriched from metastatic cancer patients yields critical information on disease progression, therapy response, and the mechanism of cancer metastasis. Here we describe in detail a label-free enrichment process of circulating tumor cells based on its unique physical properties (size and deformability). Viable circulating tumor cells can be successfully enriched and analyzed, or easily released for further characterization due to the novel separable two-layer design.

Determination of Ancylostoma caninum ova viability using metabolic profiling.

PubMed

Gyawali, P; Beale, D J; Ahmed, W; Karpe, A V; Magalhaes, R J Soares; Morrison, P D; Palombo, E A

2016-09-01

Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.

Thermoresponsive release of viable microfiltrated Circulating Tumor Cells (CTCs) for precision medicine applications

PubMed Central

Ao, Zheng; Parasido, Erika; Rawal, Siddarth; Williams, Anthony; Schlegel, Richard; Liu, Stephen; Albanese, Chris; Cote, Richard J.; Agarwal, Ashutosh; Datar, Ram H.

2015-01-01

Stimulus responsive release of Circulating Tumor Cells (CTCs), with high recovery rates from their capture platform, is highly desirable for off-chip analyses. Here, we present a temperature responsive polymer coating method to achieve both release as well as culture of viable CTCs captured from patient blood samples. PMID:26426331

Effect of high pressure treatment on liquid whole egg

NASA Astrophysics Data System (ADS)

Németh, Csaba; Dalmadi, István; Mráz, Balázs; Friedrich, László; Zeke, Ildikó; Juhász, Réka; Suhajda, Ágnes; Balla, Csaba

2012-06-01

In our tests, we artificially infected liquid whole egg samples with Salmonella enteritidis, Listeria monocytogenes, and Staphylococcus aureus bacteria, and then treated the samples in "Food Lab900" high hydrostatic pressure (HHP) instrument for 3-17 min at 200-400 MPa. Subsequently, the change of the viable cell count of the specific bacteria has been tested. In addition to the samples infected with various bacteria, non-infected samples were also treated in each test and the change in viable cell count, colour and viscosity of the samples upon the effect of the treatment. In summary, it can be concluded that in each test of our investigations, the viable cell count of S. enteritidis critical for egg products is reduced significantly, while the reduction of the total viable cell count was around two magnitudes. Additionally, based on our results, microbial destruction, reduction of enthalpy (denaturation of egg white) caused by the treatment at HPP, and colour change are primarily affected by the pressure level, while the changes in rheological properties are also significantly affected by the duration of high pressure treatment (p<0.05).

Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing

PubMed Central

Suo, Biao; Yang, Hua; Wang, Yuexia; Lv, Haipeng; Li, Zhen; Xu, Chao; Ai, Zhilu

2018-01-01

When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food. PMID:29774015

Ecabet sodium alleviates neomycin-induced hair cell damage.

PubMed

Rah, Yoon Chan; Choi, June; Yoo, Myung Hoon; Yum, Gunhwee; Park, Saemi; Oh, Kyoung Ho; Lee, Seung Hoon; Kwon, Soon Young; Cho, Seung Hyun; Kim, Suhyun; Park, Hae-Chul

2015-12-01

Ecabet sodium (ES) is currently applied to some clinical gastrointestinal disease primarily by the inhibition of the ROS production. In this study, the protective role of ES was evaluated against the neomycin-induced hair cell loss using zebrafish experimental animal model. Zebrafish larvae (5-7 dpf), were treated with each of the following concentrations of ES: 5, 10, 20, 40, and 80 μg/mL for 1 h, followed by 125 μM neomycin for 1h. The positive control group was established by 125 μM neomycin-only treatment (1h) and the negative control group with no additional chemicals was also established. Hair cells inside four neuromasts ( SO1, SO2, O1, OC1) were assessed using fluorescence microscopy (n = 10). Hair cell survival was calculated as the mean number of viable hair cells for each group. Apoptosis and mitochondrial damage were investigated using special staining (TUNEL and DASPEI assay, respectively), and compared among groups. Ultrastructural changes were evaluated using scanning electron microscopy. Pre-treatment group with ES increased the mean number of viable hair cells as a dose-dependent manner achieving almost same number of viable hair cells with 40 μM/ml ES treatment (12.98 ± 2.59 cells) comparing to that of the negative control group (14.15 ± 1.39 cells, p = 0.72) and significantly more number of viable hair cells than that of the positive control group (7.45 ± 0.91 cells, p < 0.01). The production of reactive oxygen species significantly increased by 183% with 125 μM neomycin treatment than the negative control group and significantly decreased down to 105% with the pre-treatment with 40 μM/ml ES (n = 40, p = 0.04). A significantly less number of TUNEL-positive cells (reflecting apoptosis, p < 0.01) and a significantly increased DASPEI reactivity (reflecting viable mitochondria, p < 0.01) were observed in 40 μM/ml ES pre-treatment group. Our data suggest that ES could protect against neomycin-induced hair cell loss possibly by reducing apoptosis, mitochondrial damages, and the ROS generation. Copyright © 2015 Elsevier Inc. All rights reserved.

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

PubMed Central

He, Min; Storr-Paulsen, Thomas; Wang, Annie L.; Ghezzi, Chiara E.; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P.; Islam, Rakibul; Griffith, May; Islam, M. Mirazul; Hodges, Robin R.; Wnek, Gary E.; Kaplan, David L.; Dartt, Darlene A.

2016-01-01

Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study. PMID:27832279

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion.

PubMed

He, Min; Storr-Paulsen, Thomas; Wang, Annie L; Ghezzi, Chiara E; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P; Islam, Rakibul; Griffith, May; Islam, M Mirazul; Hodges, Robin R; Wnek, Gary E; Kaplan, David L; Dartt, Darlene A

2016-11-01

We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

NASA Astrophysics Data System (ADS)

Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

2011-11-01

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

Effect of sodium hypochlorite on human pulp cells: an in vitro study

PubMed Central

Essner, Mark D.; Javed, Amjad; Eleazer, Paul D.

2014-01-01

Background The purpose of this study was to determine the effect of sodium hypochlorite (NaOCl) on human pulp cells to provide an aid in determining its optimum concentration in maintaining the viability of remaining pulp cells in the revascularization of immature permanent teeth with apical periodontitis. Study design Human pulp tissue cells taken from extracted third molars were plated, incubated, and subjected to various concentrations of NaOCl (0.33%, 0.16%, 0.08%, and 0.04%) for 5-, 10-, and 15-minute time intervals to simulate possible contact times in vivo. The Cell Titer–Glo Luminescent Cell Viability Assay was used to determine the number of viable cells present in culture following treatment. Results The results showed an increase in cell viability with the lowering of NaOCl concentration. The use of 0.04% NaOCl was similar to the control, indicating nearly complete preservation of cell viability at all time intervals tested. As sodium hypochlorite concentration increased from 0.04% to 0.33%, cell viability decreased correspondingly. Conclusions The results indicate that the lowest concentration of NaOCl tested did not affect the viability of cells. This may prove beneficial in developing a new treatment protocol to help preserve existing vital pulp cells in revascularization cases. PMID:21821446

Effects of cell condition, pH, and temperature on lead, zinc, and copper sorption to Acidithiobacillus caldus strain BC13

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

John E. Aston; William A. Apel; Brady D. Lee

2010-12-01

This study describes the effects of cell condition, pH, and temperature on lead, zinc, and copper sorption to Acidithiobacillus caldus strain BC13 with a Langmuir model. Copper exhibited the highest loading capacity, 4.76 ± 0.28 mmol g-1, to viable cells at pH 5.5. The highest kL (binding-site affinity) observed was 61.2 ± 3.0 L mmol-1 to dehydrated cells at pH 4.0. The pHs that maximized loading capacities and binding-site affinities were generally between 4.0 and 5.5, where the sum of free-proton and complexed-metal concentrations was near a minimum. Of additional importance, lead, zinc, and copper sorbed to viable cells atmore » pH values as low as 1.5. Previous studies with other acidithiobacilli did not measure viable-cell sorption below pH 4.0. In separate experiments, desorption studies showed that far less copper was recovered from viable cells than any other metal or cell condition, suggesting that uptake may play an important role in copper sorption by At. caldus strain BC13. To reflect an applied system, the sorption of metal mixtures was also studied. In these experiments, lead, zinc, and copper sorption from a tertiary mixture were 40.2 ± 4.3%, 28.7 ± 3.8%, and 91.3 ± 3.0%, respectively, of that sorbed in single-metal systems.« less

Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

PubMed

Mattos, Diogo A; Silva, Marlon V; Gaspar, Luciane P; Castilho, Leda R

2015-08-20

In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV. Copyright © 2015 Elsevier Ltd. All rights reserved.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

PubMed

Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

2011-11-10

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

A Novel Approach for Using Dielectric Spectroscopy to Predict Viable Cell Volume (VCV) in Early Process Development

PubMed Central

Downey, Brandon J; Graham, Lisa J; Breit, Jeffrey F; Glutting, Nathaniel K

2014-01-01

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam-sterilizable dielectric-spectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell-culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probe-permittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency-scanning data from the dielectric-spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single-frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed-batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency-scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape-characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:479–487, 2014 PMID:24851255

Impedance microflow cytometry for viability studies of microorganisms

NASA Astrophysics Data System (ADS)

Di Berardino, Marco; Hebeisen, Monika; Hessler, Thomas; Ziswiler, Adrian; Largiadèr, Stephanie; Schade, Grit

2011-02-01

Impedance-based Coulter counters and its derivatives are widely used cell analysis tools in many laboratories and use normally DC or low frequency AC to perform these electrical analyses. The emergence of micro-fabrication technologies in the last decade, however, provides a new means of measuring electrical properties of cells. Microfluidic approaches combined with impedance spectroscopy measurements in the radio frequency (RF) range increase sensitivity and information content and thus push single cell analyses beyond simple cell counting and sizing applications towards multiparametric cell characterization. Promising results have been shown already in the fields of cell differentiation and blood analysis. Here we emphasize the potential of this technology by presenting new data obtained from viability studies on microorganisms. Impedance measurements of several yeast and bacteria strains performed at frequencies around 10 MHz enable an easy discrimination between dead and viable cells. Moreover, cytotoxic effects of antibiotics and other reagents, as well as cell starvation can also be monitored easily. Control analyses performed with conventional flow cytometers using various fluorescent dyes (propidium iodide, oxonol) indicate a good correlation and further highlight the capability of this device. The label-free approach makes on the one hand the use of usually expensive fluorochromes obsolete, on the other hand practically eliminates laborious sample preparation procedures. Until now, online cell monitoring was limited to the determination of viable biomass, which provides rather poor information of a cell culture. Impedance microflow cytometry, besides other aspects, proposes a simple solution to these limitations and might become an important tool for bioprocess monitoring applications in the biotech industry.

Kinetics of killing Listeria monocytogenes by macrophages: rapid killing accompanying phagocytosis

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

Davies, W.A.

1983-08-01

The kinetics of bactericidal activity of activated macrophages can be precisely described by a mathematical model in which phagocytosis, killing, digestion, and release of degraded bacterial material are considered to occur continuously. To gain a better understanding of these events, I have determined the period of time between first contact of bacteria with macrophages and the onset of killing. Activated rat peritoneal macrophages were incubated for various times up to 15 min with Listeria monocytogenes previously labeled with /sup 3/H-thymidine and the unassociated bacteria removed by two centrifugations through a density interface. Both cell-associated radioactivity and cell-associated viable bacteria, determinedmore » as colony forming units after sonication of the cell pellet, increased with time of incubation. However, the specific viability of these bacteria, expressed as the ratio of number of viable bacteria per unit radioactivity declined with time, as an approximate inverse exponential, after a lag period of 2.9 +/- 0.8 min. Evidence is given that other possible causes for this decline in specific viability, other than death of the bacteria, such as preferential ingestion of dead Listeria, clumping of bacteria, variations in autolytic activity, or release of Listericidins are unlikely. I conclude therefore that activated macrophages kill Listeria approximately 3 min after the cell and the bacterium first make contact.« less

The immunomodulatory properties of viable Lactobacillus salivarius ssp. salivarius CECT5713 are not restricted to the large intestine.

PubMed

Arribas, Belén; Garrido-Mesa, Natividad; Perán, Laura; Camuesco, Desirée; Comalada, Mònica; Bailón, Elvira; Olivares, Mónica; Xaus, Jordi; Kruidenier, Laurens; Sanderson, Ian R; Zarzuelo, Antonio; Rodríguez-Cabezas, Maria Elena; Gálvez, Julio

2012-04-01

The aim of this study was to better characterise the biological effects of Lactobacillus salivarius ssp. salivarius CECT5713, a probiotic with immunomodulatory properties. Live or dead probiotic was assayed in the TNBS model of rat colitis to determine whether viability was a requisite to exert the beneficial effects. In vitro studies were also performed in Caco-2 cells to evaluate its effects on epithelial cell recovery and IL-8 production. Finally, the probiotic was assayed in the LPS model of septic shock in mice to establish its effects when there is an altered systemic immune response. The viability of the probiotic was required for its anti-inflammatory activity. The probiotic inhibited IL-8 production in stimulated Caco-2 cells and facilitated the recovery of damaged intestinal epithelium. In LPS-treated mice, the probiotic inhibited the production of TNFα in plasma and lungs and increased the hepatic glutathione content. These effects were associated with an improvement in the altered production of the T-cell cytokines in splenocytes, by reducing IL-2 and IL-5 and by increasing IL-10. Finally, it reduced the increased plasma IgG production in LPS-treated mice. The anti-inflammatory effects of viable L. salivarius ssp. salivarius CECT5713 are not restricted to the gastrointestinal tract.

Determination of viable legionellae in engineered water systems: Do we find what we are looking for?

PubMed Central

Kirschner, Alexander K.T.

2016-01-01

In developed countries, legionellae are one of the most important water-based bacterial pathogens caused by management failure of engineered water systems. For routine surveillance of legionellae in engineered water systems and outbreak investigations, cultivation-based standard techniques are currently applied. However, in many cases culture-negative results are obtained despite the presence of viable legionellae, and clinical cases of legionellosis cannot be traced back to their respective contaminated water source. Among the various explanations for these discrepancies, the presence of viable but non-culturable (VBNC) Legionella cells has received increased attention in recent discussions and scientific literature. Alternative culture-independent methods to detect and quantify legionellae have been proposed in order to complement or even substitute the culture method in the future. Such methods should detect VBNC Legionella cells and provide a more comprehensive picture of the presence of legionellae in engineered water systems. However, it is still unclear whether and to what extent these VBNC legionellae are hazardous to human health. Current risk assessment models to predict the risk of legionellosis from Legionella concentrations in the investigated water systems contain many uncertainties and are mainly based on culture-based enumeration. If VBNC legionellae should be considered in future standard analysis, quantitative risk assessment models including VBNC legionellae must be proven to result in better estimates of human health risk than models based on cultivation alone. This review critically evaluates current methods to determine legionellae in the VBNC state, their potential to complement the standard culture-based method in the near future, and summarizes current knowledge on the threat that VBNC legionellae may pose to human health. PMID:26928563

Viable bacteria associated with red blood cells and plasma in freshly drawn blood donations.

PubMed

Damgaard, Christian; Magnussen, Karin; Enevold, Christian; Nilsson, Martin; Tolker-Nielsen, Tim; Holmstrup, Palle; Nielsen, Claus Henrik

2015-01-01

Infection remains a leading cause of post-transfusion mortality and morbidity. Bacterial contamination is, however, detected in less than 0.1% of blood units tested. The aim of the study was to identify viable bacteria in standard blood-pack units, with particular focus on bacteria from the oral cavity, and to determine the distribution of bacteria revealed in plasma and in the red blood cell (RBC)-fraction. Cross-sectional study. Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA) or blue lactose plates. For identification colony PCR was performed using primers targeting 16S rDNA. Blood donors attending Capital Region Blood Bank, Copenhagen University Hospital, Rigshospitalet, Hvidovre, Denmark, October 29th to December 10th 2013. 60 donors (≥50 years old), self-reported medically healthy. Bacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations. Growth was evident in 21 (35%) of 60 RBC-fractions and in 32 (53%) of 60 plasma-fractions versus 8 of 60 negative controls (p = 0.005 and p = 2.6x10-6, respectively). Propionibacterium acnes was found in 23% of the donations, and Staphylococcus epidermidis in 38%. The majority of bacteria identified in the present study were either facultative anaerobic (59.5%) or anaerobic (27.8%) species, which are not likely to be detected during current routine screening. Viable bacteria are present in blood from donors self-reported as medically healthy, indicating that conventional test systems employed by blood banks insufficiently detect bacteria in plasma. Further investigation is needed to determine whether routine testing for anaerobic bacteria and testing of RBC-fractions for adherent bacteria should be recommended.

Determination of viable legionellae in engineered water systems: Do we find what we are looking for?

PubMed

Kirschner, Alexander K T

2016-04-15

In developed countries, legionellae are one of the most important water-based bacterial pathogens caused by management failure of engineered water systems. For routine surveillance of legionellae in engineered water systems and outbreak investigations, cultivation-based standard techniques are currently applied. However, in many cases culture-negative results are obtained despite the presence of viable legionellae, and clinical cases of legionellosis cannot be traced back to their respective contaminated water source. Among the various explanations for these discrepancies, the presence of viable but non-culturable (VBNC) Legionella cells has received increased attention in recent discussions and scientific literature. Alternative culture-independent methods to detect and quantify legionellae have been proposed in order to complement or even substitute the culture method in the future. Such methods should detect VBNC Legionella cells and provide a more comprehensive picture of the presence of legionellae in engineered water systems. However, it is still unclear whether and to what extent these VBNC legionellae are hazardous to human health. Current risk assessment models to predict the risk of legionellosis from Legionella concentrations in the investigated water systems contain many uncertainties and are mainly based on culture-based enumeration. If VBNC legionellae should be considered in future standard analysis, quantitative risk assessment models including VBNC legionellae must be proven to result in better estimates of human health risk than models based on cultivation alone. This review critically evaluates current methods to determine legionellae in the VBNC state, their potential to complement the standard culture-based method in the near future, and summarizes current knowledge on the threat that VBNC legionellae may pose to human health. Copyright © 2016 The Author. Published by Elsevier Ltd.. All rights reserved.

Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses.

PubMed

Dutta, Debarun; Cole, Nerida; Kumar, Naresh; Willcox, Mark D P

2013-01-07

To develop a stable antimicrobial contact lens, which is effective against the International Organization for Standardization (ISO) panel microorganisms, Acanthamoeba castellanii and drug resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. Melimine was covalently incorporated into etafilcon A lenses. The amount of peptide present on the lens surface was quantified using amino acid analysis. After coating, the heat stability (121°C), lens surface hydrophobicity (by captive bubble), and in vitro cytotoxicity to mouse L929 cells of the lenses were investigated. Antimicrobial activity against the micro-organisms was evaluated by viable plate count and fluorescence microscopy, measuring the proportion of cell death compared with control lenses with no melimine. The most effective concentration was determined to be 152 ± 44 μg lens(-1) melimine on the lens surface. After coating, lenses were relatively hydrophilic and were nontoxic to mammalian cells. The activity remained high after autoclaving (e.g., 3.1, 3.9, 1.2, and 1.0 log inhibition against P. aeruginosa, S. aureus, A. castellanii, and Fusarium solani, respectively). Fluorescence microscopy confirmed significantly reduced (P < 0.001) adhesion of viable bacteria to melimine contact lenses. Viable count confirmed that lenses were active against all the bacteria and fungi from the ISO panel, Acanthamoeba and gave at least 2 log inhibition against all the multidrug resistant S. aureus and P. aeruginosa strains. Melimine may offer excellent potential for development as a broad spectrum antimicrobial coating for contact lenses, showing activity against all the bacterial and fungal ISO panel microorganisms, Acanthamoeba, and antibiotic resistant strains of P. aeruginosa and S. aureus.

Classification of select category A and B bacteria by Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Samuels, Alan C.; Snyder, A. Peter; St. Amant, Diane; Emge, Darren K.; Minter, Jennifer; Campbell, Mark; Tripathi, Ashish

2008-04-01

Relatively few reports have investigated the determination and classification of pathogens such as the National Institute of Allergy and Infectious Diseases (NIAID) Category A Bacillus anthracis spores and cells (BA), Yersinia species, Francisella tularensis (FT), and Category B Brucella species from FTIR spectra. We investigated the classification ability of the Fourier transform infrared (FTIR) spectra of viable pathogenic and non-pathogenic NIAID Category A and B bacteria. The impact of different growth media, growth time and temperature, rolling circle filter of the data, and wavelength range were investigated for their microorganism differentiation. Various 2-D PC plots provided differential degrees of separation with respect to the four viable, bacterial genera including the BA sub-categories of pathogenic spores, vegetative cells, and nonpathogenic vegetative cells. FT spectra were separated from that of the three other genera. The BA pathogenic spore strains 1029, LA1, and Ames were clearly differentiated from the rest of the dataset. Yersinia species were distinctly separated from the remaining dataset and could also be classified by growth media. This work provided evidence that FTIR spectroscopy can separate the four major pathogenic bacterial genera of NIAID Category A and B biological threat agents.

Epidermal effects of tretinoin and isotretinoin: influence of isomerism.

PubMed

Tadini, K A; Gaspar, L R; Maia Campos, P M B G

2006-05-01

The efficacy of tretinoin is well established in the treatment of acne and photoaged skin, however as a typical side effect of tretinoin treatment most patients develop a low-grade irritant dermatitis. Since isotretinoin topical treatment usually shows much lower incidence and intensity of adverse effects than tretinoin topical treatment, histological studies are needed to scientifically evaluate the effects of isotretinoin application on epidermis and also to assess if it can be used in anti-aging products as an alternative to tretinoin. Thus, the aim of this study was to compare the effects of topical use of tretinoin or isotretinoin on hairless mice epidermis, using appropriate histopathological and histometric techniques, in order to evaluate the influence of isomerism on skin effects. For this, gel cream formulations containing or not 0.05% tretinoin or 0.05% isotretinoin were applied in the dorsum of hairless mice, once a day for seven days. Histopathological evaluation, viable epidermal and horny layer thicknesses as well as the number of epidermal cell layers were determined. Our results showed that tretinoin and isotretinoin were effective in the enhancement of viable epidermis thickness and number of epidermal cell layers, suggesting that they could be used for stimulation of cellular renewal. However isomerism influenced skin effects since isotretinoin had more pronounced effects than tretinoin in viable epidermis. In addition only isotretinoin treatment enhanced horny layer thickness when compared to the gel cream treatment.

Formation and resuscitation of viable but nonculturable Salmonella typhi.

PubMed

Zeng, Bin; Zhao, Guozhong; Cao, Xiaohong; Yang, Zhen; Wang, Chunling; Hou, Lihua

2013-01-01

Salmonella typhi is a pathogen that causes the human disease of typhoid fever. The aim of this study was to investigate the viable but nonculturable (VBNC) state of S. typhi. Some samples were stimulated at 4°C or -20°C, while others were induced by different concentrations of CuSO4. Total cell counts remained constant throughout several days by acridine orange direct counting; however, plate counts declined to undetectable levels within 48 hours by plate counting at -20°C. The direct viable counts remained fairly constant at this level by direct viable counting. Carbon and nitrogen materials slowly decreased which indicated that a large population of cells existed in the VBNC state and entered the VBNC state in response to exposure to 0.01 or 0.015 mmol/L CuSO4 for more than 14 or 12 days, respectively. Adding 3% Tween 20 or 1% catalase enabled cells to become culturable again, with resuscitation times of 48 h and 24 h, respectively. The atomic force microscope results showed that cells gradually changed in shape from short rods to coccoids, and decreased in size when they entered the VBNC state. Further animal experiments suggested that resuscitated cells might regain pathogenicity.

The water kefir grain inoculum determines the characteristics of the resulting water kefir fermentation process.

PubMed

Laureys, D; De Vuyst, L

2017-03-01

To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process. Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these micro-organisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii and Saccharomyces cerevisiae were always present and may be the key micro-organisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of micro-organisms, fast fermentation and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria. The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key micro-organisms was always present during fermentation. This study allows a rational selection of a water kefir grain inoculum. © 2016 The Society for Applied Microbiology.

Survival of genetically modified and self-cloned strains of commercial baker's yeast in simulated natural environments: environmental risk assessment.

PubMed

Ando, Akira; Suzuki, Chise; Shima, Jun

2005-11-01

Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (ATH1) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study, ATH1 disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains ATH1 loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of ATH1 by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.

Transvaginal ultrasound in threatened abortions with empty gestational sacs.

PubMed

Tongsong, T; Wanapirak, C; Srisomboon, J; Sirichotiyakul, S; Polsrisuthikul, T; Pongsatha, S

1994-09-01

To determine whether transvaginal ultrasound criteria alone can distinguish viable from non-viable gestational sacs at a single examination. A prospective descriptive study was undertaken and analysis performed on 211 pregnancies complicated by threatened abortion and empty gestation sacs diagnosed by transvaginal ultrasound. The main outcome measure was the final diagnosis of viable or non-viable gestation on subsequent transvaginal sonography. The study shows that a single transvaginal ultrasound examination is useful in differentiating viable from non-viable gestation sacs. The mean sac diameter (MSD) was found to be the most useful criterion for determining non-viability. An MSD of > or = 17 mm that lacked an embryo and an MSD of > or = 13 mm without visible yolk sac were reliable predictors of non-viable gestation sacs at a single examination with 100% specificity and 100% positive predictive value. An MSD > or = 13 mm without visible yolk sac was the most sensitive criterion. Using MSD criteria, 73% of non-viable gestations could be reliably identified without any false-positive diagnoses. Deformed shape, low position and thin decidual reaction are strong indicators of non-viable gestations but are not 100% accurate. There is still a significant proportion of empty sacs, where no accurate distinction between viable and non-viable can be made according to one criterion at a single examination and in these cases serial examinations should be carried out before any active management is advocated. In most cases, transvaginal sonographic criteria alone can distinguish viable from non-viable empty gestational sacs at a single examination.

Applicator for in-vitro ultrasound-activated targeted drug delivery

NASA Astrophysics Data System (ADS)

Gerold, B.; Gourevich, D.; Volovick, A.; Xu, D.; Arditti, F.; Prentice, P.; Cochran, S.; Gnaim, J.; Medan, Y.; Wang, L.; Melzer, A.

2012-10-01

Reducing toxicity and improving uptake of cancer drugs in tumors are important goals of targeted drug delivery (TDD). Ultrasonic drug release from various encapsulants has been a focus of many research groups. However, a single standard ultrasonic device, viable for use by biologists, is not currently present in the market. The device reported here is designed to allow investigation of the impact of ultrasound on cellular uptake and cell viability in-vitro. In it, single-element transducers with different operating frequencies are mounted below a standard 96-well plate. The plate is moved above the transducers, such that each line of wells can be sonicated at a different frequency. To assess the device, 96-well plates were seeded with cells and sonicated using different ultrasonic parameters, with and without doxorubicin. Cell viability was measured by colorimetric MTT assay and the uptake of doxorubicin by cells was also determined. The device proved to be highly viable in preliminary tests; it demonstrated that change in ultrasonic parameters produces different effect on cells. For example, increase in uptake of doxorubicin was demonstrated following ultrasound application. The growing interest in ultrasound-activated TDD emphasizes the need for standardization of the ultrasound device and the one reported here may offer some indications of how that may be achieved. It is planned to further improve the prototype by increasing the number of ultrasonic frequencies and degrees of freedom for each transducer.

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

PubMed Central

Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M. Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

2016-01-01

Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

Yield of Routine Image-Guided Biopsy of Renal Mass Thermal Ablation Zones: 11-Year Experience.

PubMed

Wasnik, Ashish P; Higgins, Ellen J; Fox, Giovanna A; Caoili, Elaine M; Davenport, Matthew S

2018-06-19

To determine the yield of routine image-guided core biopsy of renal cell carcinoma (RCC) thermal ablation zones. Institutional review board approval was obtained for this Health Insurance Portability and Accountability Act-compliant quality improvement effort. Routine core biopsy of RCC ablation zones was performed 2 months postablation from July 2003 to December 2014. Routine nicotinamide adenine dinucleotide staining was performed by specialized genitourinary pathologists to assess cell viability. The original purpose of performing routine postablation biopsy was to verify, in addition to imaging, whether the mass was completely treated. Imaging was stratified as negative, indeterminate, or positive for viable malignancy. Histology was stratified as negative, indeterminate, positive, or nondiagnostic for viable malignancy. Histology results were compared to prebiopsy imaging findings. Routine ablation zone biopsy was performed after 50% (146/292) of index ablations (24 cryoablations, 122 radiofrequency ablations), and postablation imaging was performed more often with multiphasic computed tomography than magnetic resonance imaging (100 vs 46, p < 0.0001). When imaging was negative (n = 117), biopsy added no additional information (92% [n = 108] negative, 0.9% [n = 1] indeterminate, 7% [n = 8] nondiagnostic). When imaging was indeterminate (n = 19), 11% (n = 2) of biopsies had viable RCC and 89% (n = 17) were negative. When imaging was positive, biopsy detected viable neoplasm in only 10% (1/10) of cases; 80% (8/10) were negative and 10% (1/10) were nondiagnostic. Routine biopsy of renal ablation zones to validate postablation imaging results was not value-added and therefore was discontinued at the study institution. Copyright © 2018. Published by Elsevier Inc.

Survivability of Salmonella cells in popcorn after microwave oven and conventional cooking.

PubMed

Anaya, I; Aguirrezabal, A; Ventura, M; Comellas, L; Agut, M

2008-01-01

The survivability of Salmonella cells in popcorn preparation was determined for two distinct cooking methods. The first method used a standard microwave oven. The second method used conventional cooking in a pan. Prior to thermal processing in independent experiments, 12 suspensions in a range between 1x10(3) and 8x10(6) colony-forming units (CFU) per gram of Salmonella cells were inoculated in both raw microwave popcorn and conventional corn kernels. The influence of the initial concentration of Salmonella cells in the raw products and the lethal effects on Salmonella by thermal treatments for cooking were studied. Survival of Salmonella cells was determined in the thermally processed material by pre-enrichment and enrichment in selective medium, in accordance with the legislation for expanded cereals and cereals in flakes. Viable experimental contaminants were recovered from the conventionally cooked popcorn with initial inoculation concentrations of 9x10(4)cells/g or greater. Salmonella cell viability was significantly reduced after microwave oven treatment, with recoveries only from initial concentrations of 2x10(6)cells/g or superior.

The nature of immunity to Snyder-Theilen fibrosarcoma virus induced tumors in cats.

PubMed

Johnson, L; Pedersen, N C; Theilen, G H

1985-07-01

Snyder-Theilen fibrosarcoma virus (ST-FeSV) induced tumors evoked a vigorous immune response in adolescent cats. The response was characterized histologically by a lymphoid and histiocytic cell infiltrate beginning around the 9th day post inoculation. Hyperemia edema, hemorrhage, and necrosis of the tumors occurred shortly thereafter. Gross regression of the tumors commenced around the 15th day. Viable fibrosarcoma cells could be recovered as almost pure cultures from tumors biopsied on the 9th day. Biopsies taken between days 9 and 15 contained progressively fewer tumor cells and increasing numbers of lymphoid cells, histiocytes, giant cells, and normal fibroblasts. Tumor cells in such mixed cultures did not replicate as fast as normal and died out within 7 to 14 days. Viable tumor cells were not recovered from biopsies taken after day 15. Fibrosarcoma regression was associated with the appearance of tumor cell specific cytotoxic lymphocytes and antibodies in the blood. Cell mediated immunity, as determined by a chromium release assay, consisted of both antibody dependent and independent mechanisms. Fluorescent and complement dependent cytolytic antibodies were detected in the blood at the same time as cytotoxic lymphocytes, but persisted after regression. In a preliminary experiment, serum from tumor regressor cats was injected into susceptible kittens, and the kittens were then challenged with ST-FeSV transformed fibroblasts or whole FeSV. Immune serum did not prevent the appearance of initial growth of tumors, but did slow their subsequent growth and increased the rate of regression. Immune serum had a much more dramatic inhibitory effect on the accompanying retrovirus infection.

Can dead bacterial cells be defined and are genes expressed after cell death?

PubMed

Trevors, J T

2012-07-01

There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

Assay of enterocin AS-48 for inhibition of foodborne pathogens in desserts.

PubMed

Martinez Viedma, Pilar; Abriouel, Hikmate; Ben Omar, Nabil; Lucas López, Rosario; Valdivia, Eva; Gálvez, Antonio

2009-08-01

Enterocin AS-48 was tested against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes in different kinds of desserts. The highest activity against S. aureus was detected in baker cream. However, in yogurt-type soy-based desserts and in gelatin pudding, AS-48 (175 arbitrary units [AU]/g) reduced viable cell counts of S. aureus by only 1.5 to 1.8 log units at most. The efficacy of AS-48 in puddings greatly depended on inoculum size, and viable S. aureus counts decreased below detection levels within 24 h for inocula lower than 4 to 5.5 log CFU/g. For L. monocytogenes, bacteriocin concentrations of 52.5 to 87.5 AU/g reduced viable counts below detection levels and avoided regrowth of survivors. The lowest activity was detected in yogurt-type desserts. For B. cereus, viable cell counts were reduced below detection levels for bacteriocin concentrations of 52.5 AU/g in instant pudding without soy or by 175 AU/g in the soy pudding. In gelatin pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10 degrees C or after 48 h at 22 degrees C. Bacteriocin addition also inhibited gelatin liquefaction caused by the proteolytic activity of B. cereus.

The growth of Propionibacterium cyclohexanicum in fruit juices and its survival following elevated temperature treatments.

PubMed

Walker, Michelle; Phillips, Carol A

2007-06-01

This study investigated the growth of Propionibacterium cyclohexanicum in orange juice over a temperature range from 4 to 40 degrees C and its ability to multiply in tomato, grapefruit, apple, pineapple and cranberry juices at 30 and 35 degrees C. Survival after 10 min exposure to 50, 60, 70, 80, 85, 90 and 95 degrees C in culture medium and in orange juice was also assessed. In orange juice the organism was able to multiply by 2 logs at temperatures from 4 to 35 degrees C and survived for up to 52 days. However, at 40 degrees C viable counts were reduced after 6 days and no viable cells isolated after 17 days. The optimum growth temperature in orange juice over 6 days was 25 degrees C but over 4 days it was 35 degrees C. The growth of P. cyclohexanicum was monitored in tomato, grapefruit, cranberry, pineapple and apple juices at 30 and 35 degrees C over 29 days. Cranberry, grapefruit and apple juice did not support the growth of P. cyclohexanicum. At 30 degrees C no viable cells were detected after 8 days in cranberry juice or after 22 days in grapefruit juice while at 35 degrees C no viable cells were detected after 5 and 15 days, respectively. However, in apple juice, although a 5 log reduction occurred, viable cells could be detected after 29 days. P. cyclohexanicum was able to multiply in both tomato and pineapple juices. In tomato juice, there was a 2 log increase in viable counts after 8 days at 30 degrees C but no increase at 35 degrees C, while in pineapple juice there was a 1 log increase in numbers over 29 days with no significant difference between numbers of viable cells present at 30 and 35 degrees C. The organism survived at 50 degrees C for 10 min in culture medium without a significant loss of viability while similar treatment at 60, 70 and 80 degrees C resulted in approximately a 3-4 log reduction, with no viable cells detected after treatment at 85 or 90 or 95 degrees C but, when pre-treated at intermediate temperatures before exposure to higher temperatures, some cells survived. However, in orange juice a proportion of cells survived at 95 degrees C for 10 min without pre-treatment and there was no significant difference between numbers surviving with and without pre-treatment. The results from this study demonstrate that P. cyclohexanicum is able to grow in a number of juices, other than orange juice, and able to survive a number of high temperature procedures. Therefore, if initially present in the raw materials P. cyclohexanicum might survive the pasteurization procedures used in the fruit juice industry, contaminate and consequently spoil the final product.

Multisite evaluation of the BD™ Stem Cell Enumeration (SCE) Kit for CD34+ cell enumeration on the BD FACSCanto II™ and BD FACSCalibur™ flow cytometers

PubMed Central

Preti, Robert A; Chan, Wai Shun; Kurtzberg, Joanne; Dornsife, Ronna E.; Wallace, Paul K.; Furlange, Rosemary; Lin, Anna; Omana-Zapata, Imelda; Bonig, Halvard; Tonn, Thorsten

2018-01-01

Background Evaluation of the BD™ Stem Cell Enumeration (SCE) Kit was conducted at four clinical sites with flow cytometry CD34+ enumeration, to assess agreement between two investigational methods, the BD FACSCanto™ II and BD FACSCalibur™ systems, and the predicate method (Beckman Coulter Stem-Kit™ reagents). Methods Leftover and delinked specimens (n = 1,032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow, and leucopheresis and cord blood anticoagulated with CPD, ACD-A, heparin, and EDTA alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing, and analysis. Results The mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (−2.81 to 4.31 ±7.1) and BD FACSCalibur (−2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared to the predicate for viable CD34+, percentage of CD34+ in CD45+, and viable CD45+ populations (or gates). Bias analyses of the distribution of the predicate low, mid, and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34+, percentage of CD34+ in CD45+, and viable CD45+. Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R2 >0.92 for both investigational methods. Discussion In conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34+, percentage of viable CD34+ in CD45+, and absolute viable CD45+ populations. PMID:24927716

Viable Cancer Cells in the Remnant Stomach are a Potential Source of Peritoneal Metastasis after Curative Distal Gastrectomy for Gastric Cancer.

PubMed

Murata, Satoshi; Yamamoto, Hiroshi; Yamaguchi, Tsuyoshi; Kaida, Sachiko; Ishida, Mitsuaki; Kodama, Hirokazu; Takebayashi, Katsushi; Shimizu, Tomoharu; Miyake, Toru; Tani, Tohru; Kushima, Ryoji; Tani, Masaji

2016-09-01

The mechanisms underlying peritoneal metastasis (PM) after curative gastrectomy for gastric cancer (GC) are not well elucidated. This study assessed whether viable cancer cells, including cancer stemlike cells (CSCs), were present in the remnant stomach immediately before gastrointestinal (GI) tract reconstruction because these could be a source of PM after gastrectomy. Saline fluid used for remnant stomach lumen irrigation before GI reconstruction was prospectively collected from 142 consecutive patients undergoing distal gastrectomy for GC and cytologically examined. Proliferative activity (Ki67 staining) and stemness (expression of the CSC surface markers CD44s or CD44v6) were evaluated in detected cancer cells. Viable cancer cells were detected in 33 (23.2 %) of the 142 remnant stomachs. These cells formed clusters and stained positively for Ki67, indicating proliferation. Cancer cells in remnant stomachs and surface cancer cells in primary GCs from 10 (30.3 %) of these 33 cases also stained positively for CD44s or CD44v6. In a multiple logistic regression analysis, advanced cancer (odds ratio [OR], 4.65; 95 % confidence interval [CI], 1.32-16.4; P = 0.017), tumor size of 40 mm or larger (OR, 3.78; 95 % CI, 1.12-12.8; P = 0.033), and histologic differentiation (OR, 3.10; 95 % CI, 1.30-7.40; P = 0.011) were associated independently with the presence of cancer cells in the remnant stomach. Viable, proliferative, and clustered cancer cells, including CSCs, were found in remnant gastric lumens immediately before GI reconstruction, indicating a possible cellular source of PM after curative gastrectomy for GC. Dissemination of gastric contents into the peritoneal cavity should be avoided during GI reconstruction.

Specific detection of viable Listeria monocytogenes in Spanish wastewater treatment plants by Fluorescent In Situ Hybridization and PCR.

PubMed

Moreno, Yolanda; Ballesteros, Lorena; García-Hernández, Jorge; Santiago, Paula; González, Ana; Ferrús, M Antonia

2011-10-01

Listeria monocytogenes detection in wastewater can be difficult because of the large amount of background microbiota and the presence of viable but non-culturable forms in this environment. The aim of this study was to evaluate a Fluorescent In Situ Hybridization (FISH) assay combined with Direct Viable Count (DVC) method for detecting viable L. monocytogenes in wastewater samples, as an alternative to conventional culture methods. 16S rRNA sequence data were used to design a specific oligonucleotide probe. In order to assess the suitability of the method, the assays were performed on naturally (n=87) and artificially (n=14) contaminated samples and results were compared to those obtained with the isolation of cells on selective media and with a PCR method. The detection limit of FISH and PCR assays was 10(4) cells/mL without enrichment and 10 cells/mL after enrichment. A total of 47 samples, including 3 samples from effluent sites, yielded FISH positive results for L. monocytogenes. Using DVC-FISH technique, the presence of viable L. monocytogenes cells was detected in 23 out of these 47 FISH positive wastewater samples. PCR and culture methods yielded 27 and 23 positive results, respectively. According to these results, FISH technique has the potential to be used as a sensitive method for the detection and enumeration of L. monocytogenes in environmental wastewater samples. Copyright © 2011 Elsevier Ltd. All rights reserved.

Calcium phosphate composite cements based on simple mixture of brushite and apatite phases

NASA Astrophysics Data System (ADS)

Egorov, A. A.; Fedotov, A. Yu; Pereloma, I. S.; Teterina, A. Yu; Sergeeva, N. S.; Sviridova, I. K.; Kirsanova, V. A.; Akhmedova, S. A.; Nesterova, A. V.; Reshetov, I. V.; Barinov, S. M.; Komlev, V. S.

2018-04-01

The composite cements based on simple mixtures brishite and apatite with ratio 70/30, 50/50, 30/70 were developed. The processes of phase formation, microstructure and mechanical properties were studied. The kinetics of degradation in simulated body fluid depending on the microstructure and the materials phase composition was carried out. The biological test in vitro were performed using the MTT-test on the human fibroblast immortalized (hFB) cell line and the human osteosarcoma cell line MG-63. The materials didn’t have acute cytoxicity and possessed surface matrix properties. It was determined that the both line of cells actively proliferated, with viable cells values higher 20-60 % then control at all observation periods.

Kinetics of killing Listeria monocytogenes by macrophages: correlation of /sup 3/H-DNA release from labeled bacteria and changes in numbers of viable organisms by mathematical model

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

Davies, W.A.

1982-12-01

Conventional methods of assessing antibacterial activities of macrophages by viable counting are limited by the precision of the statistics and are difficult to interpret quantitatively because of unrestrained extracellular growth of bacteria. An alternative technique based on the release of radioactive DNA from labeled bacteria has been offered as overcoming these drawbacks. To assess it for use with macrophages I have made a correlation with the conventional viable counting method using a mathematical model. Opsonized Listeria monocytogenes labeled with /sup 3/H-thymidine were exposed to rat macrophages for periods up to 4 hr. Numbers of viable bacteria determined after sonication increasedmore » exponentially in the absence of live cells and this growth rate was progressively inhibited by increasing numbers of macrophages. After a lag period of 30-60 min soluble /sup 3/H appeared in the supernatant, the amount increasing with time and numbers of macrophages. To correlate these data I developed a mathematical model that considered that changes in numbers of viable organisms were due to the difference between rates of 1) growth of extracellular bacteria and 2) killing within the macrophage. On the basis of this model curves of best fit to the viable counts data were used to predict the release of radioactivity, assuming that death of a bacterium led to the total release of its label. These predictions and the experimental data agreed well, the lag period of 30-60 min between death of the bacterium and release of radioactivity being consistent with intracellular digestion. Release of soluble radioactivity appears to be an accurate reflection of the number of bacteria killed within the macrophage.« less

Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy.

PubMed

Opel, Cary F; Li, Jincai; Amanullah, Ashraf

2010-01-01

Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. (c) 2010 American Institute of Chemical Engineers

EMA-qPCR to monitor the efficiency of a closed-coupled solar pasteurization system in reducing Legionella contamination of roof-harvested rainwater.

PubMed

Reyneke, B; Dobrowsky, P H; Ndlovu, T; Khan, S; Khan, W

2016-05-15

Solar pasteurization is effective in reducing the level of indicator organisms in stored rainwater to within drinking water standards. However, Legionella spp. were detected at temperatures exceeding the recommended pasteurization temperatures using polymerase chain reaction assays. The aim of the current study was thus to apply EMA quantitative polymerase chain reaction (EMA-qPCR) to determine whether the Legionella spp. detected were intact cells and therefore possibly viable at pasteurization temperatures >70°C. The BacTiter-Glo™ Microbial Cell Viability Assay was also used to detect the presence of ATP in the tested samples, as ATP indicates the presence of metabolically active cells. Chemical analysis also indicated that all anions and cations were within the respective drinking water guidelines, with the exception of iron (mean: 186.76 μg/L) and aluminium (mean: 188.13 μg/L), which were detected in the pasteurized tank water samples at levels exceeding recommended guidelines. The BacTiter-Glo™ Microbial Cell Viability Assay indicated the presence of viable cells for all pasteurized temperatures tested, with the percentage of ATP (in the form of relative light units) decreasing with increasing temperature [70-79°C (96.7%); 80- 89°C (99.2%); 90-95°C (99.7%)]. EMA-qPCR then indicated that while solar pasteurization significantly reduced (p<0.05) the genomic copy numbers of intact Legionella cells in the pasteurized tank water (~99%), no significant difference (p>0.05) in the mean copy numbers was detected with an increase in the pasteurization temperature, with 6 × 10(3) genomic copies/mL DNA sample obtained at 95°C. As intact Legionella cells were detected in the pasteurized tank water samples, quantitative microbial risk assessment studies need to be conducted to determine the potential health risk associated with using the water for domestic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells.

PubMed

Lokmic, Zerina

2016-01-01

A protocol describing the isolation of foreskin lymphatic endothelial cells (LECs) and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented herein. To isolate LECs and LM LECs, tissues are mechanically disrupted to make a single-cell suspension, which is then enzymatically digested in dispase and collagenase type II. LECs and LM LECs, in the resulting single-cell suspension, are then sequentially labeled with antibodies recognizing fibroblast and endothelial cell surface antigens CD34 and CD31 and separated from the remaining components in the cell suspension by capture with magnetic beads. Viable LECs and LM LECs are then seeded and expanded on fibronectin-coated flasks. LEC and LM LEC purity is determined immunohistochemically using cell surface markers CD31, CD34, podoplanin, VEGFR-3 and nuclear marker PROX-1. Cells whose purity is >98 % are used for experiments between passage 4 and 6.

Rational design of an improved tissue-engineered vascular graft: determining the optimal cell dose and incubation time.

PubMed

Lee, Yong-Ung; Mahler, Nathan; Best, Cameron A; Tara, Shuhei; Sugiura, Tadahisa; Lee, Avione Y; Yi, Tai; Hibino, Narutoshi; Shinoka, Toshiharu; Breuer, Christopher

2016-03-01

We investigated the effect of cell seeding dose and incubation time on tissue-engineered vascular graft (TEVG) patency. Various doses of bone marrow-derived mononuclear cells (BM-MNCs) were seeded onto TEVGs, incubated for 0 or 12 h, and implanted in C57BL/6 mice. Different doses of human BM-MNCs were seeded onto TEVGs and measured for cell attachment. The incubation time showed no significant effect on TEVG patency. However, TEVG patency was significantly increased in a dose-dependent manner. In the human graft, more bone marrow used for seeding resulted in increased cell attachment in a dose-dependent manner. Increasing the BM-MNC dose and reducing incubation time is a viable strategy for improving the performance and utility of the graft.

Metrology for Fuel Cell Manufacturing

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

Stocker, Michael; Stanfield, Eric

2015-02-04

The project was divided into three subprojects. The first subproject is Fuel Cell Manufacturing Variability and Its Impact on Performance. The objective was to determine if flow field channel dimensional variability has an impact on fuel cell performance. The second subproject is Non-contact Sensor Evaluation for Bipolar Plate Manufacturing Process Control and Smart Assembly of Fuel Cell Stacks. The objective was to enable cost reduction in the manufacture of fuel cell plates by providing a rapid non-contact measurement system for in-line process control. The third subproject is Optical Scatterfield Metrology for Online Catalyst Coating Inspection of PEM Soft Goods. Themore » objective was to evaluate the suitability of Optical Scatterfield Microscopy as a viable measurement tool for in situ process control of catalyst coatings.« less

Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems.

PubMed

Qin, Tian; Tian, Zhengan; Ren, Hongyu; Hu, Guangchun; Zhou, Haijian; Lu, Jinxing; Luo, Chengwang; Liu, Zunyu; Shao, Zhujun

2012-05-01

Legionella are prevalent in human-made water systems and cause legionellosis in humans. Conventional culturing and polymerase chain reaction (PCR) techniques are not sufficiently accurate for the quantitative analysis of live Legionella bacteria in water samples because of the presence of viable but nonculturable cells and dead cells. Here, we report a rapid detection method for viable Legionella that combines ethidium monoazide (EMA) with quantitative real-time PCR (qPCR) and apply this method to detect Legionella in a large number of water samples from different sources. Results yielded that samples treated with 5 μg/ml EMA for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting Legionella. EMA treatment before qPCR could block the signal from approximately 4 log(10) of dead cells. When investigating environmental water samples, the percent-positive rate obtained by EMA-qPCR was significantly higher than conventional PCR and culture methods, and slightly lower than qPCR. The bacterial count of Legionella determined by EMA-qPCR were mostly greater than those determined by culture assays and lower than those determined by qPCR. Acceptable correlations were found between the EMA-qPCR and qPCR results for cooling towers, piped water and hot spring water samples (r = 0.849, P < 0.001) and also found between the EMA-qPCR and culture results for hot spring water samples (r = 0.698, P < 0.001). The results indicate that EMA-qPCR could be used as a complementary tool for the detection and monitoring of Legionella in water systems, especially in hot spring water samples.

Survival and High-Hydrostatic Pressure Inactivation of Foodborne Pathogens in Salmorejo, a Traditional Ready-to-Eat Food.

PubMed

Toledo Del Árbol, Julia; Pérez Pulido, Rubén; Grande, Ma José; Gálvez, Antonio; Lucas, Rosario

2015-11-01

Salmorejo is a traditional tomato-based creamy product. Because salmorejo is not heat-processed, there is a risk of contamination with foodborne pathogens from raw materials. Even though bacterial growth in salmorejo is strongly inhibited because of its acidic pH (close to 3.9), the growth and survival of 3 foodborne pathogens in this food has not been studied before. In this study, 3 cocktails consisting of Escherichia coli O157, Salmonella enterica serovar Enteritidis, and Listeria monocytogenes strains were inoculated in freshly prepared salmorejo. The food was treated by high hydrostatic pressure (HHP) at 400, 500, or 600 MPa for 8 min, or left untreated, and stored at 4 °C for 30 d. Viable cell counts were determined on selective media and also by the triple-layer agar method in order to detect sublethally injured cells. In control samples, L. monocytogenes viable cells decreased by 2.4 log cycles at day 7 and were undetectable by day 15. S. enterica cells decreased by 0.5 or 2.4 log cycles at days 7 and 15 respectively, but still were detectable at day 30. E. coli O157 cells survived much better in salmorejo, decreasing only by 1.5 log cycles at day 30. Treatments at pressures of 400 MPa or higher reduced viable counts of L. monocytogenes and S. enterica to undetectable levels. HHP treatments significantly (P < 0.05) reduced E. coli counts by approximately 5.2 to 5.4 log cycles, but also yielded surviving cells that apparently were sublethally injured. Only samples treated at 600 MPA for 8 min were devoid of detectable E. coli cells during storage. Salmorejo is a traditional, vitamin-rich food, usually produced on a small scale. HHP treatment at 600 MPa for 8 min can be an efficient nonthermal method for industrial-scale preparation of preservative-free salmorejo with improved safety against transmission of foodborne pathogens L. monocytogenes serotyes 4a and 4b, S. enterica serovar Enteritidis, and E. coli O157. © 2015 Institute of Food Technologists®

Interaction of intact porcine spermatozoa with epithelial cells and neutrophilic granulocytes during uterine passage.

PubMed

Taylor, U; Rath, D; Zerbe, H; Schuberth, H J

2008-04-01

New insemination techniques allow a tremendous sperm reduction for successful artificial insemination (AI) if highly diluted semen is deposited in the tip of the uterine horn and close to the utero-tubal junction. High sperm losses are known to occur during uterine passage and it was the general question whether specific binding mechanisms are involved. Upon arrival in the uterus, spermatozoa are confronted with mainly two different cell types: uterine epithelial cells (UEC) and neutrophilic granulocytes (polymorphonuclear neutrophil, PMN). As cell-sperm interactions can hardly be observed in vivo, an ex vivo system was established to study the interaction between spermatozoa and the UEC. Uterine segments (10 cm) from freshly slaughtered synchronized juvenile gilts were inseminated for 60 min at 38 degrees C. Thereafter spermatozoa were recovered, counted flow cytometrically and examined for changes in viability and mitochondrial membrane potential (MMP). Significantly less spermatozoa with a functioning MMP and intact plasma membranes could be retrieved (55 +/- 7%), while the number of damaged spermatozoa hardly changed (93 +/- 12%), indicating retention of viable sperm cells in the uterine lumen. The interactions between porcine PMN and spermatozoa (motile, immotile, membrane-damaged) were studied in coincubation assays in vitro. The binding of membrane-damaged sperm cells to PMN was virtually non-existent (3 +/- 2%). Viable and motile spermatozoa attached to PMN without being phagocytosed within 60 min (45 +/- 3%), whereas binding to sodium fluoride (NaF)-immobilized spermatozoa was reduced to 20 +/- 2%. The binding of viable sperm to PMN is most likely not lectin-dependent; although both viable cell types were shown to express a broad range of different lectin-binding sugar residues, none of the lectins tested was able to selectively block PMN-sperm binding significantly. The results of the study suggest that viable spermatozoa are already subject to selective processes within the uterus before further selection is initiated at the utero-tubal junction and in the oviductal isthmus.

7 CFR 97.6 - Application for certificate.

Code of Federal Regulations, 2010 CFR

2010-01-01

... applicant shall submit with the application: (1) A declaration that at least 3,000 seeds of the viable basic... tuber propagated variety, a declaration that a viable cell culture will be deposited in a public...

7 CFR 97.6 - Application for certificate.

Code of Federal Regulations, 2012 CFR

2012-01-01

... applicant shall submit with the application: (1) A declaration that at least 3,000 seeds of the viable basic... tuber propagated variety, a declaration that a viable cell culture will be deposited in a public...

7 CFR 97.6 - Application for certificate.

Code of Federal Regulations, 2014 CFR

2014-01-01

... applicant shall submit with the application: (1) A declaration that at least 3,000 seeds of the viable basic... tuber propagated variety, a declaration that a viable cell culture will be deposited in a public...

7 CFR 97.6 - Application for certificate.

Code of Federal Regulations, 2013 CFR

2013-01-01

... applicant shall submit with the application: (1) A declaration that at least 3,000 seeds of the viable basic... tuber propagated variety, a declaration that a viable cell culture will be deposited in a public...

7 CFR 97.6 - Application for certificate.

Code of Federal Regulations, 2011 CFR

2011-01-01

... applicant shall submit with the application: (1) A declaration that at least 3,000 seeds of the viable basic... tuber propagated variety, a declaration that a viable cell culture will be deposited in a public...

Detection of viable but non-culturable Escherichia coli O157:H7 by PCR in combination with propidium monoazide.

PubMed

Zhong, Junliang; Zhao, Xihong

2018-01-01

The aim of this study was to evaluate the applicability of the conventional PCR detection method combined with propidium monoazide (PMA) treatment for the detection of viable but non-culturable (VBNC) state Escherichia coli O157:H7 in ground beef meatballs. Under low temperature, E. coli O157:H7 cells were induced into the VBNC state in ground beef meatballs at - 20 °C after 152 days. The optimal PMA concentration of 5 µg/mL was obtained in beef meatball samples, which could completely inhibit the DNA amplification on dead cells (10 6  cells/mL) but with no inhibition on viable cells. The established PMA-PCR assay revealed that the VBNC counts exceeded 10 7  CFU/mL in artificial contamination beef samples, which could be used for semi-quantitative detection of VBNC cells in beef meatball samples. This study indicated that the PMA-PCR assay might be a potential method for detection of VBNC state E . coli O157:H7 cells in food products.

Xenotransplantation of neonatal porcine liver cells.

PubMed

Garkavenko, O; Emerich, D F; Muzina, M; Muzina, Z; Vasconcellos, A V; Ferguson, A B; Cooper, I J; Elliott, R B

2005-01-01

Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

Antimicrobial-induced endotoxin and cytokine activity in an in vitro model of septicemia in foals.

PubMed

Bentley, Adrienne P; Barton, Michelle H; Lee, Margie D; Norton, Natalie A; Moore, James N

2002-05-01

To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy. Blood samples from 10 healthy foals. Escherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in tissue-culture media were incubated with 105 colony-forming units of E. coli and each antimicrobial or saline (0.9% NaCl) solution. After 6 hours, number of viable bacteria remaining was determined, and supernatant was tested for endotoxin and tumor necrosis activity. Testing in whole blood was compromised by bactericidal effects of the blood itself. In mononuclear cell suspensions, each antimicrobial significantly reduced the number of viable bacteria to low or undetectable amounts. Antimicrobials did not differ significantly in efficacy of bacterial killing. Amikacin used alone or in combination with ampicillin resulted in significantly less endotoxin activity than did ampicillin, imipenem, or ceftiofur alone. There was a correlation between TNF-alpha and endotoxin activity. Aminoglycosides appear less likely to induce endotoxemia and TNF-alpha synthesis during bactericidal treatment of E. coli septicemia, compared with beta-lactam antimicrobials. Use of ampicillin, imipenem, or ceftiofur in the treatment of septicemic neonatal foals should be accompanied by appropriate treatment for endotoxemia.

Activity of disinfectants against foodborne pathogens in suspension and adhered to stainless steel surfaces

PubMed Central

Cabeça, Tatiane Karen; Pizzolitto, Antonio Carlos; Pizzolitto, Elisabeth Loshchagin

2012-01-01

The purpose of this study was to investigate and compare the efficacy of various disinfectants on planktonic cells and biofilm cells of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Numbers of viable biofilm cells decreased after treatment with all tested disinfectants (iodine, biguanide, quaternary ammonium compounds, peracetic acid and sodium hypochlorite). Sodium hypochlorite was the most effective disinfectant against biofilm cells, while biguanide was the least effective. Scanning electron microscopy observations revealed that cells adhered on stainless steel surface after treatment with the disinfectants. No viable planktonic cells were observed after treatment with the same disinfectants. Based on our findings, we concluded that biofilm cells might be more resistant to disinfectants than plancktonic cells. PMID:24031935

Histone posttranslational modifications and cell fate determination: lens induction requires the lysine acetyltransferases CBP and p300

PubMed Central

Wolf, Louise; Harrison, Wilbur; Huang, Jie; Xie, Qing; Xiao, Ningna; Sun, Jian; Kong, Lingkun; Lachke, Salil A.; Kuracha, Murali R.; Govindarajan, Venkatesh; Brindle, Paul K.; Ashery-Padan, Ruth; Beebe, David C.; Overbeek, Paul A.; Cvekl, Ales

2013-01-01

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300−/− ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens. PMID:24038357

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

PubMed

Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

2015-09-01

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10 3  CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10 0  CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

Oxidative metabolic products released from polymorphonuclear leukocytes in middle ear fluid during experimental pneumococcal otitis media.

PubMed Central

Kawana, M; Kawana, C; Yokoo, T; Quie, P G; Giebink, G S

1991-01-01

To determine whether oxidative metabolic products of phagocytic cells are present in the middle ear during experimental pneumococcal otitis media, we measured the concentration of myeloperoxidase (MPO) in middle ear fluid (MEF) and the capacity of neutrophils isolated from MEF and peripheral blood to produce MPO and superoxide anion (O2-) after in vitro stimulation. Free MPO in MEF was significantly increased 24 and 48 h after either viable or nonviable pneumococci were inoculated into the middle ear. In vitro-stimulated production of MPO and O2- from middle ear neutrophils was significantly less than that from peripheral blood neutrophils 24 h after nonviable pneumococci were inoculated but similar to it after 48 h. Twenty-four hours after viable pneumococci were inoculated, middle ear neutrophils stimulated in vitro produced less MPO but the same amount of O2- as did blood neutrophils. Oxidative metabolic products, therefore, are released from phagocytic cells into the MEF during pneumococcal otitis media, and future studies will need to define the contribution of these products to acute and chronic middle ear tissue injury. PMID:1657782

Improvement of microbiological safety of sous-vide meals by gamma radiation

NASA Astrophysics Data System (ADS)

Farkas, J.; Polyák-Fehér, K.; Andrássy, É.; Mészáros, L.

2002-03-01

Experimental batches of smoked-cured pork in stewed beans sauce were inoculated with spores of psychrotrophic Bacillus cereus, more heat and radiation resistant than spores of non-proteolytic C. botulinum. After vacuum packaging, the meals were treated with combinations of pasteurizing heat treatments and gamma irradiation of 5 kGy. Prior and after treatments, and periodically during storage at 10°C, total aerobic and total anerobic viable cell counts, and selectively, the viable cell counts of B. cereus and sulphite-reducing clostridia have been determined. The effects of the treatment order as well as addition of nisin to enhance the preservative efficiency of the physical treatments were also studied. Heat-sensitization of bacterial spores surviving irradiation occurred. The quality-friendly sous-vide cooking in combination with this medium dose gamma irradiation and/or nisin addition increased considerably the microbiological safety and the keeping quality of the meals studied. However, approx. 40% loss of thiamin content occurred as an effect of combination treatments, and adverse sensorial effects may also limit the feasible radiation doses or the usable concentrations of nisin.

Specific detection of cultivable Helicobacter pylori cells from wastewater treatment plants.

PubMed

Moreno, Yolanda; Ferrús, M Antonía

2012-10-01

Helicobacter pylori is present in surface water and wastewater, and biofilms in drinking water systems have been reported as possible reservoirs of H. pylori. However, its ability to survive in an infectious state in the environment is hindered because it rapidly loses its cultivability. The aim of this study was to determine the presence of cultivable and therefore viable H. pylori in wastewater treatment plants to understand the role of wastewater in the pathogen's transmission. A modified filter technique was used to obtain a positive H. pylori culture, and specific detection of this pathogen was achieved with FISH and PCR techniques. A total of six positive H. pylori cultures were obtained from the water samples, and molecular techniques positively identified H. pylori in 21 culture-negative samples. The combination of a culturing procedure after sample filtration followed by the application of a molecular method, such as PCR or FISH, provides a specific tool for the detection, identification, and direct visualization of cultivable and therefore viable H. pylori cells from complex mixed communities such as water samples. © 2012 Blackwell Publishing Ltd.

Design and characterization of a biodegradable composite scaffold for ligament tissue engineering.

PubMed

Hayami, James W S; Surrao, Denver C; Waldman, Stephen D; Amsden, Brian G

2010-03-15

Herein we report on the development and characterization of a biodegradable composite scaffold for ligament tissue engineering based on the fundamental morphological features of the native ligament. An aligned fibrous component was used to mimic the fibrous collagen network and a hydrogel component to mimic the proteoglycan-water matrix of the ligament. The composite scaffold was constructed from cell-adherent, base-etched, electrospun poly(epsilon-caprolactone-co-D,L-lactide) (PCLDLLA) fibers embedded in a noncell-adherent photocrosslinked N-methacrylated glycol chitosan (MGC) hydrogel seeded with primary ligament fibroblasts. Base etching improved cellular adhesion to the PCLDLLA material. Cells within the MGC hydrogel remained viable (72 +/- 4%) during the 4-week culture period. Immunohistochemistry staining revealed ligament ECM markers collagen type I, collagen type III, and decorin organizing and accumulating along the PCLDLLA fibers within the composite scaffolds. On the basis of these results, it was determined that the composite scaffold design was a viable alternative to the current approaches used for ligament tissue engineering and merits further study. (c) 2009 Wiley Periodicals, Inc.

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

PubMed

Oback, B; Wells, D N

2007-05-01

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples▿

PubMed Central

Delgado-Viscogliosi, Pilar; Solignac, Lydie; Delattre, Jean-Marie

2009-01-01

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells. PMID:19363080

Efficacy of SYBR 14/propidium iodide viability stain for the amphibian chytrid fungus Batrachochytrium dendrobatidis.

PubMed

Stockwell, M P; Clulow, J; Mahony, M J

2010-01-25

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently described pathogen that has been implicated as a causal agent in the global decline in amphibians. Research into its biology and epidemiology has frequently involved in vitro experimentation. However, this research is currently limited by the inability to differentiate between viable and inviable zoospores. Stains are frequently used to determine cell viability, and this study tested a 2-colour fluorescence assay for the detection and quantification of viable B. dendrobatidis zoospores. The results show that the nucleic acid stains SYBR 14 and propidium iodide are effective in distinguishing live from dead zoospores, and a protocol has been optimized for their use. This viability assay provides an efficient and reliable tool that will have applications in B. dendrobatidis challenge and amphibian exposure experiments.

Inactivation kinetics of various chemical disinfectants on Aeromonas hydrophila planktonic cells and biofilms.

PubMed

Jahid, Iqbal Kabir; Ha, Sang-Do

2014-05-01

The present article focuses on the inactivation kinetics of various disinfectants including ethanol, sodium hypochlorite, hydrogen peroxide, peracetic acid, and benzalkonium chloride against Aeromonas hydrophila biofilms and planktonic cells. Efficacy was determined by viable plate count and compared using a modified Weibull model. The removal of the biofilms matrix was determined by the crystal violet assay and was confirmed by field-emission scanning electron microscope. The results revealed that all the experimental data and calculated Weibull α (scale) and β (shape) parameters had a good fit, as the R(2) values were between 0.88 and 0.99. Biofilms are more resistant to disinfectants than planktonic cells. Ethanol (70%) was the most effective in killing cells in the biofilms and significantly reduced (p<0.05) the biofilms matrix. The Weibull parameter b-value correlated (R(2)=0.6835) with the biofilms matrix removal. The present findings deduce that the Weibull model is suitable to determine biofilms matrix reduction as well as the effectiveness of chemical disinfectants on biofilms. The study showed that the Weibull model could successfully be used on food and food contact surfaces to determine the exact contact time for killing biofilms-forming foodborne pathogens.

Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

PubMed

Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph

2015-11-01

The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain

PubMed Central

Parkins, Katie M.; Hamilton, Amanda M.; Makela, Ashley V.; Chen, Yuanxin; Foster, Paula J.; Ronald, John A.

2016-01-01

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade. PMID:27767185

A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain.

PubMed

Parkins, Katie M; Hamilton, Amanda M; Makela, Ashley V; Chen, Yuanxin; Foster, Paula J; Ronald, John A

2016-10-21

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain

NASA Astrophysics Data System (ADS)

Parkins, Katie M.; Hamilton, Amanda M.; Makela, Ashley V.; Chen, Yuanxin; Foster, Paula J.; Ronald, John A.

2016-10-01

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

Aging of perennial cells and organ parts according to the programmed aging paradigm.

PubMed

Libertini, Giacinto; Ferrara, Nicola

2016-04-01

If aging is a physiological phenomenon-as maintained by the programmed aging paradigm-it must be caused by specific genetically determined and regulated mechanisms, which must be confirmed by evidence. Within the programmed aging paradigm, a complete proposal starts from the observation that cells, tissues, and organs show continuous turnover: As telomere shortening determines both limits to cell replication and a progressive impairment of cellular functions, a progressive decline in age-related fitness decline (i.e., aging) is a clear consequence. Against this hypothesis, a critic might argue that there are cells (most types of neurons) and organ parts (crystalline core and tooth enamel) that have no turnover and are subject to wear or manifest alterations similar to those of cells with turnover. In this review, it is shown how cell types without turnover appear to be strictly dependent on cells subjected to turnover. The loss or weakening of the functions fulfilled by these cells with turnover, due to telomere shortening and turnover slowing, compromises the vitality of the served cells without turnover. This determines well-known clinical manifestations, which in their early forms are described as distinct diseases (e.g., Alzheimer's disease, Parkinson's disease, age-related macular degeneration, etc.). Moreover, for the two organ parts (crystalline core and tooth enamel) without viable cells or any cell turnover, it is discussed how this is entirely compatible with the programmed aging paradigm.

[Acquirement of autologous murine cytotoxic T lymphocytes via cryopreservation of lymphocytes].

PubMed

Wang, Lei; Peng, Na; Hu, Xiaoyan; Liang, Wentao; Liang, Kai; Peng, Guizhu

2016-11-01

Objective To evaluate the effects of cryopreservation on the proliferation and killing activity of lymphocytes, and explore a novel protocol of preparing autologous mouse cytotoxic T lymphocytes (CTLs). Methods Mononuclear cells derived from spleen (5.0×10 6 /mL) were cryopreserved in CELLBANKER2 for 6 days and recovered in water bath at 39DegreesCelsius. The fresh lymphocytes and post-cryopreservation lymphocytes were induced by CD3 mAb (100 ng/mL) and recombinant mouse interleukin 2 (rmIL-2, 100 ng/mL) to obtain cytokine-induced killer cells (CIKs). Dendritic cells (DCs) were co-cultured with fresh allogenic lymphocytes and post-cryopreservation autologous lymphocytes to obtain CTLs. The viable cells were counted by trypan blue staining; the percentages of CD3 + T cells and regulatory T cells (Tregs) were determined by flow cytometry; the levels of supernatant IFN-γ were detected through ELISA and the cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. Results The rate of viable lymphocytes declined to 78% after cryopreservation, and there were no significant differences in the percentages of CD3 + T cells and Tregs between pre-cryopreservation and post-cryopreservation. There were no significant differences in the proliferation of Tregs, the level of IFN-γ and the cytotoxicity between the fresh CIKs and cryopreservation CIKs, and the similar results were get between the autologous CTLs and allogenic CTLs. Conclusion The autologous CTLs acquired via cryopreservation of lymphocytes is equivalent to the allogenic CTLs with the similar proliferation and killing activity in vitro.

Induction of Escherichia coli into a VBNC state through chlorination/chloramination and differences in characteristics of the bacterium between states.

PubMed

Chen, Sheng; Li, Xi; Wang, Yahong; Zeng, Jie; Ye, Chengsong; Li, Xianping; Guo, Lizheng; Zhang, Shenghua; Yu, Xin

2018-05-30

Many pathogens can enter into a viable but nonculturable (VBNC) state in response to harsh environmental stresses. Bacteria in this state can retain certain features of viable cells, such as cellular integrity, metabolic activity, or virulence and may present health risks associated with drinking water. In this study, we investigated the ability of chlorination and chloramination, which are widely used methods to disinfect drinking water, to induce Escherichia coli into a VBNC state. After treatment with chlorine and chloramine at concentrations of 1, 2, 3, and 4 mg/L, the counts of culturable E. coli cells decreased from 10 6  CFU/mL to 0 CFU/mL at 5-60 min post treatment. Meanwhile, viable cell counts were still approximately 10 3 -10 5  cells/mL. These viable E. coli cells may be induced into a VBNC state by chlorination and chloramination. Scanning electron microscopy and laser confocal microscopy showed that some bacteria maintained cellular integrity, but the average length of VBNC cells was less than that of culturable cells. Respiratory activity of VBNC cells decreased approximately 50% relative to that of culturable cells. We also used heavy water (D 2 O) combined with Raman microspectroscopy to show that E. coli in a VBNC state retained metabolic activity involving water (e.g. condensation reactions) at the single-cell level. Furthermore, soxR, gadA, and katG genes remained highly expressed, suggesting that VBNC cells were physiologically active. Finally, resuscitation of VBNC cells induced by chlorine in Luria-Bertani (LB) broth was identified by calculating the generation time. Results of this study will facilitate a better understanding of the health risks associated with VBNC bacteria and the development of more effective strategies for drinking water disinfection. Copyright © 2018. Published by Elsevier Ltd.

Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine.

PubMed

Willenburg, Elize; Divol, Benoit

2012-11-15

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

Optical sensor for rapid microbial detection

NASA Astrophysics Data System (ADS)

Al-Adhami, Mustafa; Tilahun, Dagmawi; Rao, Govind; Kostov, Yordan

2016-05-01

In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes' signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.

Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells.

PubMed

Wolffs, Petra; Norling, Börje; Rådström, Peter

2005-03-01

Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

PubMed

Gambihler, S; Delius, M; Ellwart, J W

1994-09-01

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.

MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted-cell viability

NASA Astrophysics Data System (ADS)

Chan, Kannie W. Y.; Liu, Guanshu; Song, Xiaolei; Kim, Heechul; Yu, Tao; Arifin, Dian R.; Gilad, Assaf A.; Hanes, Justin; Walczak, Piotr; van Zijl, Peter C. M.; Bulte, Jeff W. M.; McMahon, Michael T.

2013-03-01

Biocompatible nanomaterials and hydrogels have become an important tool for improving cell-based therapies by promoting cell survival and protecting cell transplants from immune rejection. Although their potential benefit has been widely evaluated, at present it is not possible to determine, in vivo, if and how long cells remain viable following their administration without the use of a reporter gene. Here, we report a pH-nanosensor-based magnetic resonance imaging (MRI) technique that can monitor cell death in vivo non-invasively. We demonstrate that specific MRI parameters that change on cell death of microencapsulated hepatocytes are associated with the measured bioluminescence imaging radiance. Moreover, the readout from this pH-sensitive nanosensor can be directly co-registered with high-resolution anatomical images. All of the components of these nanosensors are clinical grade and hence this approach should be a translatable and universal modification of hydrogels.

Liquid biopsy in cancer patients: advances in capturing viable CTCs for functional studies using the EPISPOT assay.

PubMed

Alix-Panabières, Catherine; Pantel, Klaus

2015-01-01

Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling 'liquid biopsies'. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarkers, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. This article discusses the current possibilities to enrich and, in particular, detect viable CTCs with emphasis on the EPithelial ImmunoSPOT technology. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate and colon cancer) and melanomas. Moreover, the article summarizes recent advances in the in vitro and in vivo expansion of CTCs from cancer patients. These functional analyses will contribute to identifying the biological properties of metastatic cells and reveal new therapeutic targets against disseminating cancer cells.

Primary cultures of astrocytes from fetal bovine brain.

PubMed

Ballarin, Cristina; Peruffo, Antonella

2012-01-01

We describe here a method to obtain primary cell cultures from the cerebral cortex and the hypothalamus of bovine fetuses. We report how tissue origin, developmental stages, and culture medium conditions influence cell differentiation and the prevalence of glial cells vs. neurons. We compare explants from early, middle, and late stages of development and two different fetal calf serum concentrations (1 and 10%) to identify the best conditions to obtain and grow viable astrocytes in culture. In addition, we describe how to cryopreserve and obtain viable cortical astrocytes from frozen fetal bovine brain samples.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells.

PubMed

Swioklo, Stephen; Constantinescu, Andrei; Connon, Che J

2016-03-01

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 10(6) cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. ©AlphaMed Press.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758).

PubMed

Borges, Alana A; Lira, Gabriela P O; Nascimento, Lucas E; Queiroz Neta, Luiza B; Santos, Maria V O; Oliveira, Moacir F; Silva, Alexandre R; Pereira, Alexsandra F

2018-04-01

Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p < 0.05). Nevertheless, for viability after the first passage, only the EG-SUC (86.9%) and DMSO-SUC (91.4%) groups maintained viable cell recovery similar to the nonvitrified group (96.3%, p > 0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

PubMed

Williams, Anna J; Cooper, Willie M; Summage-West, Christine V; Sims, Lillie M; Woodruff, Robert; Christman, Jessica; Moskal, Ted J; Ramsaroop, Shawn; Sutherland, John B; Alusta, Pierre; Wilkes, Jon G; Buzatu, Dan A

2015-12-23

The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed. Published by Elsevier B.V.

A viable circulating tumor cell isolation device with high retrieval efficiency using a reversibly deformable membrane barrier

NASA Astrophysics Data System (ADS)

Kim, Yoonji; Bu, Jiyoon; Cho, Young-Ho; Son, Il Tae; Kang, Sung-Bum

2017-02-01

Circulating tumor cells (CTCs) contain prognostic information of the tumor, since they shed from the primary tumor and invade into the bloodstream. Therefore, the viable isolation is necessary for a consequent analysis of CTCs. Here, we present a device for the viable isolation and efficient retrieval of CTCs using slanted slot filters, formed by a reversibly deformable membrane barrier. Conventional filters have difficulties in retrieving captured cells, since they easily clog the slots. Moreover, large stress concentration at the sharp edges of squared slots, causes cell lysis. In contrast, the present device shows over 94% of high retrieval efficiency, since the slots can be opened simply by relieving the pressure. Furthermore, the inflated membrane barrier naturally forms the slanted slots, thus reducing the cell damage. By using cancer cell lines, we verified that the present device successfully isolate targeted cells, even at an extremely low concentrations (~10 cells/0.1 ml). In the clinical study, 85.7% of patients initially showed CTC positive while the numbers generally decreased after the surgery. We have also proved that the number of CTCs were highly correlated with tumour invasiveness. Therefore, the present device has potential for use in cancer diagnosis, surgical validation, and invasiveness analysis.

Designing primers and evaluation of the efficiency of propidium monoazide - Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.

PubMed

Lai, Chieh-Hsien; Wu, Sih-Rong; Pang, Jen-Chieh; Ramireddy, Latha; Chiang, Yu-Cheng; Lin, Chien-Ku; Tsen, Hau-Yang

2017-07-01

The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4-5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable. Copyright © 2016. Published by Elsevier B.V.

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection.

PubMed

Chen, Jia-Yang; Tsai, Wen-Sy; Shao, Hung-Jen; Wu, Jen-Chia; Lai, Jr-Ming; Lu, Si-Hong; Hung, Tsung-Fu; Yang, Chih-Tsung; Wu, Liang-Chun; Chen, Jinn-Shiun; Lee, Wen-Hwa; Chang, Ying-Chih

2016-01-01

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) "smart coating" to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

Saliva affects the antifungal activity of exogenously added histatin 3 towards Candida albicans.

PubMed

Yamagishi, Hisako; Fitzgerald, Deirdre H; Sein, Tin; Walsh, Thomas J; O'Connell, Brian C

2005-03-01

Antifungal activity of histatin 3 against two Candida albicans clinical isolates was determined in assays containing rabbit submandibular gland saliva. Histatin 3 inhibited the cell growth and germination of both isolates dose-dependently (10-100 microg ml(-1)) with maximum inhibition occurring after 60 min incubation. Adding fresh histatin 3 after 60 min caused further reduction in the viable cell count. Higher histatin 3 concentrations (50-100 microg ml(-1)) and prolonged exposure to peptide were required to inhibit germination. Histatin 3 was rapidly degraded in rabbit submandibular gland saliva and this may explain why fresh addition of histatin 3 increases candidacidal activity.

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry.

PubMed

Foladori, P; Bruni, L; Tamburini, S; Ziglio, G

2010-07-01

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 +/- 2.4%), settled wastewater (10.7 +/- 3.1%), activated sludge (11.1 +/- 2.1%) and effluent (3.2 +/- 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant. Copyright 2010 Elsevier Ltd. All rights reserved.

The intratumoral distribution of radiolabeled 177Lu-BR96 monoclonal antibodies changes in relation to tumor histology over time in a syngeneic rat colon carcinoma model.

PubMed

Örbom, Anders; Eriksson, Sophie E; Elgström, Erika; Ohlsson, Tomas; Nilsson, Rune; Tennvall, Jan; Strand, Sven-Erik

2013-08-01

The therapeutic effect of radioimmunotherapy depends on the distribution of the absorbed dose in relation to viable cancer cells within the tumor, which in turn is a function of the activity distribution. The aim of this study was to investigate the distribution of (177)Lu-DOTA-BR96 monoclonal antibodies targeting the Lewis Y antigen over 7 d using a syngeneic rat model of colon carcinoma. Thirty-eight tumor-bearing rats were intravenously given 25 or 50 MBq of (177)Lu-DOTA-BR96 per kilogram of body weight and were sacrificed 2, 8, 24, 48, 72, 96, 120, or 168 h after injection, with activity measured in blood and tumor samples. Adjacent cryosections of each tumor were analyzed in 3 ways: imaging using a silicon-strip detector for digital autoradiography, staining for histologic characterization, or staining to determine the distribution of the antigen, vasculature, and proliferating cells using immunohistochemistry. Absorbed-dose rate distribution images at the moment of sacrifice were calculated using the activity distribution and a point-dose kernel. The correlations between antigen expression and both activity uptake and absorbed-dose rate were calculated for several regions of interest in each tumor. Nine additional animals with tumors were given unlabeled antibody to evaluate possible immunologic effects. At 2-8 h after injection, activity was found in the tumor margins; at 24 h, in viable antigen-expressing areas within the tumor; and at 48 h and later, increasingly in antigen-negative areas of granulation tissue. The correlation between antigen expression and both the mean activity and the absorbed-dose rate in regions of interest changed from positive to negative after 24 h after injection. Antigen-negative areas also increased over time in animals injected with unlabeled BR96, compared with untreated tumors. The results indicate that viable Lewis Y-expressing tumor cells are most efficiently treated during the initial uptake period. The activity then seems to remain in these initial uptake regions after the elimination of tumor cells and formation of granulation tissue. Further studies using these techniques could aid in determining the effects of the intratumoral activity distribution on overall therapeutic efficacy.

Effect of soy saponin on the growth of human colon cancer cells

PubMed Central

Tsai, Cheng-Yu; Chen, Yue-Hwa; Chien, Yi-Wen; Huang, Wen-Hsuan; Lin, Shyh-Hsiang

2010-01-01

AIM: To investigate the effect of extracted soybean saponins on the growth of human colon cancer cells. METHODS: WiDr human colon cancer cells were treated with 150, 300, 600 or 1200 ppm of soy saponin to determine the effect on cell growth, cell morphology, alkaline phosphatase (AP) and protein kinase C (PKC) activities, and P53 protein, c-Fos and c-Jun gene expression. RESULTS: Soy saponin decreased the number of viable cells in a dose-dependent manner and suppressed 12-O-tetradecanol-phorbol-13-acetate-stimulated PKC activity (P < 0.05). Cells treated with saponins developed cytoplasmic vesicles and the cell membrane became rougher and more irregular in a dose-dependent manner, and eventually disassembled. At 600 and 1200 ppm, the activity of AP was increased (P < 0.05). However, the apoptosis markers such as c-Jun and c-Fos were not significantly affected by saponin. CONCLUSION: Soy saponin may be effective in preventing colon cancer by affecting cell morphology, cell proliferation enzymes, and cell growth. PMID:20632438

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth.

PubMed

Martin, Michelle D; Fingleton, Barbara; Lynch, Conor C; Wells, Sam; McIntyre, J Oliver; Piston, David W; Matrisian, Lynn M

2008-01-01

The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.

Cytotoxic effect of indigenously fabricated dental magnets for application in prosthodontics.

PubMed

Guttal, Satyabodh Shesharaj; Nadiger, Ramesh K; Shetty, Pravinkumar

2018-01-01

Dental magnets are used for retaining removable prostheses such as a removable partial denture, complete denture, and maxillofacial prosthesis. They provide good retention for the prostheses. However, the elements released from the magnets may be cytotoxic for the tissues. Therefore, it is necessary to evaluate their cytotoxic effect on cell lines. The aim of the study is to check the cytotoxic effect of indigenously fabricated dental magnets on animal cell lines. Neodymium-iron-boron (Nd-Fe-B) magnet was tested for cytotoxicity. The magnet was encased in a teflon cylinder. Magnets were placed in the well tissue-cultured plates together with a suspension containing NIH 3T3 mouse fibroblasts (5 × 10 5 cells/ml). After 3 days of incubation at 37°C, cell viability was determined by mean transit time (MTT) assay. Cells were subsequently dissolved in 100 μl dimethyl sulfoxide with gentle shaking for 2 h at room temperature followed by measurement of absorbance at 570 nm. Eight replicate wells were used at each point in each of four separate measurements. Measured absorbance values were directly used for calculating percent of viable cells remaining after the respective treatment. Data were analyzed statistically with significance level set at P < 0.05. The control group had highest absorbance reading for the MTT assay followed by test group. The lowest values were found with bare Nd-Fe-B magnets. One-way ANOVA test was performed for the data obtained. There was a statistical significant difference seen in the positive control (bare magnets, 44.96) and the test (teflon cased magnets, 96.90) group. More number of viable cells was visible in test group cells indicating that the indigenously fabricated dental magnet did not show any cytotoxicity.

Model-based strategy for cell culture seed train layout verified at lab scale.

PubMed

Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

2016-08-01

Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

Flow Cytometry of Human Primary Epidermal and Follicular Keratinocytes

PubMed Central

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-01-01

Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis. PMID:18350110

Flow cytometry of human primary epidermal and follicular keratinocytes.

PubMed

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

Impact of endoscopic stent insertion on detection of viable circulating tumor cells from obstructive colorectal cancer

PubMed Central

Yamashita, Shinya; Tanemura, Masahiro; Sawada, Genta; Moon, Jeongho; Shimizu, Yosuke; Yamaguchi, Toshiki; Kuwai, Toshio; Urata, Yasuo; Kuraoka, Kazuya; Hatanaka, Nobutaka; Yamashita, Yoshinori; Taniyama, Kiyomi

2018-01-01

The placement of a self-expanding metallic stent (SEMS) in obstructive colorectal cancer (OCRC) is acknowledged to be a safe and effective procedure for the relief of obstruction. However, there is concern that shear forces acting on the tumor during stent expansion may release cancer cells into the circulation, resulting in a poor prognosis. The aim of the present study was to determine whether colonic stent insertion increases viable circulating tumor cells (v-CTCs). A telomerase-specific replication-selective adenovirus-expressing GFP (TelomeScanF35) detection system was used to detect v-CTCs in 8 OCRC patients with a SEMS before and after stent insertion and after surgical resection. In 7 patients, a SEMS was inserted as a bridge to surgery (BTS), and in one patient, a SEMS was inserted for palliation. Surgical resection (R0) was performed in 7 patients. Four patients had no v-CTCs before SEMS placement, two of four measurable patients had an increased number of v-CTCs after SEMS placement (1–3 v-CTCs), and one of two patients with increased v-CTCs developed distant lymphatic metastasis despite curative resection. Four patients had v-CTCs (1–19 cells) before SEMS placement, and two of these four patients had an increase in the number of v-CTCs (20–21 cells) after SEMS placement, while one of the four patients died early with distant metastasis. The present study demonstrated that endoscopic stent insertion for OCRC may result in tumor cell dissemination into the peripheral circulation and may induce distant metastases. PMID:29391884

Impact of endoscopic stent insertion on detection of viable circulating tumor cells from obstructive colorectal cancer.

PubMed

Yamashita, Shinya; Tanemura, Masahiro; Sawada, Genta; Moon, Jeongho; Shimizu, Yosuke; Yamaguchi, Toshiki; Kuwai, Toshio; Urata, Yasuo; Kuraoka, Kazuya; Hatanaka, Nobutaka; Yamashita, Yoshinori; Taniyama, Kiyomi

2018-01-01

The placement of a self-expanding metallic stent (SEMS) in obstructive colorectal cancer (OCRC) is acknowledged to be a safe and effective procedure for the relief of obstruction. However, there is concern that shear forces acting on the tumor during stent expansion may release cancer cells into the circulation, resulting in a poor prognosis. The aim of the present study was to determine whether colonic stent insertion increases viable circulating tumor cells (v-CTCs). A telomerase-specific replication-selective adenovirus-expressing GFP (TelomeScanF35) detection system was used to detect v-CTCs in 8 OCRC patients with a SEMS before and after stent insertion and after surgical resection. In 7 patients, a SEMS was inserted as a bridge to surgery (BTS), and in one patient, a SEMS was inserted for palliation. Surgical resection (R0) was performed in 7 patients. Four patients had no v-CTCs before SEMS placement, two of four measurable patients had an increased number of v-CTCs after SEMS placement (1-3 v-CTCs), and one of two patients with increased v-CTCs developed distant lymphatic metastasis despite curative resection. Four patients had v-CTCs (1-19 cells) before SEMS placement, and two of these four patients had an increase in the number of v-CTCs (20-21 cells) after SEMS placement, while one of the four patients died early with distant metastasis. The present study demonstrated that endoscopic stent insertion for OCRC may result in tumor cell dissemination into the peripheral circulation and may induce distant metastases.

A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

PubMed

Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

2012-12-01

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.

Heterotrophic bacteria in soils of Larsemann Oasis of East Antarctica

NASA Astrophysics Data System (ADS)

Churilin, Nikita; Soina, Vera

2015-04-01

The study of diversity and functional state of microorganisms in subsurface rocks layers, their participation in the biochemical weathering and formation of organic horizons of soils is important for understanding ecology and microorganisms in Antarctic soils. The study of cultured forms of microorganisms and their potential viability is still relevant to characterize the physiological state, biological activity and resilience of microorganisms involved in the initial soil formation. Improvement of isolation techniques of viable bacteria from the extreme habitats has a particular importance for rising the efficiency of environmental monitoring. The aim of the study was to investigate the viable heterotrophic bacteria involved in the formation of soils from wet valleys Larsemann Oasis, which is one of the warmest ice-free space of East Antarctica. Soil samples were taken from the intermountain humid valleys, where silt-gravelly substrates formed moss, algae, lichen cover. We used nutrient solutions (trypticase soy, R2A and glucose-peptone) to isolate cultured bacteria and study their morphological types in the light microscope. The total number of microorganisms was determined by fluorescent microscopy with acridine orange. SEM was used for morphological studies of bacterial communities in situ. To activate the growth processes we added into nutrient solutions various regulatory metabolites that have dose-dependence and operate at the community level. Physiological and functional conditions were determined by the duration of the lag phase and specific growth rate of bacterial communities in nutrient solutions containing various organic substrates. Soils form under protection of «stone pavement» and organisms leave the surface, so the forming organo-mineral horizon occurs inside of rock, thus the microprofile can form on both sides of the organic horizons. UV radiation, lack of moisture and strong wind are main limiting factors for microorganisms' growth in Antarctic soils. Primitive soils and permafrost layer have a great unevenness in the number of cultivated and potentially viable cells in different horizons. This phenomenon is characteristic for habitats with stable and alternating negative temperatures that can be attributed to the irregular migration of cells during freezing and heterogeneity of microbial populations along the depth of dormancy. One of the identified features was the lack of correlation with the organic content. SEM study of microbial communities in native Antarctic soils revealed the presence of biofilms, which can play an important role in weathering of rocks and primary soil formation, by forming organic horizon and protecting cells from environmental impact. Biofilms can also influence on distribution of bacterial cells in forming soils. Growth regulators (indoleacetic acid, wheat germ agglutinin, alkylhydroxybenzenes, pyruvate Na and serotonin) were used in experiments on the growth reactivation using soil samples with low number of microorganisms. The results obtained by this analysis can be used for further research to develop methods of the most complete selection of viable bacteria from Antarctic soils. We also determined the physiological condition of bacterial populations and their maximum specific growth rate. This method determines the functional (trophic) diversity of microbial communities and the maximum specific growth rate that reflects the environmental strategy of bacterial growth. In spite of the extreme conditions, a variety of physiological and metabolic willingness to consume polymers hydrolytic bacterial associations of endolithic soil is highest in the surface horizon and sharply decreases in the mineral horizon.

The Boston Keratoprosthesis: Comparing Corneal Epithelial Cell Compatibility with Titanium and PMMA

PubMed Central

Ament, Jared D.; Spurr-Michaud, Sandra J.; Dohlman, Claes H.; Gipson, Ilene K.

2014-01-01

Purpose To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). Methods Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant. Results HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36). Conclusion This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated. PMID:19574903

31P-nuclear magnetic resonance spectroscopy in vivo of six human melanoma xenograft lines: tumour bioenergetic status and blood supply.

PubMed Central

Lyng, H.; Olsen, D. R.; Southon, T. E.; Rofstad, E. K.

1993-01-01

Six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice were subjected to 31P-nuclear magnetic resonance (31P-NMR) spectroscopy in vivo. The following resonances were detected: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr) and nucleoside triphosphate gamma, alpha and beta (NTP gamma, alpha and beta). The main purpose of the work was to search for possible relationships between 31P-NMR resonance ratios and tumour pH on the one hand and blood supply per viable tumour cell on the other. The latter parameter was measured by using the 86Rb uptake method. Tumour bioenergetic status [the (PCr + NTP beta)/Pi resonance ratio], tumour pH and blood supply per viable tumour cell decreased with increasing tumour volume for five of the six xenograft lines. The decrease in tumour bioenergetic status was due to a decrease in the (PCr + NTP beta)/total resonance ratio as well as an increase in the Pi/total resonance ratio. The decrease in the (PCr + NTP beta)/total resonance ratio was mainly a consequence of a decrease in the PCr/total resonance ratio for two lines and mainly a consequence of a decrease in the NTP beta/total resonance ratio for three lines. The magnitude of the decrease in the (PCr + NTP beta)/total resonance ratio and the magnitude of the decrease in tumour pH were correlated to the magnitude of the decrease in blood supply per viable tumour cell. Tumour pH decreased with decreasing tumour bioenergetic status, and the magnitude of this decrease was larger for the tumour lines showing a high than for those showing a low blood supply per viable tumour cell. No correlations across the tumour lines were found between tumour pH and tumour bioenergetic status or any other resonance ratio on the one hand and blood supply per viable tumour cell on the other. The differences in the 31P-NMR spectrum between the tumour lines were probably caused by differences in the intrinsic biochemical properties of the tumour cells rather than by the differences in blood supply per viable tumour cell. Biochemical properties of particular importance included rate of respiration, glycolytic capacity and tolerance to hypoxic stress. On the other hand, tumour bioenergetic status and tumour pH were correlated to blood supply per viable tumour cell within individual tumour lines. These observations suggest that 31P-NMR spectroscopy may be developed to be a clinically useful method for monitoring tumour blood supply and parameters related to tumour blood supply during and after physiological intervention and tumour treatment. However, clinically useful parameters for prediction of tumour treatment resistance caused by insufficient blood supply can probably not be derived from a single 31P-NMR spectrum since correlations across tumour lines were not detected; additional information is needed. PMID:8260356

Extending viability of Lactobacillus plantarum and Lactobacillus johnsonii by microencapsulation in alginate microgels.

PubMed

Tiani, Kendra A; Yeung, Timothy W; McClements, D Julian; Sela, David A

2018-03-01

To investigate whether microencapsulation of Lactobacillus in alginate microbeads will lead to increased longevity during refrigerated storage or simulated digestion. Microscopy was used to confirm that Lactobacillus plantarum ATCC BAA-793 and Lactobacillus johnsonii ATCC 33200 were immobilised within the microbeads and laser scattering analysis was used to determine the mean diameter of the microbeads. The number of viable cells were enumerated throughout refrigerated storage and simulated digestion experiments. Microencapsulation was shown to have differing effects on viability depending on the species, but led to extended viability during refrigerated storage and simulated digestion in L. johnsonii and L. plantarum respectively. Fermented functional foods contain microbes beneficial to human health. However, extended shelf storage and the harsh environment of the GI tract significantly reduces the number of viable microbes reaching the consumer. Microencapsulation allows beneficial microbes to reach the gut of the consumer in higher numbers, and thus confer greater health benefits.

40 CFR 503.32 - Pathogens.

Code of Federal Regulations, 2013 CFR

2013-07-01

... determine whether the sewage sludge contains viable helminth ova. (B) When the density of viable helminth ova in the sewage sludge prior to pathogen treatment is less than one per four grams of total solids (dry weight basis), the sewage sludge is Class A with respect to viable helminth ova until the next...

40 CFR 503.32 - Pathogens.

Code of Federal Regulations, 2014 CFR

2014-07-01

... determine whether the sewage sludge contains viable helminth ova. (B) When the density of viable helminth ova in the sewage sludge prior to pathogen treatment is less than one per four grams of total solids (dry weight basis), the sewage sludge is Class A with respect to viable helminth ova until the next...

Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

EPA Science Inventory

Aims: The goal of the study was to further develop an incubation-qPCR method for quantifying viable Ascaris eggs. The specific objectives were to characterize the detection limit and number of template copies per egg, determine the specificity of the method, and test the method w...

Effect of Induced Pluripotent Stem Cell Technology in Blood Banking

PubMed Central

Focosi, Daniele

2016-01-01

Summary Population aging has imposed cost-effective alternatives to blood donations. Artificial blood is still at the preliminary stages of development, and the need for viable cells seems unsurmountable. Because large numbers of viable cells must be promptly available for clinical use, stem cell technologies, expansion, and banking represent ideal tools to ensure a regular supply. Provided key donors can be identified, induced pluripotent stem cell (iPSC) technology could pave the way to a new era in transfusion medicine, just as it is already doing in many other fields of medicine. The present review summarizes the current state of research on iPSC technology in the field of blood banking, highlighting hurdles, and promises. Significance The aging population in Western countries is causing a progressive reduction of blood donors and a constant increase of blood recipients. Because blood is the main therapeutic option to treat acute hemorrhage, cost-effective alternatives to blood donations are being actively investigated. The enormous replication capability of induced pluripotent stem cells and their promising results in many other fields of medicine could be an apt solution to produce the large numbers of viable cells required in transfusion and usher in a new era in transfusion medicine. The present report describes the potentiality, technological hurdles, and promises of induced pluripotent stem cells to generate red blood cells by redifferentiation. PMID:26819256

Death of the Escherichia coli K-12 strain W3110 in soil and water.

PubMed Central

Bogosian, G; Sammons, L E; Morris, P J; O'Neil, J P; Heitkamp, M A; Weber, D B

1996-01-01

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death. PMID:8900002

Progress Towards the Vindication of Panspermia

NASA Astrophysics Data System (ADS)

Wickramasinghe, N. C.; Wainwright, M.; Narlikar, J. V.; Rajaratnam, P.; Harris, M. J.; Lloyd, D.

Theories of panspermia are rapidly coming into vogue, with the possibility of the transfer of viable bacterial cells from one planetary abode to another being generally accepted as inevitable. The panspermia models of Hoyle and Wickramasinghe require the transfer of viable bacterial cells from interstellar dust to comets and back into interplanetary and interstellar space. In such a cycle a viable fraction of as little as 10-18 at the inception of a newly formed comet/planet system suffices for cometary panspermia to dominate over competing processes for the origin and transfer of life. The well-attested survival attributes of microbes under extreme conditions, which have recently been discovered, gives credence to the panspermia hypothesis. The prediction of the theory that comets bring microbes onto the Earth at the present time is testable if aseptic collections of stratospheric air above the tropopause can be obtained. We describe a recent collection of this kind and report microbiological analysis that shows the existence of viable cells at 41km, falling to Earth at the rate of a few tonnes per day over the entire globe. Some of these cells have been cultured in the laboratory and found to include microorganisms that are not too different from related species on the Earth. This is in fact what the Hoyle-Wickramasinghe theory predicts. The weight of evidence goes against the more conservative explanation that organisms are being lofted to the high atmosphere from the ground.

Allogeneic Cardiospheres Delivered via Percutaneous Transendocardial Injection Increase Viable Myocardium, Decrease Scar Size, and Attenuate Cardiac Dilatation in Porcine Ischemic Cardiomyopathy

PubMed Central

Tseliou, Eleni; Cheng, Ke; Luthringer, Daniel J.; Ho, Chak-Sum; Takayama, Kentaro; Minamino, Naoto; Dawkins, James F.; Chowdhury, Supurna; Duong, Doan Trang; Seinfeld, Jeffrey; Middleton, Ryan C.; Dharmakumar, Rohan; Li, Debiao; Marbán, Linda; Makkar, Raj R.; Marbán, Eduardo

2014-01-01

Background Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy. Methods and Results We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled (“dose optimization”) study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled (“pivotal”) study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo. Conclusions Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration. PMID:25460005

Expression of adhesion molecules, chemokines and matrix metallo- proteinases (MMPs) in viable and degenerating stage of Taenia solium metacestode in swine neurocysticercosis.

PubMed

Singh, Satyendra K; Singh, Aloukick K; Prasad, Kashi N; Singh, Amrita; Singh, Avinash; Rai, Ravi P; Tripathi, Mukesh; Gupta, Rakesh K; Husain, Nuzhat

2015-11-30

Neurocysticercosis (NCC) is a parasitic infection of central nervous system (CNS). Expression of adhesion molecules, chemokines and matrix metalloproteinases (MMPs) were investigated on brain tissues surrounding viable (n=15) and degenerating cysticerci (n=15) of Taenia solium in swine by real-time RT-PCR and ELISA. Gelatin gel zymography was performed for MMPs activity. ICAM-1 (intercellular adhesion molecule-1), E-selectin, MIP-1α (macrophage inflammatory protein-1α), Eotaxin-1 and RANTES (regulated on activation, normal T cell expressed and secreted) were associated with degenerating cysticerci (cysts). However, VCAM-1 (vascular cell adhesion molecule-1), MCP-1 (monocyte chemotactic protein-1), MMP-2 and MMP-9 were associated with both viable and degenerating cysts. In conclusion, viable and degenerating cysticerci have different immune molecule profiles and role of these molecules in disease pathogenesis needs to be investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

Control of aerosol contaminants in indoor air: combining the particle concentration reduction with microbial inactivation.

PubMed

Grinshpun, Sergey A; Adhikari, Atin; Honda, Takeshi; Kim, Ki Youn; Toivola, Mika; Rao, K S Ramchander; Reponen, Tiina

2007-01-15

An indoor air purification technique, which combines unipolar ion emission and photocatalytic oxidation (promoted by a specially designed RCI cell), was investigated in two test chambers, 2.75 m3 and 24.3 m3, using nonbiological and biological challenge aerosols. The reduction in particle concentration was measured size selectively in real-time, and the Air Cleaning Factor and the Clean Air Delivery Rate (CADR) were determined. While testing with virions and bacteria, bioaerosol samples were collected and analyzed, and the microorganism survival rate was determined as a function of exposure time. We observed that the aerosol concentration decreased approximately 10 to approximately 100 times more rapidly when the purifier operated as compared to the natural decay. The data suggest that the tested portable unit operating in approximately 25 m3 non-ventilated room is capable to provide CADR-values more than twice as great than the conventional closed-loop HVAC system with a rating 8 filter. The particle removal occurred due to unipolar ion emission, while the inactivation of viable airborne microorganisms was associated with photocatalytic oxidation. Approximately 90% of initially viable MS2 viruses were inactivated resulting from 10 to 60 min exposure to the photocatalytic oxidation. Approximately 75% of viable B. subtilis spores were inactivated in 10 min, and about 90% or greater after 30 min. The biological and chemical mechanisms that led to the inactivation of stress-resistant airborne viruses and bacterial spores were reviewed.

Morphology and function of lacrimal gland acinar cells in primary culture.

PubMed

Hann, L E; Tatro, J B; Sullivan, D A

1989-01-01

The objectives of the current investigation were fourfold: (1) to establish an effective procedure for the isolation of acinar cells from the rat lacrimal gland; (2) to evaluate the functional capacity of freshly isolated cells; (3) to determine defined culture conditions which permit maintenance of viable, differentiated cells, as well as secretory component (SC) production, during long-term culture; and (4) to characterize the morphological features of cultured cells. Acinar cells were isolated by serial incubation of gland fragments in chelating and enzymatic solutions, followed by centrifugation through a Ficoll gradient. The yield of viable cells/gland appeared to be age-dependent: cell recovery was inversely proportional to the age of the animals. Immunofluorescence analysis of freshly isolated cells showed the presence of SC, the IgA antibody receptor, within isolated cells. In addition, experiments with a labeled analog (Nle4-D-Phe7-alpha MSH) of alpha-melanocyte-stimulating hormone (alpha-MSH) demonstrated specific binding sites on freshly isolated cells; alpha-MSH is a known modulator of acinar protein secretion. Maximum binding of the alpha-MSH analog occurred within 30 min, was dependent upon cell density and was reduced by coincubation with unlabeled alpha-MSH. To determine the culture requirements of acinar cells, cells were cultured on a variety of substrates (plastic or modified plastic [Primaria], coated with or without extracellular matrix [Matrigel]) in the presence or absence of various supplements and/or fetal calf serum (FCS) for 0.7 to 3.5 weeks. Cell attachment, function and long-term viability required an extracellular matrix. Moreover, in long term cultures (25 days), acinar cell attachment was enhanced by the inclusion of supplements to media containing 10% FCS. Replacement of serum with fibroblast growth factor, high-density lipoprotein and an increased concentration of epidermal growth factor resulted in a distinct "cobblestone" morphology characteristic of epithelial cell cultures. Electron microscopic analysis of cells cultured in supplemented serum-free media demonstrated extensive rough endoplasmic reticulum and Golgi, intermediate filaments and numerous secretory granules, as well as tight junctions and desmosomes. In addition to cell maintenance and attachment, acinar cell synthesis and/or secretion of SC was positively influenced by inclusion of supplements in the media. In summary, we have isolated lacrimal gland acinar cells, which express receptors for IgA antibodies and alpha-MSH. In addition, we have defined culture conditions which permit the long-term maintenance of SC-secreting acinar cells.

Biocatalytically active silCoat-composites entrapping viable Escherichia coli.

PubMed

Findeisen, A; Thum, O; Ansorge-Schumacher, M B

2014-02-01

Application of whole cells in industrial processes requires high catalytic activity, manageability, and viability under technical conditions, which can in principle be accomplished by appropriate immobilization. Here, we report the identification of carrier material allowing exceptionally efficient adsorptive binding of Escherichia coli whole cells hosting catalytically active carbonyl reductase from Candida parapsilosis (CPCR2). With the immobilizates, composite formation with both hydrophobic and hydrophilized silicone was achieved, yielding advanced silCoat-material and HYsilCoat-material, respectively. HYsilCoat-whole cells were viable preparations with a cell loading up to 400 mg(E. coli) · g(-1)(carrier) and considerably lower leaching than native immobilizates. SilCoat-whole cells performed particularly well in neat substrate exhibiting distinctly increased catalytic activity.

Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications.

PubMed

Polakovič, Milan; Švitel, Juraj; Bučko, Marek; Filip, Jaroslav; Neděla, Vilém; Ansorge-Schumacher, Marion B; Gemeiner, Peter

2017-05-01

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2017-03-01

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Naturally acquired microchimerism

PubMed Central

Eikmans, Michael; van Halteren, Astrid GS; van Besien, Koen; van Rood, Jon J; Drabbels, Jos JM; Claas, Frans HJ

2014-01-01

Microchimerism represents a condition where one individual harbors genetically distinct cell populations, and the chimeric population constitutes <1% of the total number of cells. The most common natural source of microchimerism is pregnancy. The reciprocal cell exchange between a mother and her child often leads to the stable engraftment of hematopoietic and non-hematopoietic stem cells in both parties. Interaction between cells from the mother and those from the child may result in maternal immune cells becoming sensitized to inherited paternal alloantigens of the child, which are not expressed by the mother herself. Vice versa, immune cells of the child may become sensitized toward the non-inherited maternal alloantigens of the mother. The extent of microchimerism, its anatomical location, and the sensitivity of the techniques used for detecting its presence collectively determine whether microchimerism can be detected in an individual. In this review, we focus on the clinical consequences of microchimerism in solid organ and hematopoietic stem cell transplantation, and propose concepts derived from data of epidemiologic studies. Next, we elaborate on the latest molecular methodology, including digital PCR, for determining in a reliable and sensitive way the extent of microchimerism. For the first time, tools have become available to isolate viable chimeric cells from a host background, so that the challenges of establishing the biologic mechanisms and function of these cells may finally be tackled. PMID:24762743

Three-species biofilm model onto plasma-treated titanium implant surface.

PubMed

Matos, Adaias O; Ricomini-Filho, Antônio P; Beline, Thamara; Ogawa, Erika S; Costa-Oliveira, Bárbara E; de Almeida, Amanda B; Nociti Junior, Francisco H; Rangel, Elidiane C; da Cruz, Nilson C; Sukotjo, Cortino; Mathew, Mathew T; Barão, Valentim A R

2017-04-01

In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and promising technology to treat bone-integrated dental implants as the new surfaces displayed improved mechanical and biological properties with no increase in biofilm proliferation. Copyright © 2017 Elsevier B.V. All rights reserved.

Post-Flight Microbial Analysis of Samples from the International Space Station Water Recovery System and Oxygen Generation System

NASA Technical Reports Server (NTRS)

Birmele, Michele N.

2011-01-01

The Regenerative, Environmental Control and Life Support System (ECLSS) on the International Space Station (ISS) includes the the Water Recovery System (WRS) and the Oxygen Generation System (OGS). The WRS consists of a Urine Processor Assembly (UPA) and Water Processor Assembly (WPA). This report describes microbial characterization of wastewater and surface samples collected from the WRS and OGS subsystems, returned to KSC, JSC, and MSFC on consecutive shuttle flights (STS-129 and STS-130) in 2009-10. STS-129 returned two filters that contained fluid samples from the WPA Waste Tank Orbital Recovery Unit (ORU), one from the waste tank and the other from the ISS humidity condensate. Direct count by microscopic enumeration revealed 8.38 x 104 cells per mL in the humidity condensate sample, but none of those cells were recoverable on solid agar media. In contrast, 3.32 x lOs cells per mL were measured from a surface swab of the WRS waste tank, including viable bacteria and fungi recovered after S12 days of incubation on solid agar media. Based on rDNA sequencing and phenotypic characterization, a fungus recovered from the filter was determined to be Lecythophora mutabilis. The bacterial isolate was identified by rDNA sequence data to be Methylobacterium radiotolerans. Additional UPA subsystem samples were returned on STS-130 for analysis. Both liquid and solid samples were collected from the Russian urine container (EDV), Distillation Assembly (DA) and Recycle Filter Tank Assembly (RFTA) for post-flight analysis. The bacterium Pseudomonas aeruginosa and fungus Chaetomium brasiliense were isolated from the EDV samples. No viable bacteria or fungi were recovered from RFTA brine samples (N= 6), but multiple samples (N = 11) from the DA and RFTA were found to contain fungal and bacterial cells. Many recovered cells have been identified to genus by rDNA sequencing and carbon source utilization profiling (BiOLOG Gen III). The presence of viable bacteria and fungi from WRS and OGS subsystems demonstrates the need for continued monitoring of ECLSS during future ISS operations and investigation of advanced antimicrobial controls.

Extended survival times of Mycoplasma gallisepticum and Mycoplasma synoviae on kanekalon synthetic hair fibres.

PubMed

Abolnik, Celia; Gouws, Johan

2014-01-01

The survival times of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) on washed and unwashed natural and synthetic kanekalon hair samples over a 5-d period were evaluated using the color changing unit method for comparison with results of previous studies conducted on natural hair. Regardless of whether synthetic or natural hair samples prewashed with a disinfectant shampoo were spiked with Mg or Ms, all viable organisms rapidly dropped below a count of 1 × 10(1)/mL of culture. Unwashed natural hair seeded with a titer of approximately 1 × 10(6)/mL of viable Mg or Ms decreased to 6 × 10(5)/mL and 6 × 10(3)/mL, respectively, by 4 h postseeding, but no viable Mg or Ms were detected on natural hair from 8 h onwards. By contrast, the titers of Mg and Ms on synthetic hair did not decline from the initial 1 × 10(6)/mL seed dose up to 96 h postseeding, and, in fact, viable Mg and Ms was still detectable at 9 d postinfection. Application of a real-time quantitative single-tube duplex PCR assay confirmed that no proliferation of Mg or Ms had occurred on the synthetic hair samples, the cells simply remained viable. The unexpected finding that Mg and Ms survive for extended periods on synthetic kanekalon hair fibers raises the question of whether attachment to a surface is a prerequisite for the survival and persistence of Mg and Ms in the extra-host environment. Future studies should be aimed at determining whether other synthetic hair types or indeed other types of plastics commonly found in the poultry house offer similar survival advantages to mycoplasmas.

No difference in mitochondrial distribution is observed in human oocytes after cryopreservation.

PubMed

Stimpfel, Martin; Vrtacnik-Bokal, Eda; Virant-Klun, Irma

2017-08-01

The primary aim of this study was to determine if any difference in mitochondrial distribution can be observed between fresh and cryopreserved (slow-frozen/thawed and vitrified/warmed) oocytes when oocytes are stained with Mitotracker Red CMXRos and observed under a conventional fluorescent microscope. Additionally, the influence of cryopreservation procedure on the viable rates of oocytes at different maturation stages was evaluated. The germinal vesicle (GV) and MII oocytes were cryopreserved with slow-freezing and vitrification. After thawing/warming, oocytes were stained using Mitotracker Red CMXRos and observed under a conventional fluorescent microscope. Mitotracker staining revealed that in GV oocytes the pattern of mitochondrial distribution appeared as aggregated clusters around the whole oocyte. In mature MII oocytes, three different patterns of mitochondrial distribution were observed; a smooth pattern around the polar body with aggregated clusters at the opposite side of the polar body, a smooth pattern throughout the whole cell, and aggregated clusters as can be seen in GV oocytes. There were no significant differences in the observed patterns between fresh, vitrified/warmed and frozen/thawed oocytes. When comparing the viable rates of oocytes after two different cryopreservation procedures, the results showed no significant differences, although the trend of viable MII oocytes tends to be higher after vitrification/warming and for viable GV oocytes it tends to be higher after slow-freezing/thawing. Mitotracker Red CMXRos staining of mitochondria in oocytes did not reveal differences in mitochondrial distribution between fresh and cryopreserved oocytes at different maturity stages. Additionally, no difference was observed in the viable rates of GV and MII oocytes after slow-freezing/thawing and vitrification/warming.

Comparison of coconut water, propolis, HBSS, and milk on PDL cell survival.

PubMed

Gopikrishna, Velayutham; Baweja, Parvinder Singh; Venkateshbabu, Nagendrababu; Thomas, Toby; Kandaswamy, Deivanayagam

2008-05-01

Coconut water is biologically pure and sterile, with a rich presence of amino acids, proteins, vitamins, and minerals. The purpose of this study was to use a collagenase-dispase assay to investigate the potential of a new storage medium, coconut water, in comparison with propolis, Hank's balanced salt solution (HBSS), and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Seventy freshly extracted human teeth were divided into 4 experimental groups and 2 control groups. The positive and negative controls corresponded to 0-minute and 8-hour dry times, respectively. The experimental teeth were stored dry for 30 minutes and then immersed in 1 of the 4 media (coconut water, propolis, HBSS, and milk). The teeth were then treated with dispase grade II and collagenase for 30 minutes. The number of viable PDL cells was counted with a hemocytometer and analyzed. Statistical analysis showed that coconut water kept significantly more PDL cells viable compared with propolis, HBSS, or milk. Coconut water can be used as a superior transport medium for avulsed teeth.

Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities.

PubMed

Checinska, Aleksandra; Probst, Alexander J; Vaishampayan, Parag; White, James R; Kumar, Deepika; Stepanov, Victor G; Fox, George E; Nilsson, Henrik R; Pierson, Duane L; Perry, Jay; Venkateswaran, Kasthuri

2015-10-27

The International Space Station (ISS) is a unique built environment due to the effects of microgravity, space radiation, elevated carbon dioxide levels, and especially continuous human habitation. Understanding the composition of the ISS microbial community will facilitate further development of safety and maintenance practices. The primary goal of this study was to characterize the viable microbiome of the ISS-built environment. A second objective was to determine if the built environments of Earth-based cleanrooms associated with space exploration are an appropriate model of the ISS environment. Samples collected from the ISS and two cleanrooms at the Jet Propulsion Laboratory (JPL, Pasadena, CA) were analyzed by traditional cultivation, adenosine triphosphate (ATP), and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. The 16S rRNA gene Illumina iTag sequencing was used to elucidate microbial diversity and explore differences between ISS and cleanroom microbiomes. Statistical analyses showed that members of the phyla Actinobacteria, Firmicutes, and Proteobacteria were dominant in the samples examined but varied in abundance. Actinobacteria were predominant in the ISS samples whereas Proteobacteria, least abundant in the ISS, dominated in the cleanroom samples. The viable bacterial populations seen by PMA treatment were greatly decreased. However, the treatment did not appear to have an effect on the bacterial composition (diversity) associated with each sampling site. The results of this study provide strong evidence that specific human skin-associated microorganisms make a substantial contribution to the ISS microbiome, which is not the case in Earth-based cleanrooms. For example, Corynebacterium and Propionibacterium (Actinobacteria) but not Staphylococcus (Firmicutes) species are dominant on the ISS in terms of viable and total bacterial community composition. The results obtained will facilitate future studies to determine how stable the ISS environment is over time. The present results also demonstrate the value of measuring viable cell diversity and population size at any sampling site. This information can be used to identify sites that can be targeted for more stringent cleaning. Finally, the results will allow comparisons with other built sites and facilitate future improvements on the ISS that will ensure astronaut health.

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

PubMed Central

Chatelus, C; Carrier, P; Saignes, P; Libert, M F; Berlier, Y; Lespinat, P A; Fauque, G; Legall, J

1987-01-01

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena. PMID:3310883

Evaluation of the adequacy of the consume-by date of rice balls sold at convenience stores.

PubMed

Sato, Jun; Yokokawa, Kana

2014-01-01

The adequacy of the consume-by date was validated for rice balls sold at convenience stores (CVSs),taking into account the process of distribution. The results indicated that the increase in the viable cell counts differed significantly depending on the type of rice balls and the storage temperature. At 19 h after delivery, Staphylococcus spp. were detected in 4 samples (26.7%) of the Tunamayo samples of Company A and in the majority of the Plum samples of Company B. Results showed there was a strong correlation between the elapsed time after delivery and the viable cell counts for all samples except for the Plum samples of Company B. The regression equations varied for the different types of rice balls and the different storage temperatures. Using the obtained regression equations and assuming a safety factor of 0.7, the appropriate consume-by date was determined to be 11 h for the Tunamayo and 38 h for the Plum of Company A, and 21 h for the Tunamayo of Company B. Among 14 strains of isolated Gram-negative bacteria, 11 strains (78.6%) belonged to the genus Serratia.

Antibacterial activity of antibacterial cutting boards in household kitchens.

PubMed

Kounosu, Masayuki; Kaneko, Seiichi

2007-12-01

We examined antibacterial cutting boards with antibacterial activity values of either "2" or "4" in compliance with the JIS Z 2801 standard, and compared their findings with those of cutting boards with no antibacterial activity. These cutting boards were used in ten different households, and we measured changes in the viable cell counts of several types of bacteria with the drop plate method. We also identified the detected bacterial flora and measured the minimum antimicrobial concentrations of several commonly used antibacterial agents against the kinds of bacteria identified to determine the expected antibacterial activity of the respective agents. Cutting boards with activity values of both "2" and "4" proved to be antibacterial in actual use, although no correlation between the viable cell counts and the antibacterial activity values was observed. In the kitchen environment, large quantities of Pseudomonas, Flavobacterium, Micrococcus, and Bacillus were detected, and it was confirmed that common antibacterial agents used in many antibacterial products are effective against these bacterial species. In addition, we measured the minimum antimicrobial concentrations of the agents against lactobacillus, a typical good bacterium, and discovered that this bacterium is less sensitive to these antibacterial agents compared to more common bacteria.

Toward investigating changes in cell mechanoelastic properties in response to nanosecond pulsed electric fields

NASA Astrophysics Data System (ADS)

Coker, Zachary; Troyanova-Wood, Maria; Traverso, Andrew; Meng, Zhaokai; Ballmann, Charles; Petrov, Georgi; Ibey, Bennett L.; Yakovlev, Vladislav

2017-02-01

Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique microscopy system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 50kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.

Label-free Rapid Viable Enrichment of Circulating Tumor Cell by Photosensitive Polymer-based Microfilter Device.

PubMed

Kang, Yoon-Tae; Doh, Il; Byun, Jiyoung; Chang, Hee Jin; Cho, Young-Ho

2017-01-01

We present a clinical device for simple, rapid, and viable isolation of circulating tumor cells (CTCs) from cancer patient bloods. In spite of the clinical importance of CTCs, the lack of easy and non-biased isolation methods is a big hurdle for implementing CTC into clinical use. The present device made of photosensitive polymer was designed to attach to conventional syringe to isolate the CTCs at minimal resources. Its unique tapered-slits on the filter are capable not only to isolate the cell based on their size and deformability, but also to increase sample flow rate, thus achieving label-free rapid viable CTC isolation. We verified our device performance using 9 different types of cancer cells at the cell concentration from 5 to 100cells/ml, showing that the device capture 77.7% of the CTCs while maintaining their viability of 80.6%. We extended our study using the 18 blood samples from lung, colorectal, pancreatic and renal cancer patients and captured 1-172 CTCs or clustered CTCs by immunofluorescent or immunohistochemical staining. The captured CTCs were also molecularly assayed by RT-PCR with three cancer-associated genes (CK19, EpCAM, and MUC1). Those comprehensive studies proved to use our device for cancer study, thereby inaugurating further in-depth CTC-based clinical researches.

The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus.

PubMed

Green, Charlotte J; Charlton, Catriona A; Wang, Lai-Mun; Silva, Michael; Morten, Karl J; Hodson, Leanne

2017-12-01

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of "healthy" resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

PubMed

Stein, G H

1985-10-01

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

Tumour T1 changes in vivo are highly predictive of response to chemotherapy and reflect the number of viable tumour cells – a preclinical MR study in mice

PubMed Central

2014-01-01

Background Effective chemotherapy rapidly reduces the spin–lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus. Methods Mice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo. Results Everolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the %Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and %Ki67+. In B16/BL6 tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97). Conclusion These studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic. PMID:24528602

In situ Raman spectroscopy for simultaneous monitoring of multiple process parameters in mammalian cell culture bioreactors.

PubMed

Whelan, Jessica; Craven, Stephen; Glennon, Brian

2012-01-01

In this study, the application of Raman spectroscopy to the simultaneous quantitative determination of glucose, glutamine, lactate, ammonia, glutamate, total cell density (TCD), and viable cell density (VCD) in a CHO fed-batch process was demonstrated in situ in 3 L and 15 L bioreactors. Spectral preprocessing and partial least squares (PLS) regression were used to correlate spectral data with off-line reference data. Separate PLS calibration models were developed for each analyte at the 3 L laboratory bioreactor scale before assessing its transferability to the same bioprocess conducted at the 15 L pilot scale. PLS calibration models were successfully developed for all analytes bar VCD and transferred to the 15 L scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Clinical Application of Stem Cells in the Cardiovascular System

NASA Astrophysics Data System (ADS)

Stamm, Christof; Klose, Kristin; Choi, Yeong-Hoon

Regenerative medicine encompasses "tissue engineering" - the in vitro fabrication of tissues and/or organs using scaffold material and viable cells - and "cell therapy" - the transplantation or manipulation of cells in diseased tissue in vivo. In the cardiovascular system, tissue engineering strategies are being pursued for the development of viable replacement blood vessels, heart valves, patch material, cardiac pacemakers and contractile myocardium. Anecdotal clinical applications of such vessels, valves and patches have been described, but information on systematic studies of the performance of such implants is not available, yet. Cell therapy for cardiovascular regeneration, however, has been performed in large series of patients, and numerous clinical studies have produced sometimes conflicting results. The purpose of this chapter is to summarize the clinical experience with cell therapy for diseases of the cardiovascular system, and to analyse possible factors that may influence its outcome.

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

PubMed

Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María Del Carmen; Alaminos, Miguel

2012-01-01

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Demonstrating a Total Transit Solution for Fuel Cell Electric Buses in Boston

DOT National Transportation Integrated Search

2017-05-01

The Federal Transit Administrations (FTA) National Fuel Cell Bus Program (NFCBP) focuses on developing commercially viable fuel cell bus technologies. Nuvera is leading the Massachusetts Fuel Cell Bus project to demonstrate a complete transit solu...

Functional Reconstruction of Tracheal Defects by Protein-Loaded, Cell-Seeded, Fibrous Constructs in Rabbits

PubMed Central

Ott, Lindsey M.; Vu, Cindy H.; Farris, Ashley L.; Fox, Katrina D.; Galbraith, Richard A.; Weiss, Mark L.; Weatherly, Robert A.

2015-01-01

Tracheal stenosis is a life-threatening disease and current treatments include surgical reconstruction with autologous rib cartilage and the highly complex slide tracheoplasty surgical technique. We propose using a sustainable implant, composed of a tunable, fibrous scaffold with encapsulated chondrogenic growth factor (transforming growth factor-beta3 [TGF-β3]) or seeded allogeneic rabbit bone marrow mesenchymal stromal cells (BMSCs). In vivo functionality of these constructs was determined by implanting them in induced tracheal defects in rabbits for 6 or 12 weeks. The scaffolds maintained functional airways in a majority of the cases, with the BMSC-seeded group having an improved survival rate and the Scaffold-only group having a higher occurrence of more patent airways as determined by microcomputed tomography. The BMSC group had a greater accumulation of inflammatory cells over the graft, while also exhibiting normal epithelium, subepithelium, and cartilage formation. Overall, it was concluded that a simple, acellular scaffold is a viable option for tracheal tissue engineering, with the intraoperative addition of cells being an optional variation to the scaffolds. PMID:26094554

The use of flow cytometry to accurately ascertain total and viable counts of Lactobacillus rhamnosus in chocolate.

PubMed

Raymond, Yves; Champagne, Claude P

2015-04-01

The goals of this study were to evaluate the precision and accuracy of flow cytometry (FC) methodologies in the evaluation of populations of probiotic bacteria (Lactobacillus rhamnosus R0011) in two commercial dried forms, and ascertain the challenges in enumerating them in a chocolate matrix. FC analyses of total (FC(T)) and viable (FC(V)) counts in liquid or dried cultures were almost two times more precise (reproducible) than traditional direct microscopic counts (DCM) or colony forming units (CFU). With FC, it was possible to ascertain low levels of dead cells (FC(D)) in fresh cultures, which is not possible with traditional CFU and DMC methodologies. There was no interference of chocolate solids on FC counts of probiotics when inoculation was above 10(7) bacteria per g. Addition of probiotics in chocolate at 40 °C resulted in a 37% loss in viable cells. Blending of the probiotic powder into chocolate was not uniform which raised a concern that the precision of viable counts could suffer. FCT data can serve to identify the correct inoculation level of a sample, and viable counts (FCV or CFU) can subsequently be better interpreted. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Alpha-2 agonist attenuates ischemic injury in spinal cord neurons.

PubMed

Freeman, Kirsten A; Puskas, Ferenc; Bell, Marshall T; Mares, Joshua M; Foley, Lisa S; Weyant, Michael J; Cleveland, Joseph C; Fullerton, David A; Meng, Xianzhong; Herson, Paco S; Reece, T Brett

2015-05-01

Paraplegia secondary to spinal cord ischemia-reperfusion injury remains a devastating complication of thoracoabdominal aortic intervention. The complex interactions between injured neurons and activated leukocytes have limited the understanding of neuron-specific injury. We hypothesize that spinal cord neuron cell cultures subjected to oxygen-glucose deprivation (OGD) would simulate ischemia-reperfusion injury, which could be attenuated by specific alpha-2a agonism in an Akt-dependent fashion. Spinal cords from perinatal mice were harvested, and neurons cultured in vitro for 7-10 d. Cells were pretreated with 1 μM dexmedetomidine (Dex) and subjected to OGD in an anoxic chamber. Viability was determined by MTT assay. Deoxyuridine-triphosphate nick-end labeling staining and lactate dehydrogenase (LDH) assay were used for apoptosis and necrosis identification, respectively. Western blot was used for protein analysis. Vehicle control cells were only 59% viable after 1 h of OGD. Pretreatment with Dex significantly preserves neuronal viability with 88% viable (P < 0.05). Dex significantly decreased apoptotic cells compared with that of vehicle control cells by 50% (P < 0.05). Necrosis was not significantly different between treatment groups. Mechanistically, Dex treatment significantly increased phosphorylated Akt (P < 0.05), but protective effects of Dex were eliminated by an alpha-2a antagonist or Akt inhibitor (P < 0.05). Using a novel spinal cord neuron cell culture, OGD mimics neuronal metabolic derangement responsible for paraplegia after aortic surgery. Dex preserves neuronal viability and decreases apoptosis in an Akt-dependent fashion. Dex demonstrates clinical promise for reducing the risk of paraplegia after high-risk aortic surgery. Copyright © 2015 Elsevier Inc. All rights reserved.

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

PubMed Central

Brayton, P R; Tamplin, M L; Huq, A; Colwell, R R

1987-01-01

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality. PMID:3324967

In vitro evaluation of the viability of vaginal cells (VK2/E6E7) and probiotic Lactobacillus species in lemon juice.

PubMed

Anukam, Kingsley C; Reid, Gregor

2009-03-01

Women, especially in developing countries, most often bear the brunt of HIV infections. The continued lack of viable vaccines and microbicides has made some women resort to using natural products such as lemon or lime juice to avoid infection. Few in vitro studies have been done on the effect of lemon juice on vaginal cells and lactobacilli that constitute the major microbiota in healthy women. The objective of the present study was to evaluate in vitro the effect of lemon juice on the viability of vaginal cells (VK2/E6E7) and vaginal Lactobacillus species. Vaginal cells were exposed to different concentrations (0-30%) of lemon juice at pH 2.3 and 4.5 for 10 min. Viability was determined by staining the cells with propidium iodide and analysing them by flow cytometry. Lactobacillus organisms were dispensed into microplates with vaginally defined medium + peptone (VDMP) containing different concentrations of lemon juice ranging from 0 to 100%. Lemon juice at pH 2.3 had a significant (P = 0.03) toxic effect on the vaginal cell line used. At 30% concentration, the vaginal cells were practically non-viable, typified by a 95% loss of viability, whereas at pH 4.5 there was only 5% cell loss. Lemon juice had varying growth inhibitory effects on the Lactobacillus species tested. At pH 4.5 and using 10-30% lemon juice, there was a stimulatory growth effect on certain Lactobacillus species. Lemon juice (20-30%) at pH 2.3 was highly toxic to VK2/E6E7 cells, and at pH 4.5 there was no significant effect on the viability of the cells within 10 min. Lemon juice above 10% at pH 2.3 was found to be detrimental to the growth of vaginal lactobacilli. Although lemon juice may be useful in other applications, its use in the vaginal region should be discouraged.

Survival of Deinococcus geothermalis in Biofilms under Desiccation and Simulated Space and Martian Conditions

NASA Astrophysics Data System (ADS)

Frösler, Jan; Panitz, Corinna; Wingender, Jost; Flemming, Hans-Curt; Rettberg, Petra

2017-05-01

Biofilm formation represents a successful survival strategy for bacteria. In biofilms, cells are embedded in a matrix of extracellular polymeric substances (EPS). As they are often more stress-tolerant than single cells, biofilm cells might survive the conditions present in space and on Mars. To investigate this topic, the bacterium Deinococcus geothermalis was chosen as a model organism due to its tolerance toward desiccation and radiation. Biofilms cultivated on membranes and, for comparison, planktonically grown cells deposited on membranes were air-dried and exposed to individual stressors that included prolonged desiccation, extreme temperatures, vacuum, simulated martian atmosphere, and UV irradiation, and they were exposed to combinations of stressors that simulate space (desiccation + vacuum + UV) or martian (desiccation + Mars atmosphere + UV) conditions. The effect of sulfatic Mars regolith simulant on cell viability during stress was investigated separately. The EPS produced by the biofilm cells contained mainly polysaccharides and proteins. To detect viable but nonculturable (VBNC) cells, cultivation-independent viability indicators (membrane integrity, ATP, 16S rRNA) were determined in addition to colony counts. Desiccation for 2 months resulted in a decrease of culturability with minor changes of membrane integrity in biofilm cells and major loss of membrane integrity in planktonic bacteria. Temperatures between -25°C and +60°C, vacuum, and Mars atmosphere affected neither culturability nor membrane integrity in both phenotypes. Monochromatic (254 nm; ≥1 kJ m-2) and polychromatic (200-400 nm; >5.5 MJ m-2 for planktonic cells and >270 MJ m-2 for biofilms) UV irradiation significantly reduced the culturability of D. geothermalis but did not affect cultivation-independent viability markers, indicating the induction of a VBNC state in UV-irradiated cells. In conclusion, a substantial proportion of the D. geothermalis population remained viable under all stress conditions tested, and in most cases the biofilm form proved advantageous for surviving space and Mars-like conditions.

Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells.

PubMed

Costa, Patricia Marçal da; Ferreira, Paulo Michel Pinheiro; Bolzani, Vanderlan da Silva; Furlan, Maysa; de Freitas Formenton Macedo Dos Santos, Vânia Aparecida; Corsino, Joaquim; de Moraes, Manoel Odorico; Costa-Lotufo, Letícia Veras; Montenegro, Raquel Carvalho; Pessoa, Cláudia

2008-06-01

Pristimerin has been shown to be cytotoxic to several cancer cell lines. In the present work, the cytotoxicity of pristimerin was evaluated in human tumor cell lines and in human peripheral blood mononuclear cells (PBMC). This work also examined the effects of pristimerin (0.4; 0.8 and 1.7 microM) in HL-60 cells, after 6, 12 and 24h of exposure. Pristimerin reduced the number of viable cells and increased number of non-viable cells in a concentration-dependent manner by tripan blue test showing morphological changes consistent with apoptosis. Nevertheless, pristimerin was not selective to cancer cells, since it inhibited PBMC proliferation with an IC50 of 0.88 microM. DNA synthesis inhibition assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation in HL-60 cells was 70% and 83% for the concentrations of 0.4 and 0.8 microM, respectively. Pristimerin (10 and 20 microM) was not able to inhibit topoisomerase I. In AO/EB (acridine orange/ethidium bromide) staining, all tested concentrations reduced the number of HL-60 viable cells, with the occurrence of necrosis and apoptosis in a concentration-dependent manner, results in agreement with trypan blue exclusion findings. The analysis of membrane integrity and internucleosomal DNA fragmentation by flow cytometry in the presence of pristimerin indicated that treated cells underwent apoptosis. The present data point to the importance of pristimerin as representative of an emerging class of potential anticancer chemicals, exhibiting an antiproliferative effect by inhibiting DNA synthesis and triggering apoptosis.

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods.

PubMed

Gyawali, P; Ahmed, W; Sidhu, J P S; Nery, S V; Clements, A C; Traub, R; McCarthy, J S; Llewellyn, S; Jagals, P; Toze, S

2016-09-01

In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79-80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 10(5) ± 6.4 × 10(5) and 6.3 × 10(5) ± 4.7 × 10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 10(5) ± 1.5 × 10(4) (determined by qPCR) compared to 4.9 × 10(4) ± 3.7 × 10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

PubMed Central

Lowder, M.; Unge, A.; Maraha, N.; Jansson, J. K.; Swiggett, J.; Oliver, J. D.

2000-01-01

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost. PMID:10919764

Agouti signaling protein stimulates cell division in "viable yellow" (A vy/a) mouse liver

USDA-ARS?s Scientific Manuscript database

Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti Avy/- mice (1); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in Avy/a or a...

In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells.

PubMed

Abengózar-Vela, Antonio; Arroyo, Cristina; Reinoso, Roberto; Enríquez-de-Salamanca, Amalia; Corell, Alfredo; González-García, María Jesús

2015-01-01

To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlue(®) assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H(2)DCF-DA. Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals.

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates.

PubMed

Dowd, Jason E; Jubb, Anthea; Kwok, K Ezra; Piret, James M

2003-05-01

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of approximately 5 x 10(6) cells mL(-1). Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell(-1) day(-1). Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized ( approximately 40 mg L(-1)) at 0.2 nL cell(-1) day(-1). The volumetric protein productivity ( approximately 60 mg L(-1) day(-1) was maximized above 0.3 nL cell(-1) day(-1). The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.

Tumor Infiltration in Enhancing and Non-Enhancing Parts of Glioblastoma: A Correlation with Histopathology.

PubMed

Eidel, Oliver; Burth, Sina; Neumann, Jan-Oliver; Kieslich, Pascal J; Sahm, Felix; Jungk, Christine; Kickingereder, Philipp; Bickelhaupt, Sebastian; Mundiyanapurath, Sibu; Bäumer, Philipp; Wick, Wolfgang; Schlemmer, Heinz-Peter; Kiening, Karl; Unterberg, Andreas; Bendszus, Martin; Radbruch, Alexander

2017-01-01

To correlate histopathologic findings from biopsy specimens with their corresponding location within enhancing areas, non-enhancing areas and necrotic areas on contrast enhanced T1-weighted MRI scans (cT1). In 37 patients with newly diagnosed glioblastoma who underwent stereotactic biopsy, we obtained a correlation of 561 1mm3 biopsy specimens with their corresponding position on the intraoperative cT1 image at 1.5 Tesla. Biopsy points were categorized as enhancing (CE), non-enhancing (NE) or necrotic (NEC) on cT1 and tissue samples were categorized as "viable tumor cells", "blood" or "necrotic tissue (with or without cellular component)". Cell counting was done semi-automatically. NE had the highest content of tissue categorized as viable tumor cells (89% vs. 60% in CE and 30% NEC, respectively). Besides, the average cell density for NE (3764 ± 2893 cells/mm2) was comparable to CE (3506 ± 3116 cells/mm2), while NEC had a lower cell density with 2713 ± 3239 cells/mm2. If necrotic parts and bleeds were excluded, cell density in biopsies categorized as "viable tumor tissue" decreased from the center of the tumor (NEC, 5804 ± 3480 cells/mm2) to CE (4495 ± 3209 cells/mm2) and NE (4130 ± 2817 cells/mm2). The appearance of a glioblastoma on a cT1 image (circular enhancement, central necrosis, peritumoral edema) does not correspond to its diffuse histopathological composition. Cell density is elevated in both CE and NE parts. Hence, our study suggests that NE contains considerable amounts of infiltrative tumor with a high cellularity which might be considered in resection planning.

Membrane currents in the oocyte of the toad Bufo arenarum.

PubMed

Kotsias, Basilio A; Damiano, Alicia E; Godoy, Sebastian; Assef, Yanina; Ibarra, Cristina; Cantiello, Horacio F

2002-03-01

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes. Copyright 2002 Wiley-Liss, Inc.

Novel paths towards neural cellular products for neurological disorders.

PubMed

Daadi, Marcel M

2011-11-01

The prospect of using neural cells derived from stem cells or from reprogrammed adult somatic cells provides a unique opportunity in cell therapy and drug discovery for developing novel strategies for brain repair. Cell-based therapeutic approaches for treating CNS afflictions caused by disease or injury aim to promote structural repair of the injured or diseased neural tissue, an outcome currently not achieved by drug therapy. Preclinical research in animal models of various diseases or injuries report that grafts of neural cells enhance endogenous repair, provide neurotrophic support to neurons undergoing degeneration and replace lost neural cells. In recent years, the sources of neural cells for treating neurological disorders have been rapidly expanding and in addition to offering therapeutic potential, neural cell products hold promise for disease modeling and drug discovery use. Specific neural cell types have been derived from adult or fetal brain, from human embryonic stem cells, from induced pluripotent stem cells and directly transdifferentiated from adult somatic cells, such as skin cells. It is yet to be determined if the latter approach will evolve into a paradigm shift in the fields of stem cell research and regenerative medicine. These multiple sources of neural cells cover a wide spectrum of safety that needs to be balanced with efficacy to determine the viability of the cellular product. In this article, we will review novel sources of neural cells and discuss current obstacles to developing them into viable cellular products for treating neurological disorders.

Connecticut Nutmeg Fuel Cell Bus Project : Demonstrating Advanced-Design Hybrid Fuel Cell Buses in Connecticut

DOT National Transportation Integrated Search

2011-07-01

The Federal Transit Administrations (FTA) National Fuel Cell Bus Program (NFCBP) focuses on developing commercially viable fuel cell bus technologies. The Northeast Advanced Vehicle Consortium (NAVC) is one of three non-profit consortia chosen to ...

Within-population variation in ejaculate characteristics in a prolonged breeder, Peron's tree frog, Litoria peronii

NASA Astrophysics Data System (ADS)

Sherman, Craig D. H.; Uller, Tobias; Wapstra, Erik; Olsson, Mats

2008-11-01

Sperm number is often a good predictor of success in sperm competition; however, it has become increasingly clear that, for some species, variation in probability of paternity cannot be explained by sperm number alone. Intraspecific variation in ejaculate characteristics, such as the number of viable sperm and sperm longevity, may play an equally important role in determining fertilization success. Here, we assess variation among ejaculates in three factors that may contribute to fertilization success (number of sperm per ejaculate, viability, and longevity), in a population of Peron’s tree frog ( Litoria peronii). We detected large variation among males in the number of sperm per ejaculate and the proportion of viable sperm within ejaculates, which could not be explained by variation in either male size or body condition. However, the proportion of viable sperm released by males increased over the season. Finally, we assessed sperm longevity (proportion viable sperm determined using a dual-fluorochrome vital dye) at two different temperatures. At 23°C, on average, 75% of sperm remained viable after 2 h, but there were significant differences amongst males with the percentage of viable sperm ranging from 43% to 95%. For sperm incubated at 4°C, ejaculates varied fivefold in sperm longevity with some males having 50% viable sperm after 5 days. Our data suggest that ejaculate characteristics (sperm number, viability, and longevity) vary widely in Peron’s tree frog and may therefore play an important role in determining siring success both in the presence and absence of sperm competition. We discuss the results in relation to selection on ejaculate traits via natural and sexual selection in this and other amphibians.

Microdevice for the isolation and enumeration of cancer cells from blood.

PubMed

Tan, Swee Jin; Yobas, Levent; Lee, Gabriel Yew Hoe; Ong, Choon Nam; Lim, Chwee Teck

2009-08-01

Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between the disease development and number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. Here, we present a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is at least 80% for tests performed on breast and colon cancer cells. Viable isolated cells are also obtained which may give further insights to the understanding of the metastatic process. Contrasting with conventional biochemical techniques, the uniqueness of this microdevice lies in the mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting.

Cutting Edge: Protection by Antiviral Memory CD8 T Cells Requires Rapidly Produced Antigen in Large Amounts.

PubMed

Remakus, Sanda; Ma, Xueying; Tang, Lingjuan; Xu, Ren-Huan; Knudson, Cory; Melo-Silva, Carolina R; Rubio, Daniel; Kuo, Yin-Ming; Andrews, Andrew; Sigal, Luis J

2018-05-15

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design. Copyright © 2018 by The American Association of Immunologists, Inc.

Nanosphere-based one-step strategy for efficient and nondestructive detection of circulating tumor cells.

PubMed

Wu, Ling-Ling; Wen, Cong-Ying; Hu, Jiao; Tang, Man; Qi, Chu-Bo; Li, Na; Liu, Cui; Chen, Lan; Pang, Dai-Wen; Zhang, Zhi-Ling

2017-08-15

Detecting viable circulating tumor cells (CTCs) without disruption to their functions for in vitro culture and functional study could unravel the biology of metastasis and promote the development of personalized anti-tumor therapies. However, existing CTC detection approaches commonly include CTC isolation and subsequent destructive identification, which damages CTC viability and functions and generates substantial CTC loss. To address the challenge of efficiently detecting viable CTCs for functional study, we develop a nanosphere-based cell-friendly one-step strategy. Immunonanospheres with prominent magnetic/fluorescence properties and extraordinary stability in complex matrices enable simultaneous efficient magnetic capture and specific fluorescence labeling of tumor cells directly in whole blood. The collected cells with fluorescent tags can be reliably identified, free of the tedious and destructive manipulations from conventional CTC identification. Hence, as few as 5 tumor cells in ca. 1mL of whole blood can be efficiently detected via only 20min incubation, and this strategy also shows good reproducibility with the relative standard deviation (RSD) of 8.7%. Moreover, due to the time-saving and gentle processing and the minimum disruption of immunonanospheres to cells, 93.8±0.1% of detected tumor cells retain cell viability and proliferation ability with negligible changes of cell functions, capacitating functional study on cell migration, invasion and glucose uptake. Additionally, this strategy exhibits successful CTC detection in 10/10 peripheral blood samples of cancer patients. Therefore, this nanosphere-based cell-friendly one-step strategy enables viable CTC detection and further functional analyses, which will help to unravel tumor metastasis and guide treatment selection. Copyright © 2017 Elsevier B.V. All rights reserved.

[Initial stages of steel biocorrosion].

PubMed

Zhigletsova, S K; Rodin, V B; Kobelev, V S; Aleksandrova, N V; Rasulova, G E; Kholodenko, V P

2000-01-01

Initial stages of corrosion of mild steel induced by Klebsiela rhinoscleromatis BO2 were studied in various media. The effect of the microorganism was detected 8-10 h after inoculation. The number of viable cells were virtually unchanged within one month in all media, but the corrosive activity of the strain decreased. The corrosive activity of microorganisms can be determined by spectrophotometry even only after incubation for 24 h. At a low level of organic substrate, even strong colonization with microorganisms does not inevitably result in a significant damage to metals.

Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

PubMed

Cook, James L; Stannard, James P; Stoker, Aaron M; Bozynski, Chantelle C; Kuroki, Keiichi; Cook, Cristi R; Pfeiffer, Ferris M

2016-05-01

Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Controlled laboratory study. After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable chondrocytes with infiltration of fibroblasts from the surrounding recipient tissues. In situ polymerase chain reaction (fluorescence in situ hybridization [FISH]) assays were performed in an attempt to distinguish donor (male) cells from recipient (female) cells. Unfortunately, this technique was exceptionally difficult to perform on intact articular cartilage sections, and consistent, repeatable data could not be obtained from this testing. However, the data did support histologic and live-dead data, which strongly suggested that successful grafts retained viable donor (male) chondrocytes and unsuccessful grafts degraded and were replaced by fibrous tissue populated with recipient (female) fibroblasts. Viable chondrocytes in OCAs at the time of transplantation are primarily responsible for maintenance of donor articular cartilage health in the long term. Optimizing chondrocyte viability in all aspects of OCA transplantation-including procurement, processing, storage, transportation, and surgical implantation-needs to be a primary focus for OCA clinical use. © 2016 The Author(s).

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion*

PubMed Central

Li, Dan; Peng, Shi-yun; Zhang, Zhen-wu; Feng, Rui-cheng; Li, Lu; Liang, Jie; Tai, Sheng; Teng, Chun-bo

2013-01-01

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells. PMID:23825145

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

PubMed Central

Hughes, Andrew D.; Mattison, Jeff; Powderly, John D.; Greene, Bryan T.; King, Michael R.

2012-01-01

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies. PMID:22733259

Regulatory effect and mechanism of gastrin and its antagonists on colorectal carcinoma

PubMed Central

He, Shuang-Wu; Shen, Kang-Qiang; He, Yu-Jun; Xie, Bin; Zhao, Yan-Ming

1999-01-01

AIM: To explore the effect and mechanism of gastrin and its an tagonists proglumide and somatostatin on colorectal carcinoma and their clinical significance. METHODS: A model of transplanted human colonic carcinoma was established from SW480 cell line in gymnomouse body. The volume and weight of transplanted carcinoma was observed under the effect of pentagatrin (PG), proglumide (PGL) and octapeptide somotostatin (SMS201-995, SMS). The cAMP content of carcinoma cell was determined by radioimmunoassay and the DNA, protein content and cell cycle were determined by flow-cytometry. The amount of viable cells was determined by MTT colorimetric analysis, IP3 content was determined by radioimmuno assay, Ca2+ concentration in cell by fluorometry and PKC activity by isotopic enzymolysis. The expression of gastrin, c-myc, c-fos and rasP21 in 48 case s of colorectal carcinoma tissue was detected by the immuno-cytochemistry SP method. Argyrophilia nucleolar organizer regions was determined with argyrophilia stain. RESULTS: The volume, weight, cAMP, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in PG group were all significantly higher than those of control group. When PG was at the concentration of 25 mg/L, the amount of viable cells, IP3 content and Ca2+ concentration in cell and membrane PKC activity in PG group were significantly higher than those in control group; when PGL was at a concentration of 32 mg/L, they dropped to the lowest level in PG (25 mg/L) + PGL group, but without significant difference from the control group. The positive expression rate of gastrin, c-myc, c-fos and rasP21 in carcinoma tissue was 39.6%, 54.2%, 47.9% and 54.2% respectively and significantly higher than that in mucosa 3 cm and 6 cm adjacent to carcinoma tissue and normal colorectal mucosa. The positive expression rate of gastrin of highly-differentiated adenocarcinoma group was significantly higher than that of poorly-differentiated and mucinous adenoc arcinoma groups. The AgNORs count of carcinoma tissue was significantly higher than that in mucosa 3 cm and 6 cm adjacent to carcinoma tissue and norm al colorectal mucosa; and the positive expression of c-myc and c-fos and the A gNORs count in gastrin-positive group was significantly higher than those in gastrin-negative group. CONCLUSION: Pentagastrin has a promoting effect on the growth of transplanted human colonic carcinoma from SW480 cell line. PGL has no obvious effect on the growth of human colonic carcinoma SW480 cell line, but could inhibit the growth promoting effect of PG on transplanted carcinoma. Somatostatin can not only inhibit the growth of transplanted human colonic carcinoma from SW480 cell line directly but also depress the growth-promoting effect of gastrin on the transplanted carcinoma. Some colorectal carcinoma cells can produce and secrete gastrin through autocrine, highly-differentiated adenocarcinoma express the highest level gastrin. Endogenous gastrin can stimulate the cell division and proliferation of carcinoma cell and promote the growth of colorectal carcinoma regulating the expression of oncogene c-myc, c-fos. Our study has provided experimental basis for the adjuvant treatment using gastrin antagonist such as PGL, so matostatin of patients with colorectal carcinoma. PMID:11819478

Cellular injury evidenced by impedance technology and infrared microspectroscopy

NASA Astrophysics Data System (ADS)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2015-03-01

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915 cm-1, 933 cm-1, 989 cm-1, 1192 cm-1, 1369 cm-1, 1437 cm-1, 1450 cm-1, 1546 cm-1, 1634 cm-1, 1679 cm-1 1772 cm-1, 2874 cm-1 and 2962 cm-1) associated with cytotoxicity were significantly (p value < 0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic responses was evident while clear separation was linked to cytotoxicity. RT-CES detected morphological changes as indicators of cell injury and could distinguish between viable, cytostatic and cytotoxic responses. FTIR microspectroscopy confirmed that cytostatic cells were viable and could still recover while also describing early cellular stress related responses on a molecular level.

Targeting stromal glutamine synthetase in tumors disrupts tumor microenvironment-regulated cancer cell growth

USDA-ARS?s Scientific Manuscript database

Reactive stromal cells are an integral part of tumor microenvironment (TME) and interact with cancer cells to regulate their growth. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets that make canc...

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

PubMed

Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

2013-09-01

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Cannabidiol Reduces Leukemic Cell Size - But Is It Important?

PubMed

Kalenderoglou, Nikoletta; Macpherson, Tara; Wright, Karen L

2017-01-01

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro . However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.

Cannabidiol Reduces Leukemic Cell Size – But Is It Important?

PubMed Central

Kalenderoglou, Nikoletta; Macpherson, Tara; Wright, Karen L.

2017-01-01

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent. PMID:28392768

A Ratio of Spore to Viable Organisms: A Case Study of the JPL-SAF Cleanroom

NASA Technical Reports Server (NTRS)

Hendrickson, Ryan; Urbaniak, Camilla; Malli Mohan, Ganesh Babu; Aronson, Heidi; Venkateswaran, Kasthuri

2017-01-01

Spacecraft surfaces that are destined to land on potential life-harboring celestial bodies are required to be rigorously cleaned and continuously monitored for spore bioburden as a proxy for spacecraft cleanliness. The NASA standard assay (NSA), used for spacecraft bioburden estimates, specifically measures spores that are cultivable, aerobic, resistant to heat shock, and grow at 30 C in a nutrient-rich medium. Since the vast majority of microorganisms cannot be cultivated using the NSA, it is necessary to utilize state-of-the art molecular techniques to better understand the presence of all viable microorganisms, not just those measured with the NSA. In this study, the nutrient-deprived low biomass cleanrooms, where spacecraft are assembled, were used as a surrogate for spacecraft surfaces to measure the ratio of NSA spores in relation to the total viable microorganism population in order to make comparisons with the 2006 Space Studies Board (SSB) estimate of 1 spore per approximately 50,000 viable organisms. Ninety-eight surface wipe samples were collected from the Spacecraft Assembly Facility (SAF) cleanroom at the Jet Propulsion Laboratory (JPL) over a 6-month period. The samples were processed and analyzed using classical microbiology along with molecular methodology. Traditional microbiology plating methods were used to determine the cultivable bacterial, fungal, and spore populations. Molecular assays were used to determine the total organisms (TO, dead and live) and the viable organisms (VO, live). The TO was measured using adenosine triphosphate (ATP) and quantitative polymerase chain reaction (qPCR) assays. The VO was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry (after staining for viable microorganisms) assays. Based on the results, it was possible to establish a ratio between spore counts and VO for each viability assay. The ATP-based spore to VO ratio ranged from 149-746, and the bacterial PMA-qPCR assay-based ratio ranged from 314-1,491 VO, per spore. The most conservative estimate came from fluorescent-assisted cell sorting (FACS), which estimated the ratio to be 12,091 VO per 1 NSA spore. Since archaeal (less than 1%) and fungal (approximately 2%) populations were negligible, the spore to VO ratios were based on bacterial population estimates. The most conservative ratio from this study can be used as a replacement for the SSB estimate on nutrient-deprived (oligotrophic) desiccated spacecraft surfaces, to estimate the VO from NSA measurements without utilizing state-of-the art molecular methods that are costly and require more biomass than is typically found on spacecraft surfaces.

Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

USDA-ARS?s Scientific Manuscript database

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such a...

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2).

PubMed

Kim, Joo-Shin; Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 microg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 microg/mL of each polysaccharide isolate to the cell line containing 80 microg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity.

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2)

PubMed Central

Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 µg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 µg/mL of each polysaccharide isolate to the cell line containing 80 µg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity. PMID:20368935

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

PubMed

Keserue, Hans-Anton; Baumgartner, Andreas; Felleisen, Richard; Egli, Thomas

2012-11-01

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Entry of Yersinia pestis into the viable but nonculturable state in a low-temperature tap water microcosm.

PubMed

Pawlowski, David R; Metzger, Daniel J; Raslawsky, Amy; Howlett, Amy; Siebert, Gretchen; Karalus, Richard J; Garrett, Stephanie; Whitehouse, Chris A

2011-03-16

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.

Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

PubMed Central

Pawlowski, David R.; Metzger, Daniel J.; Raslawsky, Amy; Howlett, Amy; Siebert, Gretchen; Karalus, Richard J.; Garrett, Stephanie; Whitehouse, Chris A.

2011-01-01

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism. PMID:21436885

Application of plow-tillage as an innovative technique for eliminating overwintering cyanobacteria in eutrophic lake sediments.

PubMed

Zhou, Qilin; Liu, Cheng; Fan, Chengxin

2016-12-01

Surface sediment in eutrophic lakes is both a destination and a habitat for overwintering cyanobacteria. The resuspension and recovery of viable, overwintering cyanobacteria from the surface sediment during warm spring weather is usually the primary stage of cyanobacterial blooms (CBs) in shallow eutrophic lakes. Therefore, the elimination of overwintering cyanobacteria in sediment is vital to control CBs. In the present study, sediment plow-tillage (PT) was introduced as an innovative technique for eliminating overwintering cyanobacteria in sediments from Lake Chaohu. Four depths of PT (2, 5, 10, and 15 cm) were tested during the 42-day experiment. The results showed that rapid cell death during the first 0-7 d after PT was accompanied by high oxygen uptake rates. The viable cells in deeper sediment died more quickly and at a higher rate after PT. A PT depth of >10 cm effectively eliminated viable cyanobacteria (with a removal rate of 82.8%) from the sediment and prevented their resuspension. The activity of the viable cyanobacteria also decreased quickly as cyanobacteria were eliminated. It appears that the dark, anoxic environment of the deeper sediment after PT was responsible for the elimination of viable cells. Although high release rates of nitrogen and phosphorus were found to accompany the dying and decomposition of cyanobacteria during days 0-7 of the experiment, greater depth of PT was found to decrease nutrient concentrations in the overlying water. In conclusion, we recommend sediment PT as a new technique for eliminating overwintering algae in sediments. However, the release of nutrients from the sediment and the in situ control of CBs in lakes after PT should be further studied. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana).

PubMed

Jones, A Maxwell P; Chattopadhyay, Abhishek; Shukla, Mukund; Zoń, Jerzy; Saxena, Praveen K

2012-05-30

Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

PubMed Central

2012-01-01

Background Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. Results This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. Conclusions This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts. PMID:22646730

Meniscal repair using engineered tissue.

PubMed

Peretti, G M; Caruso, E M; Randolph, M A; Zaleske, D J

2001-03-01

In this study, devitalized meniscal tissue pre-seeded with viable cultured chondrocytes was used to repair a bucket-handle incision in meniscal tissue transplanted to nude mice. Lamb knee menisci were devitalized by cyclic freezing and thawing. Chips measuring four by two by one-half millimeters were cut from this devitalized tissue to serve as scaffolds. These chips were then cultured either with or without viable allogeneic lamb chondrocytes. From the inner third of the devitalized meniscal tissue, rectangles were also cut approximately 8 x 6 mm. A 4 mm bucket-handle type incision was made in these blocks. The previously prepared chips either with (experimental group) or without viable chondrocytes (control group) were positioned into the incisions and secured with suture. Further control groups included blocks of devitalized menisci with incisions into which no chips were positioned and either closed with suture or left open with no suture. Specimens were transplanted to subcutaneous pouches of nude mice for 14 weeks. After 14 weeks, seven of eight experimental specimens (chips with viable chondrocytes) demonstrated bridging of the incision assessed by gross inspection and manual distraction. All the control groups were markedly different from the experimental group in that the incision remained grossly visible. Histological analysis was consistent with the differences apparent at the gross level. Only the experimental specimens (chips with viable chondrocytes) with gross bridging demonstrated obliteration of the interface between incision and scaffold. None of the control specimens revealed any cells or tissue filling the incision. Tissue engineering using scaffolds and viable cells may have an application in meniscal repair in vivo.

Optimization of Culture Medium Enhances Viable Biomass Production and Biocontrol Efficacy of the Antagonistic Yeast, Candida diversa.

PubMed

Liu, Jia; Li, Guangkun; Sui, Yuan

2017-01-01

Viable biomass production is a key determinant of suitability of antagonistic yeasts as potential biocontrol agents. This study investigated the effects of three metal ions (magnesium, ferrous, and zinc) on biomass production and viability of the antagonistic yeast, Candida diversa . Using response surface methodology to optimize medium components, a maximum biomass was obtained, when the collective Mg 2+ , Fe 2+ , and Zn 2+ concentrations were adjusted in a minimal mineral (MM) medium. Compared with the unmodified MM, and three ion-deficient MM media, yeast cells cultured in the three ion-modified MM medium exhibited a lower level of cellular oxidative damage, and a higher level of antioxidant enzyme activity. A biocontrol assay indicated that C. diversa grown in the ion-modified MM exhibited the greatest level of control of gray mold on apple fruit. These results provide new information on culture medium optimization to grow yeast antagonists in order to improve biomass production and biocontrol efficacy.

Optimization of Culture Medium Enhances Viable Biomass Production and Biocontrol Efficacy of the Antagonistic Yeast, Candida diversa

PubMed Central

Liu, Jia; Li, Guangkun; Sui, Yuan

2017-01-01

Viable biomass production is a key determinant of suitability of antagonistic yeasts as potential biocontrol agents. This study investigated the effects of three metal ions (magnesium, ferrous, and zinc) on biomass production and viability of the antagonistic yeast, Candida diversa. Using response surface methodology to optimize medium components, a maximum biomass was obtained, when the collective Mg2+, Fe2+, and Zn2+ concentrations were adjusted in a minimal mineral (MM) medium. Compared with the unmodified MM, and three ion-deficient MM media, yeast cells cultured in the three ion-modified MM medium exhibited a lower level of cellular oxidative damage, and a higher level of antioxidant enzyme activity. A biocontrol assay indicated that C. diversa grown in the ion-modified MM exhibited the greatest level of control of gray mold on apple fruit. These results provide new information on culture medium optimization to grow yeast antagonists in order to improve biomass production and biocontrol efficacy. PMID:29089939

A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium

PubMed Central

Rashid, Mohammed H.; Revazishvili, Tamara; Dean, Timothy; Butani, Amy; Verratti, Kathleen; Bishop-Lilly, Kimberly A.; Sozhamannan, Shanmuga; Sulakvelidze, Alexander; Rajanna, Chythanya

2012-01-01

Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a “phage cocktail” (tentatively designated “YPP-100”) capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 109, 108 and 107 PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 106 PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis. PMID:23275868

Measuring Thermodynamic Properties of Metals and Alloys With Knudsen Effusion Mass Spectrometry

NASA Technical Reports Server (NTRS)

Copland, Evan H.; Jacobson, Nathan S.

2010-01-01

This report reviews Knudsen effusion mass spectrometry (KEMS) as it relates to thermodynamic measurements of metals and alloys. First, general aspects are reviewed, with emphasis on the Knudsen-cell vapor source and molecular beam formation, and mass spectrometry issues germane to this type of instrument are discussed briefly. The relationship between the vapor pressure inside the effusion cell and the measured ion intensity is the key to KEMS and is derived in detail. Then common methods used to determine thermodynamic quantities with KEMS are discussed. Enthalpies of vaporization, the fundamental measurement, are determined from the variation of relative partial pressure with temperature using the second-law method or by calculating a free energy of formation and subtracting the entropy contribution using the third-law method. For single-cell KEMS instruments, measurements can be used to determine the partial Gibbs free energy if the sensitivity factor remains constant over multiple experiments. The ion-current ratio method and dimer-monomer method are also viable in some systems. For a multiple-cell KEMS instrument, activities are obtained by direct comparison with a suitable component reference state or a secondary standard. Internal checks for correct instrument operation and general procedural guidelines also are discussed. Finally, general comments are made about future directions in measuring alloy thermodynamics with KEMS.

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

PubMed

Chavda, S C; Griffin, P; Han-Liu, Z; Keys, B; Vekony, M A; Cann, A J

1994-11-01

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

Fermentative preparation of functional drink from Punica granatum using lactic acid bacteria and exploring its anti-tumor potential

NASA Astrophysics Data System (ADS)

Murthy, Shruthi N.; Patnaik, Amie; Srinivasan, Nandini; Selvarajan, E.; Nivetha, A.; Mohanasrinivasan, V.

2017-11-01

In the present research work probiotic pomegranate juice production by fermentation was carried out using two different strains such as Lactobacillus plantarum VITES07 and Lactobacillus acidophilus NCIM2903 (Lactic acid bacteria). Fermented pomegranate juice was carried out at room temperature for 72h. During the fermentation period at regular intervals viable cells was determined. Efficiency of the fermented juice was analysed for 4 weeks under refrigerated condition at 4˚C. Total phenolics, sugar concentration, antioxidant potential, and antibacterial activity were determined. Organic acid concentration was determined by HPLC with retention time of a compound at 9.1 can be suspected to be Kaempferol hexoside and functional group was determined by FTIR also LCMS analysis was carried out to enumerate the chemical composition of the fermented juice.

Anti-bacterial effect of essential oil from Xanthium strumarium against shiga toxin-producing Escherichia coli.

PubMed

Sharifi-Rad, J; Soufi, L; Ayatollahi, S A M; Iriti, M; Sharifi-Rad, M; Varoni, E M; Shahri, F; Esposito, S; Kuhestani, K; Sharifi-Rad, M

2016-09-19

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human pathogenic microorganisms, which can cause life-threatening infections. Xanthium strumarium L. is a plant with anti-bacterial activity against gram-negative and gram-positive bacteria. This study aims to demonstrate in vitro efficacy of the essential oil (EO) extracted from Xanthium strumarium L. against E. coli O157:H7. Using the agar test diffusion, the effect of Xanthium strumarium L. EO (5, 10, 15, 30, 60, and 120 mg/mL) was verified at each of the four different growth phases of E. coli O157:H7. Cell counts of viable cells and colony forming unit (CFU) were determined at regular time points using Breed's method and colony counting method, respectively. No viable cell was detectable after the 1 hour-exposure to X. strumarium EO at 30, 60, and 120 mg/mL concentrations. No bacterial colony was formed after 1 h until the end of the incubation period at 24 h. At lower concentrations, the number of bacteria cells decreased and colonies could be observed only after incubation. At the exponential phase, the EO at 15 mg/mL was only bacteriostatic, while from 30 mg/mL started to be bactericidal. X. strumarium EO antibacterial activity against Shiga toxin-producing E. coli O157:H7 is dependent on EO concentration and physiological state of the microorganisms tested. The best inhibitory activity was achieved during the late exponential and the stationary phases.

Low temperature aqueous precipitation of needle-like nanophase hydroxyapatite.

PubMed

Cox, Sophie C; Jamshidi, Parastoo; Grover, Liam M; Mallick, Kajal K

2014-01-01

The use of tissue engineered biodegradable porous scaffolds has become an important focus of the biomedical research field. The precursor materials used to form these structures play a vital role in their overall performance thus making the study and synthesis of these selected materials imperative. The authors present a comparison and characterisation of hydroxyapatite (HA), a popular calcium phosphate (CaP) biomaterial, synthesised by an aqueous precipitation (AP) method. The influence of various reaction conditions on the phase, crystallinity, particle size as well as morphology, molecular structure, potential in-vivo bioactivity and cell viability were assessed by XRD, SEM and TEM, FTIR, a simulated body fluid (SBF) test and a live/dead assay using MC3T3 osteoblast precursor cells, respectively. Naturally carbonated nanoparticles of HA with typically needle-like morphology were synthesised by the reported AP method. Initial pH was found to influence the crystallisation process and determine the CaP phase formed as well as the resultant particle and crystallite sizes. A marked change in particle morphology was also observed above pH 9. The use of toluene as a replacement solvent for water up to 60% was found to reduce the crystallinity of as-synthesised HA. This has marked influence on the effect of ethanolamine (5 wt%), which was found to improve HA crystallinity. SEM and EDS were used to confirm the growth of carbonated apatite on the surface of HA pellets immersed in SBF for up to 28 days. Cell culture results revealed viable cells on all samples where pH was controlled and maintained at 10-11 during precipitation, including those that used ethanolamine and toluene in preparation. When the initial alkali pH was not maintained non-viable cells were observed on HA substrates.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

PubMed Central

Possoz, Christophe; Durand, Adeline; Desfontaines, Jean-Michel; Barre, François-Xavier; Leach, David R. F.

2018-01-01

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations. PMID:29522563

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes.

PubMed

Pol, I E; Smid, E J

1999-09-01

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.

[A Case of Duodenal Invasive Advanced Gastric Cancer in Which the Primary Tumor Achieved pCR, but Viable Cancer Cells Remained in Lymph Node No.13 after Neoadjuvant Chemotherapy].

PubMed

Kubota, Tetsushi; Choda, Yasuhiro; Morito, Toshiaki; Miyake, Soichiro; Ishida, Michihiro; Sato, Daisuke; Sumitani, Daisuke; Nakano, Kanyu; Harano, Masao; Matsukawa, Hiroyoshi; Ojima, Yasutomo; Idani, Hitoshi; Shiozaki, Shigehiro; Okajima, Masazumi

2017-11-01

A woman approximately 70-years-old with duodenal invasive advanced gastric cancer was referred to our hospital. Meta- stasis to lymph node(LN)No.13 was suspected based on FDG/PET-CT. For better curability, we selected neoadjuvant chemotherapy( NAC)with S-1 plus oxaliplatin(SOX therapy). After 3 courses of SOX, distal gastrectomy with D2(+No.13) lymphadenectomy was performed. Upon pathological evaluation, no viable cancer cells were found in the primary tumor, but viable cancer cells were identified in LN No.6 and 13. LN No.13 was defined as M1 according to the current Japanese classification of gastric carcinoma. On the other hand, the 2014 Japanese gastric cancer treatment guidelines(ver. 4)mentioned that D2(+No.13)lymphadenectomy may be an option in potentially curative gastrectomy for tumors invading the duodenum. This case suggests that No.13 lymphadenectomy is necessary as a curative operation for duodenal invasive advanced gastric cancer, even if the primary tumor has achieved pCR after NAC.

Luminous Enteric Bacteria of Marine Fishes: a Study of Their Distribution, Densities, and Dispersion †

PubMed Central

Ruby, E. G.; Morin, J. G.

1979-01-01

Three taxa of luminous bacteria (Photobacterium fischeri, P. phosphoreum, and Beneckea spp.) were found in the enteric microbial populations of 22 species of surface- and midwater-dwelling fishes. These bacteria often occurred in concentrations ranging between 105 and 107 colony-forming units per ml of enteric contents. By using a genetically marked strain, it was determined that luminous cells entering the fish during ingestion of seawater or contaminated particles traversed the alimentary tract and survived the digestive processes. After excretion, luminous bacteria proliferated extensively on the fecal material and became distributed into the surrounding seawater. Thus, this enteric habitat may serve as an enrichment of viable cells entering the planktonic luminous population. PMID:16345429

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schumacher, Joanne; Lewis, Teresa D.; Leong, Jo-Ann C.; Casey, Rufina N.; Casey, James W.

2009-01-01

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

Developing and Demonstrating the Next-Generation Fuel Cell Electric Bus Made in America

DOT National Transportation Integrated Search

2012-02-01

The Federal Transit Administrations (FTA) National Fuel Cell Bus Program (NFCBP) focuses on developing commercially viable fuel cell bus technologies. CALSTART is one of three non-profit consortia chosen to manage projects competitively selected u...

Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum.

PubMed

Tan, Ching Giap; Ideris, Aini; Omar, Abdul R; Yii, Chen Pei; Kleven, Stanley H

2014-09-02

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

Manufacture of Autologous CD34+ Selected Grafts in the NIAID-Sponsored HALT-MS and SCOT Multicenter Clinical Trials for Autoimmune Diseases.

PubMed

Keever-Taylor, Carolyn A; Heimfeld, Shelly; Steinmiller, Kaitlyn C; Nash, Richard A; Sullivan, Keith M; Czarniecki, Christine W; Granderson, Tomeka C; Goldstein, Julia S; Griffith, Linda M

2017-09-01

To ensure comparable grafts for autologous hematopoietic cell transplantation (HCT) in the National Institute of Allergy and Infectious Diseases-sponsored Investigational New Drug protocols for multiple sclerosis (HALT-MS) and systemic sclerosis (SCOT), a Drug Master File approach to control manufacture was implemented, including a common Master Production Batch Record and site-specific standard operating procedures with "Critical Elements." We assessed comparability of flow cytometry and controlled rate cryopreservation among sites and stability of cryopreserved grafts using hematopoietic progenitor cells (HPCs) from healthy donors. Hematopoietic Progenitor Cells, Apheresis-CD34+ Enriched, for Autologous Use (Auto-CD34 + HPC) graft specifications included ≥70% viable CD34 + cells before cryopreservation. For the 2 protocols, 110 apheresis collections were performed; 121 lots of Auto-CD34 + HPC were cryopreserved, and 107 of these (88.4%) met release criteria. Grafts were infused at a median of 25 days (range, 17 to 68) post-apheresis for HALT-MS (n = 24), and 25 days (range, 14 to 78) for SCOT (n = 33). Subjects received precryopreservation doses of a median 5.1 × 10 6 viable CD34 + cells/kg (range, 3.9 to 12.8)  for HALT-MS and 5.6 × 10 6 viable CD34 + cells/kg (range, 2.6 to 10.2) for SCOT. Recovery of granulocytes occurred at a median of 11 days (range, 9 to 15) post-HCT for HALT-MS and 10 days (range, 8 to 12) for SCOT, independent of CD34 + cell dose. Subjects received their last platelet transfusion at a median of 9 days (range, 6 to 16) for HALT-MS and 8 days (range, 6 to 23) for SCOT; higher CD34 + /kg doses were associated with faster platelet recovery. Stability testing of cryopreserved healthy donor CD34 + HPCs over 6 months of vapor phase liquid nitrogen storage demonstrated consistent 69% to 73% recovery of viable CD34 + cells. Manufacturing of Auto-CD34 + HPC for the HALT-MS and SCOT protocols was comparable across all sites and supportive for timely recovery of granulocytes and platelets. Published by Elsevier Inc.

The impact of enterocin AS-48 on the shelf-life and safety of sardines (Sardina pilchardus) under different storage conditions.

PubMed

Ananou, S; Zentar, H; Martínez-Bueno, M; Gálvez, A; Maqueda, M; Valdivia, E

2014-12-01

The purpose of this study was to determine the effect of enterocin AS-48, packaged under normal atmosphere (NA), vacuum (VP) or modified atmosphere (MAP) on the shelf life and safety of fresh sardines (Sardina pilchardus) stored at 5 °C. We studied the effect of these hurdles, alone or combined, on the relevant autochthonous bacterial populations. Total volatile basic nitrogen (TVB-N) content was used as indicative of freshness. Levels of biogenic amines cadaverine, putrescine, tyramine, and histamine were also determined. The application of AS-48 did not reduce the mesophilic, psychrotrophic, or Gram negative bacteria viable cell counts under any of the storage conditions tested. AS-48 did cause significant reductions in viable staphylococci counts, especially under VP. In sardines under NA treated with AS-48, the populations of histamine- and tyramine-forming total and lactic acid bacteria (LAB) showed no significant reductions. MAP or VP with AS-48 allowed reductions (significant at some storage times) in histamine- and tyramine-forming LAB. The TVB-N content was also reduced under normal atmosphere and, especially, in sardines stored under MAP. The most interesting results are those concerning the decrease (by several fold) in the levels of the biogenic amines cadaverine, putrescine, tyramine, and histamine determined after treatment with AS-48. Copyright © 2014 Elsevier Ltd. All rights reserved.

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

PubMed Central

Yango, Pamela; Altman, Eran; Smith, James F.; Klatsky, Peter C.; Tran, Nam D.

2015-01-01

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications. PMID:25241367

Molecular Viability Testing of UV-Inactivated Bacteria.

PubMed

Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

2017-05-15

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli , Aeromonas hydrophila , and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment. IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection. Copyright © 2017 American Society for Microbiology.

Effect of benzalkonium chloride on viability and energy metabolism in exponential- and stationary-growth-phase cells of Listeria monocytogenes.

PubMed

Luppens, S B; Abee, T; Oosterom, J

2001-04-01

The difference in killing exponential- and stationary-phase cells of Listeria monocytogenes by benzalkonium chloride (BAC) was investigated by plate counting and linked to relevant bioenergetic parameters. At a low concentration of BAC (8 mg liter(-1)), a similar reduction in viable cell numbers was observed for stationary-phase cells and exponential-phase cells (an approximately 0.22-log unit reduction), although their membrane potential and pH gradient were dissipated. However, at higher concentrations of BAC, exponential-phase cells were more susceptible than stationary-phase cells. At 25 mg liter(-1), the difference in survival on plates was more than 3 log units. For both types of cells, killing, i.e., more than 1-log unit reduction in survival on plates, coincided with complete inhibition of acidification and respiration and total depletion of ATP pools. Killing efficiency was not influenced by the presence of glucose, brain heart infusion medium, or oxygen. Our results suggest that growth phase is one of the major factors that determine the susceptibility of L. monocytogenes to BAC.

Multimodality molecular imaging and extracellular vesicle release based genetic profiling with porphyrin nanodroplets (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zemp, Roger J.; Paproski, Robert J.

2017-03-01

For emerging tissue-engineering applications, transplants, and cell-based therapies it is important to assess cell viability and function in vivo in deep tissues. Bioluminescence and fluorescence methods are poorly suited to deep monitoring applications with high resolution and require genetically-engineered reporters which are not always feasible. We report on a method for imaging cell viability using deep, high-resolution photoacoustic imaging. We use an exogenous dye, Resazurin, itself weakly fluorescent until it is reduced from blue to a pink color with bright red fluorescence. Upon cell death fluorescence is lost and an absorption shift is observed. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration. We detect colorimetric absorption shifts using multispectral photoacoustic imaging and quantify the fraction of viable cells. SKOV-3 cells with and without ±80oC heat treatment were imaged after Resazurin treatment. High 575nm:620nm ratiometric absorption and photoacoustic signals in viable cells were observed with a much lower ratio in low-viability populations.

Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

NASA Astrophysics Data System (ADS)

Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; Vannieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

2016-06-01

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

NASA Technical Reports Server (NTRS)

Behar, Alberto E.; Stam, Christina N.; Smiley, Ronald

2013-01-01

This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the sample can be plated. Using a photoaffinity label would remove this step from the current assay as the label readily penetrates both live and dead bacterial cells. Secondly, the photoaffinity label can only penetrate dead bacterial spores, leaving behind the viable spore population. This would allow for rapid bacterial spore detection in a matter of hours compared to the several days that it takes for the NASA standard assay.

Comparison of Techniques for Preimplantation Treatment of Osteochondral Allograft Bone.

PubMed

Baumann, Charles A; Baumann, John R; Bozynski, Chantelle C; Stoker, Aaron M; Stannard, James P; Cook, James L

2018-03-07

Articular defects are a major problem with few effective treatment options. Osteochondral allograft (OCA) transplantation can be an effective treatment; however, lack of OCA bone integration can cause failure. This controlled laboratory study was designed to compare clinically applicable methods for marrow element removal and enhanced delivery of bone marrow aspirate concentrate (BMC) to OCA bone. We hypothesized that compressed carbon dioxide (CO 2 ) treatment of OCA bone would result in significantly better marrow element removal, significantly more retention and distribution of viable osteoprogenitor cells, and significantly higher osteoinductive protein elution from OCAs compared with other preimplantation treatments. Fresh humeral heads ( n  = 24) were harvested and stored for 14 days, then randomly assigned to treatment based on marrow element removal and bone treatment: (standard of care [SOC]) ( n  = 4) - SOC high-pulse saline lavage, no BMC; (BMC) ( n  = 5) - saline lavage then canine BMC; (Drill + BMC) ( n  = 5) - 1.1 mm drill-hole immediately subchondral then saline lavage then BMC injection through drill hole; (Carb + BMC) ( n  = 5) - saline lavage then CO 2 then BMC; or (Saline-Carb + BMC) ( n  = 5) - saline lavage and CO 2 together then BMC. Treated OCAs were cultured for 14 days. On day 3, media were collected, centrifuged to isolate cells, and replaced. Cells were cultured for 11 days for colony forming unit (CFU) determination. OCA media were collected on days 7 and 14 of culture for analysis. On day 14, each graft was assessed for viable cell retention and distribution, and bone marrow element removal. BMC had significantly higher ( p  = 0.001) viable cell distribution compared with the SOC, Drill + BMC, Carb + BMC, and Saline-Carb + BMC groups. BMC and Drill + BMC had significantly higher ( p  < 0.05) CFUs than SOC, Carb + BMC, and Saline-Carb + BMC. Drill + BMC and Carb + BMC had the highest media concentrations of the osteoinductive biomarkers. The Carb + BMC and Saline-Carb + BMC groups were associated with significantly superior marrow element removal ( p  < 0.02) compared with the SOC, Drill + BMC, and BMC groups. Saline irrigation plus saturation with autogenous BMC appears to be the most advantageous preimplantation treatment for OCA transplantation. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Microbial Condition of Water Samples from Foreign Fuel Storage Facilities

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

Berry, C.J.; Fliermans, C.B.; Santo Domingo, J.

1997-10-30

In order to assess the microbial condition of foreign nuclear fuel storage facilities, fourteen different water samples were received from facilities outside the United States that have sent spent nuclear fuel to SRS for wet storage. Each water sample was analyzed for microbial content and activity as determined by total bacteria, viable aerobic bacteria, viable anaerobic bacteria, viable sulfate- reducing bacteria, viable acid-producing bacteria and enzyme diversity. The results for each water sample were then compared to other foreign samples and to data from the receiving basin for off- site fuel (RBOF) at SRS.

In vitro cytotoxicity evaluation of elemental ions released from different prosthodontic materials.

PubMed

Elshahawy, Waleed M; Watanabe, Ikuya; Kramer, Phillip

2009-12-01

This study investigated the cytotoxicity of elemental ions contained in four fixed prosthodontic materials (gold, nickel-chromium, stainless-steel alloys and CAD-CAM ceramics). According to the determination of elements released from prosthodontic materials by using inductively coupled plasma mass spectroscopy, similar amounts of elements Pd, Ag, Zn, Cu, Ni, Cr, Mo, Be, Fe, Al, and K were prepared as salt solutions. Wells with a tenfold higher concentration of the tested elements were used as positive controls, while a well without any tested element was used as a negative control. These salt solutions were tested for cytotoxicity by culturing mouse L-929 fibroblasts in the salt solutions for a 7-day period of incubation. Then, the percentage of viable cells for each element was measured using trypan blue exclusion assay. The data (n=5) were statistically analyzed by ANOVA/Tukey test (p<0.05). The results showed a statistically significant difference for the cytotoxic effect of the tested elements salt solutions. For the released element concentrations the lowest percentage of viable cells (mean+/-SD) was evident with Zn, Cu or Ni indicating that they are the highly toxic elements. Be and Ag were found to be intermediate in cytotoxic effect. Fe, Cr, Mo, Al, Pd or K were found to be the least cytotoxic elements. Zn and Cu released from gold alloys, and Ni released from nickel-chromium alloys, which are commonly used as fixed prosthodontic restorations, show evidence of a high cytotoxic effect on fibroblast cell cultures.

Surface chemistry of Ti6Al4V components fabricated using selective laser melting for biomedical applications.

PubMed

Vaithilingam, Jayasheelan; Prina, Elisabetta; Goodridge, Ruth D; Hague, Richard J M; Edmondson, Steve; Rose, Felicity R A J; Christie, Steven D R

2016-10-01

Selective laser melting (SLM) has previously been shown to be a viable method for fabricating biomedical implants; however, the surface chemistry of SLM fabricated parts is poorly understood. In this study, X-ray photoelectron spectroscopy (XPS) was used to determine the surface chemistries of (a) SLM as-fabricated (SLM-AF) Ti6Al4V and (b) SLM fabricated and mechanically polished (SLM-MP) Ti6Al4V samples and compared with (c) traditionally manufactured (forged) and mechanically polished Ti6Al4V samples. The SLM-AF surface was observed to be porous with an average surface roughness (Ra) of 17.6±3.7μm. The surface chemistry of the SLM-AF was significantly different to the FGD-MP surface with respect to elemental distribution and their existence on the outermost surface. Sintered particles on the SLM-AF surface were observed to affect depth profiling of the sample due to a shadowing effect during argon ion sputtering. Surface heterogeneity was observed for all three surfaces; however, vanadium was witnessed only on the mechanically polished (SLM-MP and FGD-MP) surfaces. The direct and indirect 3T3 cell cytotoxicity studies revealed that the cells were viable on the SLM fabricated Ti6Al4V parts. The varied surface chemistry of the SLM-AF and SLM-MP did not influence the cell behaviour. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity.

PubMed

Oliveira, Maria Alcionéia Carvalho de; Borges, Aline Chiodi; Brighenti, Fernanda Lourenção; Salvador, Marcos José; Gontijo, Aline Vidal Lacerda; Koga-Ito, Cristiane Yumi

2017-11-06

The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

Establishing Caenorhabditis elegans as a model for Mycobacterium avium subspecies hominissuis infection and intestinal colonization

PubMed Central

Everman, Jamie L.; Ziaie, Navid R.; Bechler, Jessica; Bermudez, Luiz E.

2015-01-01

ABSTRACT The nematode Caenorhabditis elegans has become a model system for studying the disease interaction between pathogens and the host. To determine whether the transparent nematode could serve as a useful model for Mycobacterium avium subspecies hominissuis (MAH) infection of the intestinal tract, worms were fed MAH and assayed for the effects of the bacterial infection on the worm. It was observed during feeding that viable MAH increases in the intestinal lumen in a time dependent manner. Ingestion of MAH was deemed non-toxic to worms as MAH-fed populations have similar survival curves to those fed E. coli strain OP50. Pulse-chase analysis using E. coli strain OP50 revealed that MAH colonize the intestinal tract, as viable MAH remain within the intestine after the assay. Visualization of intestinal MAH using histology and transmission electron microscopy demonstrates that MAH localizes to the intestinal lumen, as well as establishes direct contact with intestinal epithelium. Bacterial colonization appears to have a detrimental effect on the microvilli of the intestinal epithelial cells. The MAH ΔGPL/4B2 strain with a mutation in glycopeptidolipid production is deficient in binding to human epithelial cells (HEp-2), as well as deficient in its ability to bind to and colonize the intestinal tract of C. elegans as efficiently as wild-type MAH. These data indicate the C. elegans may serve as a useful model system for MAH pathogenesis and in determining the mechanisms used by MAH during infection and colonization of the intestinal epithelium. PMID:26405050

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene.

PubMed

Fotou, K; Tzora, A; Voidarou, Ch; Alexopoulos, A; Plessas, S; Avgeris, I; Bezirtzoglou, E; Akrida-Demertzi, K; Demertzis, P G

2011-12-01

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk. The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection). During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey's manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10(3) to 2.4 × 10(4) cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10(2). Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found. From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%. Copyright © 2011 Elsevier Ltd. All rights reserved.

Adipose-Derived Stem Cell Delivery into Collagen Gels Using Chitosan Microspheres

DTIC Science & Technology

2010-02-17

Porous CSM of uniform size and composition were prepared and used as a stem cell carrier. ASC were allowed to attach to the microspheres and infiltrate...and viable, could be retrieved from the spheres, and maintained expression of stem - cell -specific markers. Electron microscopic evaluation of the cell

Influence of Waveform on Cell Viability during Ultrasound Exposure

NASA Astrophysics Data System (ADS)

Saliev, Timur; Feril, Loreto B.; McLean, Donald A.; Tachibana, Katsuro; Campbell, Paul A.

2011-09-01

We examined the role of ultrasound standing waves, and their travelling wave counterparts, on cell viability in an in-vitro insonation apparatus. Furthermore, the effect of distinct waveforms (sine and top-hat) was also explored, together with the role of microbubble presence. Measurements of cell viability in standing wave scenarios demonstrated a relatively higher rate of lysis (63.13±10.89% remaining viable) compared with the travelling wave data, where 96.22±4.0% remained viable. Significant differences were also seen as a function of waveform, where insonations employing top-hat wave shapes resulted in an average end stage viability of 30.31±5.71% compared with 61.94±14.28% in the sinusoidal counterparts.

Persistence of Mycobacterium avium subsp. paratuberculosis at a Farm-Scale Biogas Plant Supplied with Manure from Paratuberculosis-Affected Dairy Cattle▿

PubMed Central

Slana, I.; Pribylova, R.; Kralova, A.; Pavlik, I.

2011-01-01

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (101 cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (102 cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk. PMID:21398476

Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes.

PubMed

Moulavi, F; Hosseini, S M; Tanhaie-Vash, N; Ostadhosseini, S; Hosseini, S H; Hajinasrollah, M; Asghari, M H; Gourabi, H; Shahverdi, A; Vosough, A D; Nasr-Esfahani, M H

2017-03-01

Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at -20 °C and in vitro matured domestic cat oocytes. No cells growth was detected in primary culture of skin and tendon pieces or following culture of singled cells prepared by enzymatic digestion. Furthermore, no live cells were detected following differential viable staining and almost all cells had ruptured membrane. Therefore, direct injection of donor nuclei into enucleated cat oocytes matured in vitro was carried out for SCNT experiments. Early signs of nuclear remodeling were observed as early as 2 h post-iSCNT and significantly increased at 4 h post-iSCNT. The percentages of iSCNT reconstructs that cleaved and developed to 4-16 cell and morula stages were 32.3 ± 7.3, 18.2 ± 9.8 and 5.9 ± 4.3%, respectively. However, none of the iSCNT reconstructs developed to the blastocyst stage. When domestic cat somatic and oocytes were used for control SCNT and parthenogenetic activation, the respective percentages of oocytes that cleaved (51.3 ± 13.9 and 77.3 ± 4.0%) and further developed to the blastocyst stage (11.3 ± 3.3 and 16.8 ± 3.8%) were comparable. In summary, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate non-viable nuclei of Asiatic cheetah and support its reprogramming back to the embryonic stage. To our knowledge, this is the first report of iSCNT in cheetah using non-viable frozen cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Bactericidal activities of woven cotton and nonwoven polypropylene fabrics coated with hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite “Earth-plus”

PubMed Central

Kasuga, Eriko; Kawakami, Yoshiyuki; Matsumoto, Takehisa; Hidaka, Eiko; Oana, Kozue; Ogiwara, Naoko; Yamaki, Dai; Sakurada, Tsukasa; Honda, Takayuki

2011-01-01

Background Bacteria from the hospital environment, including linens and curtains, are often responsible for hospital-associated infections. The aim of the present study was to evaluate the bactericidal effects of fabrics coated with the hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite “Earth-plus”. Methods Bactericidal activities of woven and nonwoven fabrics coated with Earth-plus were investigated by the time-kill curve method using nine bacterial strains, including three Staphylococcus aureus, three Escherichia coli, and three Pseudomonas aeruginosa strains. Results The numbers of viable S. aureus and E. coli cells on both fabrics coated with Earth-plus decreased to below 2 log10 colony-forming units/mL in six hours and reached the detection limit in 18 hours. Viable cell counts of P. aeruginosa on both fabrics coated with Earth-plus could not be detected after 3–6 hours. Viable cells on woven fabrics showed a more rapid decline than those on nonwoven fabrics. Bacterial cell counts of the nine strains on fabrics without Earth-plus failed to decrease even after 18 hours. Conclusion Woven cotton and nonwoven polypropylene fabrics were shown to have excellent antibacterial potential. The woven fabric was more bactericidal than the nonwoven fabric. PMID:21931489

Modelling biofilm-induced formation damage and biocide treatment in subsurface geosystems

PubMed Central

Ezeuko, C C; Sen, A; Gates, I D

2013-01-01

Biofilm growth in subsurface porous media, and its treatment with biocides (antimicrobial agents), involves a complex interaction of biogeochemical processes which provide non-trivial mathematical modelling challenges. Although there are literature reports of mathematical models to evaluate biofilm tolerance to biocides, none of these models have investigated biocide treatment of biofilms growing in interconnected porous media with flow. In this paper, we present a numerical investigation using a pore network model of biofilm growth, formation damage and biocide treatment. The model includes three phases (aqueous, adsorbed biofilm, and solid matrix), a single growth-limiting nutrient and a single biocide dissolved in the water. Biofilm is assumed to contain a single species of microbe, in which each cell can be a viable persister, a viable non-persister, or non-viable (dead). Persisters describe small subpopulation of cells which are tolerant to biocide treatment. Biofilm tolerance to biocide treatment is regulated by persister cells and includes ‘innate’ and ‘biocide-induced’ factors. Simulations demonstrate that biofilm tolerance to biocides can increase with biofilm maturity, and that biocide treatment alone does not reverse biofilm-induced formation damage. Also, a successful application of biological permeability conformance treatment involving geologic layers with flow communication is more complicated than simply engineering the attachment of biofilm-forming cells at desired sites. PMID:23164434

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

PubMed

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive

2015-06-01

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CYTOTOXIC EFFECTS OF SOME MINERAL DUSTS ON SYRIAN HAMSTER PERITONEAL MACROPHAGES

PubMed Central

Bey, Elke; Harington, J. S.

1971-01-01

Hamster peritoneal macrophages were grown in cell culture and their response to various conditions was examined. The cultures responded favorably to high concentrations of serum and to medium which had been preconditioned by contact with tumor cells. After 2–3 days of adaptation, they entered into a period of stability which lasted from the 4th to the 9th day. Macrophage cultures in this stable phase were treated with various samples of mineral dusts and their response determined by counting the number of viable macrophages/cm2 at intervals over a period of 72 hr. Crystalline silica Snowit was found to be nontoxic. Amorphous silica Fransil caused a characteristic cytotoxic effect and a rapid decline in cell population at doses less than 150 µg/5 x 105 cells. Of the three different kinds of asbestos used, chrysotile was toxic and amosite and crocidolite nontoxic at equivalent concentrations. A comparison of two preparations of chrysotile which differed in surface area showed that weight rather than surface area determines toxicity. Pretreatment of chrysotile with tryptose phosphate broth under drastic conditions accelerated but did not increase the final intensity of the cytotoxic effect. PMID:4101804

Survival and persistence of fecal host-specific Bacteroidales cells and their DNA assessed by PMA-qPCR

NASA Astrophysics Data System (ADS)

Bae, S.; Bombardelli, F.; Wuertz, S.

2008-12-01

Understanding and managing microbial pollutions in water is one of the foremost challenges of establishing effective managements and remediation strategies to impaired water bodies polluted by uncharacterized fecal sources. Quantitative microbial source tracking (MST) approaches using fecal Bacteroidales and quantitative PCR (qPCR) assays to measure gene copies of host-specific 16S rRNA genetic markers are promising because they can allow for identifying and quantifying fecal loadings from a particular animal host and understanding the fate and transport of host-specific Bacteroidales over a range of conditions in water bodies. Similar to the case of traditional fecal indicator bacteria, a relatively long persistence of target DNA may hamper applied MST studies, if genetic markers cannot be linked to recent fecal pollution in water. We report a successful approach to removing the qPCR signal derived from free DNA and dead host-specific Bacteroidales cells by selectively binding the DNA and consequently inhibiting PCR amplification using light- activated propidium monoazide (PMA). Optimal PMA-qPCR conditions were determined as 100 µM of PMA concentration and a 10-min light exposure time at different solids concentrations in order to mimic a range of water samples. Under these conditions, PMA-qPCR resulted in the selective exclusion of DNA from heat- treated cells of non-culturable Bacteroidales in human feces and wastewater influent and effluent samples. Also, the persistence of feces-derived host-specific Bacteroidales DNA and their cells (determined by universal, human-, cow- and dog-specific Bacteroidales qPCR assays) in seawater was investigated in microcosms at environmental conditions. The average T99 (two log reduction) value for host-specific viable Bacteroidales cells was 28 h, whereas that for total host-specific Bacteroidales DNA was 177 h. Natural sunlight did not have a strong influence on the fate of fecal Bacteroidales cells and their DNA, presumably because the presence of oxygen significantly affected the viability and persistence of these obligate anaerobes. In conclusion, measuring Bacteroidales DNA in viable cells is recommended in applied MST studies because extracellular Bacteroidales DNA persists longer in the environment. The methods and results presented are helpful to improve the accuracy of MST applications, to develop a model of fate and transport of host-specific Bacteroidales, and to implement management practices to protect water quality.

Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts.

PubMed

Zhang, Ning; Fan, Yuxuan; Li, Chen; Wang, Qiming; Leksawasdi, Noppol; Li, Fuli; Wang, Shi'an

2018-05-01

Non-model yeasts within basidiomycetes have considerable importance in agriculture, industry, and environment, but they are not as well studied as ascomycetous yeasts. Serving as a basic technique, nuclear DNA staining is widely used in physiology, ecology, cell biology, and genetics. However, it is unclear whether the classical nuclear DNA staining method for ascomycetous yeasts is applicable to basidiomycetous yeasts. In this study, 5 yeasts ineffectively stained by the classical propidium iodide (PI) staining method were identified from 23 representative basidiomycetous yeasts. Pretreatment of cells using dimethyl sulfoxide (DMSO) or snailase markedly improved cell penetration to PI and thus enabled DNA content determination by flow cytometry on the recalcitrant yeasts. The pretreatments are efficient, simple, and fast, avoiding tedious mutagenesis or genetic engineering used in previous reports. The heterogeneity of cell penetration to PI among basidiomycetous yeasts was attributed to the discrepancy in cell wall polysaccharides instead of capsule or plasma membrane. This study also indicated that care must be taken in attributing PI-negative staining as viable cells when studying non-model microorganisms.

Simulation experiments to elucidate variable fluorescence as a potential proxy for bulk microalgal viability from natural water, sediments and biofilms: Implication in ships ballast water management.

PubMed

Patil, Jagadish S; Anil, Arga Chandrashekar

2018-05-30

The variable fluorescence fluorometry measuring microalgal biomass (initial fluorescence - F 0 , a chl-a proxy) and photosynthetic efficiency (F v /F m ) has been suggested as a potential tool in ballast-water assessment. In ballast tank, microalgae can be found in contiguous compartments i.e., in water, sediment, and biofilms. Therefore the utility of F 0 and F v /F m depends upon proper background corrections, which is straightforward for water samples but not for sediment and biofilms. This study proposes procedures for correcting F 0 values from sediment and biofilms. Irrespective of the saturation flash protocol used on any sample types the outcome of the results from viable and non-viable microalgae will remain same. Stress experiments (continuous darkness and biocide treatments) confirm that variable fluorescence (F v ) can be used as a potential proxy for viable cells as the values were negligible for non-viable cells and increased with an increase in abundance. Through this study, the utility of F v and σ PSII (functional-absorption-cross-section of photosystem II) along with F 0 and F v /F m in providing additional information on cell-viability and algal-size group during assessment is discussed. The findings will have implications not only from the perspective of ballast water but also in testing/assays of specific interest (e.g. toxicity, water treatments, antifouling) and ecological studies involving microalgae. Copyright © 2018 Elsevier Ltd. All rights reserved.

Correlation between apparent diffusion coefficient and histopathology subtypes of osteosarcoma after neoadjuvant chemotherapy.

PubMed

Wang, Jifei; Sun, Meili; Liu, Dawei; Hu, Xiaoshu; Pui, Margaret H; Meng, Quanfei; Gao, Zhenhua

2017-08-01

Background Neoadjuvant chemotherapy has made limb-salvage surgery possible for the patients with osteosarcoma. Diffusion-weighted magnetic resonance imaging (DWI) has been used to monitor chemotherapy response. Purpose To correlate the apparent diffusion coefficient (ADC) values with histopathology subtypes of osteosarcoma after neoadjuvant chemotherapy. Material and Methods Twelve patients with osteoblastic (n = 7), chondroblastic (n = 4), and fibroblastic (n = 1) osteosarcomas underwent post-chemotherapy DWI before limb-salvage surgery. ADCs corresponding to 127 histological tissue samples from the 12 resected specimens were compared to histological features. Results The mean ADC value of non-cartilaginous viable tumor (38/91, ADC = 1.22 ± 0.03 × 10 -3  mm 2 /s) was significantly ( P < 0.001) lower than that of non-cartilaginous tumor cell necrosis without stroma disintegration (25/91, ADC =1.77 ± 0.03 × 10 -3  mm 2 /s), cartilaginous viable tumor (14/91, ADC = 2.19 ± 0.04 × 10 -3  mm 2 /s), and cystic areas including liquefied necrosis, blood space, and secondary aneurysmal bone cyst (14/91, ADC = 2.29 ± 0.05 × 10 -3  mm 2 /s). The mean ADC value of non-cartilaginous tumor cell necrosis was also significantly ( P < 0.001) smaller than those of viable cartilaginous tumor and cystic/hemorrhagic necrosis whereas the mean ADC values were not significantly ( P > 0.05) different between viable cartilaginous tumor and cystic/hemorrhagic necrosis. Conclusion DWI allows assessment of tumor necrosis after neoadjuvant chemotherapy by ADC differences between viable tumor and necrosis in fibroblastic and osteoblastic osteosarcomas whereas viable chondroblastic osteosarcoma has high ADC and cannot be distinguished reliably from necrosis.

Smartphone-based rapid quantification of viable bacteria by single-cell microdroplet turbidity imaging.

PubMed

Cui, Xiaonan; Ren, Lihui; Shan, Yufei; Wang, Xixian; Yang, Zhenlong; Li, Chunyu; Xu, Jian; Ma, Bo

2018-05-18

Standard plate count (SPC) has been recognized as the golden standard for the quantification of viable bacteria. However, SPC usually takes one to several days to grow individual cells into a visible colony, which greatly hampers its application in rapid bacteria enumeration. Here we present a microdroplet turbidity imaging based digital standard plate count (dSPC) method to overcome this hurdle. Instead of cultivating on agar plates, bacteria are encapsulated in monodisperse microdroplets for single-cell cultivation. Proliferation of the encapsulated bacterial cell produced a detectable change in microdroplet turbidity, which allowed, after just a few bacterial doubling cycles (i.e., a few hours), enumeration of viable bacteria by visible-light imaging. Furthermore, a dSPC platform integrating a power-free droplet generator with smartphone-based turbidity imaging was established. As proof-of-concept demonstrations, a series of Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Bacillus subtilis) samples were quantified via the smartphone dSPC accurately within 6 hours, representing a detection sensitivity of 100 CFU ml-1 and at least 3 times faster. In addition, Enterobacter sakazakii (E. sakazakii) in infant milk powder as a real sample was enumerated within 6 hours, in contrast to the 24 hours needed in traditional SPC. Results with high accuracy and reproducibility were achieved, with no difference in counts found between dSPC and SPC. By enabling label-free, rapid, portable and low-cost enumeration and cultivation of viable bacteria onsite, smartphone dSPC forms the basis for a temporally and geographically trackable network for surveying live microbes globally where every citizen with a cellphone can contribute anytime and anywhere.

Application of the broad-spectrum bacteriocin enterocin AS-48 to inhibit Bacillus coagulans in canned fruit and vegetable foods.

PubMed

Lucas, R; Grande, M A J; Abriouel, H; Maqueda, M; Ben Omar, N; Valdivia, E; Martínez-Cañamero, M; Gálvez, A

2006-10-01

The enterococcal bacteriocin (enterocin) AS-48 is a broad-spectrum cyclic peptide. Enterocin AS-48 was tested against Bacillus coagulans in three vegetable canned foods: tomato paste (pH 4.64), syrup from canned peaches (pH 3.97), and juice from canned pineapple (pH 3.65). When vegetative cells of B. coagulans CECT (Spanish Type Culture Collection) 12 were inoculated in tomato paste supplemented with 6 microg/ml AS-48 and stored at different temperatures, viable cell counts were reduced by approximately 2.37 (4 degrees C), 4.3 (22 degrees C) and 3.0 (37 degrees C) log units within 24 h storage. After 15-days storage, no viable cells were detected in any sample. Strain B. coagulans CECT 561 showed a poor survival in tomato paste, but surviving cells were also killed by AS-48. The bacteriocin was also very active against B. coagulans CECT 12 vegetative cells in juice from canned pineapple stored at 22 degrees C, and slightly less active in syrup from canned peaches. In food samples supplemented with 1.5% lactic acid, enterocin AS-48 (6 microg/ml) rapidly reduced viable counts of vegetative cells below detection limits within 24 h storage. Addition of glucose and sucrose (10% and 20%) significantly increased bacteriocin activity against vegetative cells of B. coagulans CECT 12. Enterocin AS-48 had no significant effect on B. coagulans CECT 12 spores. However, the combined application of AS-48 and heat (80-95 degrees C for 5 min) significantly increased the effect of thermal treatments on spores.

Comparison of soymilk, powdered milk, Hank's balanced salt solution and tap water on periodontal ligament cell survival.

PubMed

Moazami, Fariborz; Mirhadi, Hosein; Geramizadeh, Bita; Sahebi, Safoura

2012-04-01

The purpose of this study was to evaluate the ability of soymilk, powdered milk, and Hank's balanced salt solution (HBSS) to maintain human periodontal ligament (PDL) cell viability in vitro. PDL cells were obtained from extracted healthy third molars and cultured in Dulbecco's modified Eagles medium (DMEM). The cultures were exposed for 1, 2, 4, and 8 h to experimental solutions (tap water served as negative control and DMEM as positive control) at 37°C. The viable cells were then counted using the trypan blue exclusion technique. Data were analyzed by using one-way anova, post hoc Scheffe and two-way anova test. Statistical analysis showed that HBSS, powdered baby formula, and soymilk maintain cell viability equally well in different periods of times. Tap water cannot keep cells viable as well as other solutions. Soymilk and powdered baby formula can be recommended as suitable storage media for avulsed teeth for up to 8 h. © 2011 John Wiley & Sons A/S.

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.

The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil.

PubMed

Sánchez-Aragó, María; Cuezva, José M

2011-02-08

Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU). The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells. We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable. Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells

PubMed Central

Swioklo, Stephen; Constantinescu, Andrei

2016-01-01

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C–23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 106 cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. Significance Despite considerable advancement in the clinical application of cell-based therapies, major logistical challenges exist throughout the cell therapy supply chain associated with the storage and distribution of cells between the sites of manufacture and the clinic. A simple, low-cost system capable of preserving the viability and functionality of human adipose-derived stem cells (a cell with substantial clinical interest) at hypothermic temperatures (0°C–32°C) is presented. Such a system has considerable potential for extending the shelf life of cell therapy products at multiple stages throughout the cell therapy supply chain. PMID:26826163

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

PubMed

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena; Mundkur, Lakshmi

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

PubMed Central

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856. PMID:29474436

Inactivation of selected bacterial pathogens in dairy cattle manure by mesophilic anaerobic digestion (balloon type digester).

PubMed

Manyi-Loh, Christy E; Mamphweli, Sampson N; Meyer, Edson L; Okoh, Anthony I; Makaka, Golden; Simon, Michael

2014-07-14

Anaerobic digestion of animal manure in biogas digesters has shown promise as a technology in reducing the microbial load to safe and recommended levels. We sought to treat dairy manure obtained from the Fort Hare Dairy Farm by investigating the survival rates of bacterial pathogens, through a total viable plate count method, before, during and after mesophilic anaerobic digestion. Different microbiological media were inoculated with different serial dilutions of manure samples that were withdrawn from the biogas digester at 3, 7 and 14 day intervals to determine the viable cells. Data obtained indicated that the pathogens of public health importance were 90%-99% reduced in the order: Campylobacter sp. (18 days) < Escherichia coli sp. (62 days) < Salmonella sp. (133 days) from a viable count of 10.1 × 103, 3.6 × 105, 7.4 × 103 to concentrations below the detection limit (DL = 102 cfu/g manure), respectively. This disparity in survival rates may be influenced by the inherent characteristics of these bacteria, available nutrients as well as the stages of the anaerobic digestion process. In addition, the highest p-value i.e., 0.957 for E. coli showed the statistical significance of its model and the strongest correlation between its reductions with days of digestion. In conclusion, the results demonstrated that the specific bacterial pathogens in manure can be considerably reduced through anaerobic digestion after 133 days.

Supply and Demand of Energy in the Oocyte and the Role of Mitochondria.

PubMed

Martin, Wilding

2017-01-01

The sole purpose of any mammalian oocyte is to combine with a spermatozoon and form a viable embryo that implants into the uterus and forms a viable foetus. Most of the structures and mechanisms for this reside within the oocyte itself. The sperm limits itself to fertilisation of the oocyte; apart from this, its only contribution is the male genome and the centrosome, required for cell division. Both intrinsic and extrinsic factors determine the formation of a viable embryo. However, the fundamental necessity for successful reproduction resides within the capacity for the developing embryo to generate sufficient levels of energy for optimal development to occur. Energy is generated principally within mitochondria. In this chapter, we discuss some of the fundamental processes of preimplantation embryo development and the role of mitochondria in providing sufficient energy for the successful completion of these processes. We discuss mitochondrial genetics, replication and energy production. Ageing appears to affect the capacity of the mitochondrion to produce sufficient energy to balance the requirements of the embryo. We discuss some of the theories of the effect of maternal age on mitochondrial physiology and the role this plays in reproduction. We propose that maternal age has longer-term effects on individuals than simply on the efficiency of reproduction. We also discuss some of the procedures assisted reproduction has proposed to alleviate the effect of maternal age on reproduction.

Spatiotemporal Dynamics of Total Viable Vibrio spp. in a NW Mediterranean Coastal Area.

PubMed

Girard, Léa; Peuchet, Sébastien; Servais, Pierre; Henry, Annabelle; Charni-Ben-Tabassi, Nadine; Baudart, Julia

2017-09-27

A cellular approach combining Direct Viable Counting and Fluorescent In Situ Hybridization using a one-step multiple-probe technique and Solid Phase Cytometry (DVC-FISH-SPC) was developed to monitor total viable vibrios and cover the detection of a large diversity of vibrios. FISH combined three probes in the same assay and targeted sequences located at different positions on the 16S rRNA of Vibrio and Aliivibrio members. We performed a 10-month in situ study to investigate the weekly dynamics of viable vibrios relative to culturable counts at two northwestern Mediterranean coastal sites, and identified the key physicochemical factors for their occurrence in water using a multivariate analysis. Total viable and culturable cell counts showed the same temporal pattern during the warmer season, whereas the ratios between both methods were inverted during the colder seasons (<15°C), indicating that some of the vibrio community had entered into a viable but non-culturable (VBNC) state. We confirmed that Seawater Surface Temperature explained 51-62% of the total variance in culturable counts, and also showed that the occurrence of viable vibrios is controlled by two variables, pheopigment (15%) and phosphate (12%) concentrations, suggesting that other unidentified factors play a role in maintaining viability.

Importance of airborne algae and protozoa

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

Schlichting, H.E. Jr.

1969-12-01

Membrane filters, bubblers, and exposed culture media were used to sample viable algae and protozoa from the atmosphere in Michigan, Texas, and North Carolina from 1956 to 1967. Aerial algae and protozoa were most abundant and diverse in North Central Texas, 0-8 cells/ft/sup 3/, less abundant and diverse in Michigan, 0-1.8 cells/ft/sup 3/, and least abundant in Coastal North Carolina, less than 0.41 cells/ft/sup 3/. Other significant research from 1910 to 1968 is reviewed. A total of 187 taxa of algae and protozoa has been sampled and cultured through this period. The importance of airborne algae and protozoa to manmore » is shown as related to allergies, radioactivity, clogging of air filters, an aid in determining the origin of hurricanes and other storms and adding to the understanding of the dispersal of these microorganisms throughout the world. 15 references, 2 tables.« less

The Viable But Nonculturable State of Ralstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection

PubMed Central

Grey, Brian E.; Steck, Todd R.

2001-01-01

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections. PMID:11525979

Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2016-06-01

Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

Method for procuring specific populations of viable human prostate cells for research.

PubMed

Fischer, A H; Philips, A; Taysavang, P; McKenney, J K; Amin, M B

2001-04-01

A wider range of research can be conducted on viable tissue samples than on fixed or frozen samples. A major obstacle to studying viable prostate tissue samples is the inability to accurately identify cancer on direct examination of unembedded tissue. We used a dissecting microscope to identify cancer in unfixed prostate tissue samples stained on the cut surface with 0.5% aqueous toluidine blue. We measured the diagnostic accuracy of this technique in 25 consecutive prostatectomies, determined the viability of procured samples, and estimated the effect on final pathologic assessment. Both surfaces of a 3- to 5-mm thick cross-section taken midway between base and apex of the prostate were examined. A 4-mm punch biopsy was directed to one benign and one malignant area when clearly present. The dissecting microscope allowed clearcut recognition of carcinoma in 17 of the 25 cross-sections, and carcinoma was confirmed in all 17 (100%). In 8 of 25 cases, no procurement was attempted because no carcinoma was evident in the one cross-section studied. Twenty of 25 cross-sections were adequate for benign tissue procurement; five of the cross-sections were not suitable for procurement because of the presence of extensive carcinoma or atrophy. Seventeen of the 20 were accurately diagnosed as benign (85%); one showed pseudohyperplastic adenocarcinoma, one showed focal high-grade prostatic intraepithelial neoplasia, and one showed urothelial carcinoma in situ. Prostatic epithelium obtained with the technique remains viable and can be separated from stroma. The dissecting microscope technique appears to facilitate rather than interfere with accurate pathologic assessment: extraprostatic extension or positive margins were correctly identified during tissue procurement in three cases. The procedure takes only about 30 minutes.

Evaluation of results of cell electrophoresis experiments on space shuttle STS-3 including pre-flight and post-flight laboratory experiments

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

The objectives of the red blood cell experiments were to provide a visual check on the electrophoretic process and especially electroosmotic flow in space as well as to provide test separations of non-degradable standard particles for comparison with the separations of the three viable cell types studied on the Apollo-Soyuz Test Project. Determination of the maximum concentrations of cells that can be separated in column electrophore was a significant goal. Two of the eight columns were available for red cell experiments, so two concentrations of human and rabbit RBC mixtures were used. The objectives of another experiment were to evaluate the reproducibility of microgravity electrophoretic separation of living kidney cells, to separate cells with highly viability despite two freeze-thaw cycles, and to optimize the physical conditions of cell separation. Owing to the uncertain heterogeneity of the starting material, the experimental design does not assess resolution in microgravity, but improved separability was sought in comparison to density-gradient electrophoresis or continuous-flow electrophoresis. Efforts were made to increase cell yield and cell viability and to assess reproducibility directly.

The inhibition of Caco-2 proliferation by astaxanthin from Xanthophyllomyces dendrorhous.

PubMed

Wayakanon, Kornchanok; Rueangyotchanthana, Kanjana; Wayakanon, Praween; Suwannachart, Chatrudee

2018-04-01

To investigate the efficiency of natural astaxanthin that has been extracted from Xanthophyllomyces dendrorhous in inhibiting the proliferation and viability of colorectal adenocarcinoma cell line (Caco-2; colon cancer cells). Caco-2 cells and normal human oralkeratinocytes (NOKs) were treated with different concentrations of extracted astaxanthin, ranging from 0.075 to 10 mg ml -1 , for 24, 48 and 72 h. The number of cells was determined via MTS assay and the proliferating cells were investigated by bromodeoxyuridine (BrdU) assay.Results/Key findings. Of the Caco-2 cells, 30-50 % remained viable, while the NOKs showed 110-120 % survival when treated with 5 mg ml -1 astaxanthin. The Caco-2 cells showed distinct structural shrinkage when treated with the same concentration of astaxanthin. Fluorescent labelling of the DNA of the proliferative cells with BrdU showed a significant decrease in the number of the proliferative Caco-2 cells when the concentration of astaxanthin was increased to 5 mg ml -1 . The natural astaxanthin from X. dendrorhous, at an appropriate concentration, is effective in terminating the viability of, or retarding the proliferative activity of, Caco-2 cells, without harmful effects on NOKs.

Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

PubMed Central

de la Cruz, J; Vioque, A

2001-01-01

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628

The BioStent: novel concept for a viable stent structure.

PubMed

Weinandy, Stefan; Rongen, Lisanne; Schreiber, Fabian; Cornelissen, Christian; Flanagan, Thomas Cormac; Mahnken, Andreas; Gries, Thomas; Schmitz-Rode, Thomas; Jockenhoevel, Stefan

2012-09-01

Percutaneous stenting of occluded peripheral vessels is a well-established technique in clinical practice. Unfortunately, the patency rates of small-caliber vessels after stenting remain unsatisfactory. The aim of the BioStent concept is to overcome in-stent restenosis by excluding the diseased vessel segment entirely from the blood stream, in addition to providing an intact endothelial cell layer. The concept combines the principles of vascular tissue engineering with a self-expanding stent: casting of the stent within a cellularized fibrin gel structure, followed by bioreactor conditioning, allows complete integration of the stent within engineered tissue. Small-caliber BioStents (Ø=6 mm; n=4) were produced by casting a nitinol stent within a thin fibrin/vascular smooth muscle cell (vSMC) mixture, followed by luminal endothelial cell seeding, and conditioning of the BioStent within a bioreactor system. The potential remodeling of the fibrin component into tissue was analyzed using routine histological methods. Scanning electron microscopy was used to assess the luminal endothelial cell coverage following the conditioning phase and crimping of the stent. The BioStent was shown to be noncytotoxic, with no significant effect on cell proliferation. Gross and microscopic analysis revealed complete integration of the nitinol component within a viable tissue structure. Hematoxylin and eosin staining revealed a homogenous distribution of vSMCs throughout the thickness of the BioStent, while a smooth, confluent luminal endothelial cell lining was evident and not significantly affected by the crimping/release process. The BioStent concept is a platform technology offering a novel opportunity to generate a viable, self-expanding stent structure with a functional endothelial cell lining. This platform technology can be transferred to different applications depending on the luminal cell lining required.

The effects of temperature upon the electrophysiological properties of Tetrahymena pyriformis-NT1.

PubMed

Connolly, J G; Brown, I D; Lee, A G; Kerkut, G A

1985-01-01

Cells of Tetrahymena pyriformis--NT1 were cultured at 38 degrees C (Tg 38 degrees C) and 20 degrees C (Tg 20 degrees C) and their properties investigated over the range 0-40 degrees C. Tg 20 degrees C cells were viable in the range 3-33 degrees C and changes in their properties were readily reversible between 10 degrees C and 30 degrees C. Tg 38 degrees cells were viable in the range 40-10 degrees C and their property changes were immediately reversible in the range 40-23 degrees C. The I-V relations of Tg 38 degrees C cells showed increased excitability as the cells were cooled from 40 degrees C. At 10 degrees C there was a considerable loss of excitability and slope resistance. Cooling Tg 20 degrees C cells from 20 degrees C gave a similar pattern, although over a narrower temperature range. Warming Tg 20 degrees C Tetrahymena above 20 degrees C led to a progressive loss of excitability and the cells were markedly less viable above 35 degrees C. Within physiological limits the regenerative spike magnitude, repolarization time, time to peak and input resistance increased as temperature was lowered, whereas resting potential was diminished. When compared at their growth temperatures and most intermediate temperatures, the value of the various parameters monitored were generally different for the two cultures. The Q10 value for resting potential changes of Tg 20 degrees C cells about 20 degrees C was 1.20. As in T. vorax this was significantly (P less than 0.01) greater than that predicted for a diffusion potential and suggested that T. pyriformis--NT1 may have an electrogenic pump component in its membrane potential.

A Quantitative Analysis of a Probiotic Storage Media for Avulsed Teeth

PubMed Central

2015-01-01

Aim The aim of the present in vitro study was to investigate the potential of a storage medium, probiotic yogurt (Bifidibacterium animalis DN 173010) in comparison with Hank's balanced salt solution (HBSS), saline and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Materials and methods Thirty-six freshly extracted single-rooted human teeth with closed apices were divided into six experimental groups (N=6). The teeth were extracted as atraumatically as possible and washed in sterile saline solution to eliminate residual blood. Following extractions, the coronal 3 mm of PDL tissues were scraped with a #15 scalpel to remove cells that may have been damaged. The positive and negative controls corresponded to 0 minutes and an 8-hour dry time, respectively. After extraction, the positive control teeth were immediately treated with dispase and collagenase. The negative control teeth were bench-dried for 8 h, with no follow-up storage solution time, and then placed in the dispase and collagenase. The number of viable protective least significant difference PDL cells were counted under a light microscope with a hemocytometer at 20× magnification and analyzed. Statistical analysis of the data was accomplished using Nonparametric ANOVA complemented by Kruskal-Wallis Test and Dunn's Multiple Comparisons Test. Results Positive control was found to be significantly better than the others, there were statistically significant differences between positive control and other test groups (p=0.000). The teeth stored in positive control demonstrated the highest number of viable PDL cells followed in order by probiotic yogurt, HBSS, saline and milk. Conclusion Bifidibacterium animalis DN 173010 seems to be an alternative for the temporary storage of avulsed teeth, due to high number of viable PDL cells. Probiotics may be suitable transport media for avulsed teeth, but further research is warranted using the commercially available products. PMID:27688382

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

PubMed

Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

Microbiological and experimental-histological investigations of lunar samples returned by the Lunar 16 automatic station

NASA Technical Reports Server (NTRS)

Kaulen, D. R.; Bulatova, T. I.; Fridenshteyn, A. Y.; Skvortsova, Y. B.

1974-01-01

Lunar surface material was studied for its content of viable microorganisms (aerobic and anaerobic, fungi, and viruses); the effect of the lunar surface material on the growth of microorganisms and its interaction with somatic cells of mammals was also observed. No viable microorganisms were detected; the samples exhibited neither stimulant or inhibitory action on the growth of microorganisms, and also showed no cytopathogenic action on tissue cultures. A suspension of lunar surface material particles was not toxic when parenterally administered to certain laboratory animals. The particles were subjected to intense phagocytosis by connective tissue cells in vivo and in vitro.

Frex and FrexH: Indicators of metabolic states in living cells.

PubMed

Zhao, Yuzheng; Yang, Yi

2012-01-01

Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form play central roles in energy and redox metabolisms. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level in living cells. We recently reported a series of genetically encoded fluorescent sensors highly specific for NADH. These sensors allow real-time, quantitative measurement of this significant molecule in different subcellular compartments. In this study, we provide a more detailed discussion of the benefits and limitations of these genetically encoded fluorescent sensors. These sensors are utilized in most laboratories without the need for sophisticated instruments because of their superior sensitivity and specificity. They are also viable alternatives to existing techniques for measuring the endogenous fluorescence of intracellular NAD(P)H.

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

PubMed

Agustí, Gemma; Fittipaldi, Mariana; Codony, Francesc

2018-06-01

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

Successful liver allografts in mice by combination with allogeneic bone marrow transplantation.

PubMed Central

Nakamura, T; Good, R A; Yasumizu, R; Inoue, S; Oo, M M; Hamashima, Y; Ikehara, S

1986-01-01

Successful liver allografts were established by combination with allogeneic bone marrow transplantation. When liver tissue of BALB/c (H-2d) or C57BL/6J (H-2b) mice was minced and grafted under the kidney capsules of C3H/HeN (H-2k) mice, it was rejected. However, when C3H/HeN mice were irradiated and reconstituted with T-cell-depleted BALB/c or BALB/c nu/nu bone marrow cells, or with fetal liver cells of BALB/c mice, they accepted both donor (stem-cell)-type (BALB/c) and host (thymus)-type (C3H/HeN) liver tissue. Assays for both mixed-lymphocyte reaction and induction of cytotoxic T lymphocytes revealed that the newly developed T cells were tolerant of both donor (stem-cell)-type and host (thymus)-type major histocompatibility complex determinants. We propose that liver allografts combined with bone marrow transplantation should be considered as a viable therapy for patients with liver disease such as liver cirrhosis and hepatoma. Images PMID:3520575

[Enhanced ε-poly-L-lysine production by improving cellular activity during fermentation].

PubMed

Liu, Shengrong; Wu, Qingping; Zhang, Jumei; Yang, Xiaojuan; Cai, Shuzhen

2015-06-04

To assess the effect of cellular activity on ε-poly-1-lysine (ε-PL) biosynthesis and thereby to rationally improve the production, we studied the cellular activity, ε-PL formation and other parameters cross flask fermentation by Streptomyces ahygroscopicus. Laser scanning confocal microscopy and a colorimetric method were used to determine cellular activity using BacLight Live/Dead and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as viable stains. To enhance the activity of the cells in the ε-PL production period, yeast extract was added. During ε-PL submerged fermentation in flasks, most cells were active in the growth period (0 - 16 h); cells had metabolic activity in the growth and earlier ε-PL production periods between 0 and 30 h fermentation. Almost no activity was detected after 48 h fermentation when no ε-PL was produced. The improved fermentation achieved 2. 24 g/L ε-PL from 1.04 g/L. Biosynthesis of ε-PL can be boosted by up-regulating cell activity in its production phase.

Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

PubMed Central

Webb, Jeremy S.; Barratt, Sarah R.; Sabev, Hristo; Nixon, Marianne; Eastwood, Ian M.; Greenhalgh, Malcolm; Handley, Pauline S.; Robson, Geoffrey D.

2001-01-01

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r2 > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml−1 and 500 μg ml−1, respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r2 > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds. PMID:11722914

Survivability of Psychrobacter cryohalolentis K5 Under Simulated Martian Surface Conditions

NASA Technical Reports Server (NTRS)

Smith, David J.; Schuerger, Andrew C.; Davidson, Mark M.; Pacala, Stephen W.; Bakermans, Corien; Onstott, Tullis

2008-01-01

Spacecraft launched to Mars can retain viable terrestrial microorganisms on board that may survive the interplanetary transit. Such biota might compromise the search for life beyond Earth if capable of propagating on Mars. The current study explored the survivability of Psychrobacter cryohalolentis K5, a psychrotolerant microorganism obtained from a Siberian permafrost cryopeg, under simulated martian surface conditions of high ultraviolet irradiation, high desiccation, low temperature, and low atmospheric pressure. First, a desiccation experiment compared the survival of P. cryohalolentis cells embedded, or not embedded, within a medium/salt matrix (MSM) maintained at 25 degrees C for 24 hr within a laminar flow hood. Results indicate that the presence of the MSM enhanced survival of the bacterial cells by 1 to 3 orders of magnitude. Second, tests were conducted in a Mars Simulation Chamber to determine the UV tolerance of the microorganism. No viable vegetative cells of P. cryohalolentis were detected after 8 hr of exposure to Mars-normal conditions of 4.55 W/m(2) UVC irradiation (200-280 nm), -12.5 degrees C, 7.1 mbar, and a Mars gas mix composed of CO2 (95.3%), N2 (2.7%), Ar (1.6%), O2 (0.2%), and H(2)O (0.03%). Third, an experiment was conducted within the Mars chamber in which total atmospheric opacities were simulated at tau = 0.1 (dust-free CO2 atmosphere at 7.1 mbar), 0.5 (normal clear sky with 0.4 = dust opacity and 0.1 = CO2-only opacity), and 3.5 (global dust storm) to determine the survivability of P. cryohalolentis to partially shielded UVC radiation. The survivability of the bacterium increased with the level of UVC attenuation, though population levels still declined several orders of magnitude compared to UVC-absent controls over an 8 hr exposure period.

Survivability of Psychrobacter cryohalolentis K5 Under Simulated Martian Surface Conditions

NASA Astrophysics Data System (ADS)

Smith, David J.; Schuerger, Andrew C.; Davidson, Mark M.; Pacala, Stephen W.; Bakermans, Corien; Onstott, Tullis C.

2009-03-01

Spacecraft launched to Mars can retain viable terrestrial microorganisms on board that may survive the interplanetary transit. Such biota might compromise the search for life beyond Earth if capable of propagating on Mars. The current study explored the survivability of Psychrobacter cryohalolentis K5, a psychrotolerant microorganism obtained from a Siberian permafrost cryopeg, under simulated martian surface conditions of high ultraviolet irradiation, high desiccation, low temperature, and low atmospheric pressure. First, a desiccation experiment compared the survival of P. cryohalolentis cells embedded, or not embedded, within a medium/salt matrix (MSM) maintained at 25°C for 24 h within a laminar flow hood. Results indicate that the presence of the MSM enhanced survival of the bacterial cells by 1 to 3 orders of magnitude. Second, tests were conducted in a Mars Simulation Chamber to determine the UV tolerance of the microorganism. No viable vegetative cells of P. cryohalolentis were detected after 8 h of exposure to Mars-normal conditions of 4.55 W/m2 UVC irradiation (200-280 nm), -12.5°C, 7.1 mbar, and a Mars gas mix composed of CO2 (95.3%), N2 (2.7%), Ar (1.6%), O2 (0.2%), and H2O (0.03%). Third, an experiment was conducted within the Mars chamber in which total atmospheric opacities were simulated at τ = 0.1 (dust-free CO2 atmosphere at 7.1 mbar), 0.5 (normal clear sky with 0.4 = dust opacity and 0.1 = CO2-only opacity), and 3.5 (global dust storm) to determine the survivability of P. cryohalolentis to partially shielded UVC radiation. The survivability of the bacterium increased with the level of UVC attenuation, though population levels still declined several orders of magnitude compared to UVC-absent controls over an 8 h exposure period.

Toward An Affordable Commercial Fuel Cell (LBNL Summer Lecture Series)

ScienceCinema

Visco, Steve [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Materials Sciences Division

2018-02-16

Steve Visco, a materials scientist, has come up with a solid oxide fuel cell that promises to generate electricity as cheaply as the most efficient gas turbine engine. But there's a lot more work to do before commercially viable fuel cells and pollution-free power generators become reality.

21 CFR 1271.75 - How do I screen a donor?

Code of Federal Regulations, 2010 CFR

2010-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES..., you must screen a donor of cells or tissue by reviewing the donor's relevant medical records for: (1...) Communicable disease risks associated with xenotransplantation. (b) Donors of viable, leukocyte-rich cells or...

21 CFR 1271.75 - How do I screen a donor?

Code of Federal Regulations, 2014 CFR

2014-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES..., you must screen a donor of cells or tissue by reviewing the donor's relevant medical records for: (1...) Communicable disease risks associated with xenotransplantation. (b) Donors of viable, leukocyte-rich cells or...

Cellular and soluble components decrease the viable pathogen counts in milk from dairy cows with subclinical mastitis.

PubMed

Koshiishi, Tomoko; Watanabe, Masako; Miyake, Hajime; Hisaeda, Keiichi; Isobe, Naoki

2017-08-10

The present study was undertaken to clarify the factors that reduce the viable pathogen count in milk collected from the udders of subclinical mastitic cows during preservation. Milk was centrifuged to divide somatic cells (cellular components, precipitates) and antimicrobial peptides (soluble components, supernatants without fat layer); each fraction was cultured with bacteria, and the number of viable bacteria was assessed prior to and after culture. In 28.8% of milk samples, we noted no viable bacteria immediately after collection; this value increased significantly after a 5-hr incubation of milk with cellular components but not with soluble components (48.1 and 28.8%, respectively). After culture with cellular components, the numbers of bacteria (excluding Staphylococcus aureus and Streptococcus uberis) and yeast decreased dramatically, although the differences were not statistically significant. After cultivation with soluble components, only yeasts showed a tendency toward decreased mean viability, whereas the mean bacterial counts of S. uberis and T. pyogenes tended to increase after 5-hr preservation with soluble components. These results suggest that most pathogens in high somatic cell count (SCC) milk decreased during preservation at 15 to 25°C, due to both the cellular components and antimicrobial components in the milk. Particularly, the cellular components more potently reduced bacterial counts during preservation.

Viable but non-culturable Listeria monocytogenes on parsley leaves and absence of recovery to a culturable state.

PubMed

Dreux, N; Albagnac, C; Federighi, M; Carlin, F; Morris, C E; Nguyen-the, C

2007-10-01

To investigate the presence of viable but non-culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. Under low RH (47-69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (10(9) L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3-4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. Enumeration on culture media presumably under-estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

The prognostic value of functional and anatomical parameters for the selection of patients receiving yttrium-90 microspheres for the treatment of liver cancer

NASA Astrophysics Data System (ADS)

Mesoloras, Geraldine

Yttrium-90 (90Y) microsphere therapy is being utilized as a treatment option for patients with primary and metastatic liver cancer due to its ability to target tumors within the liver. The success of this treatment is dependent on many factors, including the extent and type of disease and the nature of prior treatments received. Metabolic activity, as determined by PET imaging, may correlate with the number of viable cancer cells and reflect changes in viable cancer cell volume. However, contouring of PET images by hand is labor intensive and introduces an element of irreproducibility into the determination of functional target/tumor volume (FTV). A computer-assisted method to aid in the automatic contouring of FTV has the potential to substantially improve treatment individualization and outcome assessment. Commercial software to determine FTV in FDG-avid primary and metastatic liver tumors has been evaluated and optimized. Volumes determined using the automated technique were compared to those from manually drawn contours identified using the same cutoff in the standard uptake value (SUV). The reproducibility of FTV is improved through the introduction of an optimal threshold value determined from phantom experiments. Application of the optimal threshold value from the phantom experiments to patient scans was in good agreement with hand-drawn determinations of the FTV. It is concluded that computer-assisted contouring of the FTV for primary and metastatic liver tumors improves reproducibility and increases accuracy, especially when combined with the selection of an optimal SUV threshold determined from phantom experiments. A method to link the pre-treatment assessment of functional (PET based) and anatomical (CT based) parameters to post-treatment survival and time to progression was evaluated in 22 patients with colorectal cancer liver metastases treated using 90Y microspheres and chemotherapy. The values for pre-treatment parameters that were the best predictors of response were determined for FTV, anatomical tumor volume, total lesion glycolysis, and the tumor marker, CEA. Of the parameters considered, the best predictors of response were found to be pre-treatment FTV ≤153 cm3, ATV ≤163 cm3, TLG ≤144 g in the chemo-SIRT treated field, and CEA ≤11.6 ng/mL.

Piper betle extracts exhibit antitumor activity by augmenting antioxidant potential

PubMed Central

ALAM, BADRUL; MAJUMDER, RAJIB; AKTER, SHAHINA; LEE, SANG-HAN

2015-01-01

The present study was conducted to evaluate the methanolic extract of Piper betle leaves (MPBL) and its organic fractions with regard to antitumor activity against Ehrlich ascites carcinoma (EAC) in Swiss albino mice and to confirm their antioxidant activities. At 24 h post-intraperitoneal inoculation of tumor cells into mice, extracts were administered at 25, 50 and 100 mg/kg body weight for nine consecutive days. The antitumor effects of the extracts were then assessed according to tumor volume, packed cell count, viable and non-viable tumor cell count, median survival time and increase in life span of EAC-bearing mice. Next, hematological profiles and serum biochemical parameters were calculated, and antioxidant properties were assessed by estimating lipid peroxidation, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels. MPBL and the ethylacetate fraction (EPBL) at a dose of 100 mg/kg induced a significant decrease in tumor volume, packed cell volume and viable cell count and increased the life span of the EAC-bearing mice (P<0.05). Hematological and serum biochemical profiles were restored to normal levels in the extract-treated mice compared with the EAC control mice. MPBL and EPBL treatment significantly decreased lipid peroxidation (P<0.05) and restored GSH, SOD and CAT levels towards normal compared with the EAC control. Taken together, the results of the present study demonstrated that Piper betle extracts exhibit significant antitumor activity, which may be attributed to the augmentation of endogenous antioxidant potential. PMID:25624910

Piper betle extracts exhibit antitumor activity by augmenting antioxidant potential.

PubMed

Alam, Badrul; Majumder, Rajib; Akter, Shahina; Lee, Sang-Han

2015-02-01

The present study was conducted to evaluate the methanolic extract of Piper betle leaves (MPBL) and its organic fractions with regard to antitumor activity against Ehrlich ascites carcinoma (EAC) in Swiss albino mice and to confirm their antioxidant activities. At 24 h post-intraperitoneal inoculation of tumor cells into mice, extracts were administered at 25, 50 and 100 mg/kg body weight for nine consecutive days. The antitumor effects of the extracts were then assessed according to tumor volume, packed cell count, viable and non-viable tumor cell count, median survival time and increase in life span of EAC-bearing mice. Next, hematological profiles and serum biochemical parameters were calculated, and antioxidant properties were assessed by estimating lipid peroxidation, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels. MPBL and the ethylacetate fraction (EPBL) at a dose of 100 mg/kg induced a significant decrease in tumor volume, packed cell volume and viable cell count and increased the life span of the EAC-bearing mice (P<0.05). Hematological and serum biochemical profiles were restored to normal levels in the extract-treated mice compared with the EAC control mice. MPBL and EPBL treatment significantly decreased lipid peroxidation (P<0.05) and restored GSH, SOD and CAT levels towards normal compared with the EAC control. Taken together, the results of the present study demonstrated that Piper betle extracts exhibit significant antitumor activity, which may be attributed to the augmentation of endogenous antioxidant potential.

Three-Dimensional Bioreactor Technologies for the Cocultivation of Human Mesenchymal Stem/Stromal Cells and Beta Cells

PubMed Central

Petry, Florian; Weidner, Tobias; Salzig, Denise

2018-01-01

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy. PMID:29731775

Reproduction of Varroa destructor and offspring mortality in worker and drone brood cells of Africanized honey bees.

PubMed

Calderón, R A; Ureña, S; van Veen, J W

2012-04-01

Varroa destructor is known to be the most serious parasite of Apis mellifera worldwide. In order to reproduce varroa females enter worker or drone brood shortly before the cell is sealed. From March to December 2008, the reproductive rate and offspring mortality (mature and immature stages), focusing on male absence and male mortality of V. destructor, was investigated in naturally infested worker and drone brood of Africanized honey bees (AHB) in Costa Rica. Data were obtained from 388 to 403 single infested worker and drone brood cells, respectively. Mite fertility in worker and drone brood cells was 88.9 and 93.1%, respectively. There was no difference between the groups (X(2) = 3.6, P = 0.06). However, one of the most significant differences in mite reproduction was the higher percentage of mites producing viable offspring in drone cells (64.8%) compared to worker cells (37.6%) (X(2) = 57.2, P < 0.05). A greater proportion of mites in worker brood cells produced non-viable female offspring. Mite offspring mortality in both worker and drone cells was high in the protonymph stage (mobile and immobile). A significant finding was the high rate of male mortality. The worker and drone brood revealed that 23.9 and 6.9%, respectively, of the adult male offspring was found dead. If the absence (missing) of the male and adult male mortality are taken together the percentage of cells increased to 40.0 and 21.3% in worker and drone cells, respectively (X(2) = 28.8, P < 0.05). The absence of the male or male mortality in a considerable number of worker cells naturally infested with varroa is the major factor in our study which reduces the production of viable daughters in AHB colonies in Costa Rica.

Application of orange essential oil as an antistaphylococcal agent in a dressing model.

PubMed

Muthaiyan, Arunachalam; Biswas, Debabrata; Crandall, Philip G; Wilkinson, Brian J; Ricke, Steven C

2012-08-16

Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

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

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.

1987-05-01

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cellsmore » (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.« less

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

PubMed

Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

2016-06-01

Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

Use of donor bladder tissues for in vitro research.

PubMed

Garthwaite, Mary; Hinley, Jennifer; Cross, William; Warwick, Ruth M; Ambrose, Anita; Hardaker, Henry; Eardley, Ian; Southgate, Jennifer

2014-01-01

To evaluate deceased non-heart beating (DNHB) donors and deceased heart beating (DHB) brain-stem dead donors, as sources of viable urological tissue for use in biomedical research. To identify sources of viable human bladder tissue as an essential resource for cell biological research aimed at understanding human diseases of the bladder and for developing new tissue engineering and regenerative medicine strategies for bladder reconstruction. Typically, normal human urinary tract tissue is obtained from adult or paediatric surgical patients with benign urological conditions, but few surgical procedures yield useful quantities of healthy bladder tissue for research. Research ethics committee approval was obtained for collection of donor bladder tissue. Consent for DHB donors was undertaken by the Donor Transplant Coordinators. Tissue Donor Coordinators were responsible for consent for DNHB donors and the retrieval of bladders was coordinated through the National Blood Service Tissue Banking Service. All retrievals were performed by practicing urologists and care was taken to maintain sterility and to minimise bacterial contamination. Two bladders were retrieved from DNHB donors and four were retrieved from DHB donors. By histology, DNHB donor bladder tissue exhibited marked urothelial tissue damage and necrosis, with major loss or absence of urothelium. No cell cultures could be established from these specimens, as the urothelial cells were not viable in primary culture. Bladder urothelium from DHB donors was intact, but showed some damage, including loss of superficial cells and variable separation from the basement membrane. All four DHB bladder specimens yielded viable urothelial cells that attached in primary culture, but cell growth was slow to establish and cultures showed a limited capacity to form a functional barrier epithelium and a propensity to senesce early. We have shown that normal human bladder urothelial cell cultures can be established and serially propagated from DHB donor bladders. However, our study suggests that rapid post-mortem changes to the bladder affect the quality and viability of the urothelium, rendering tissue from DNHB donors an inadequate source for urothelial cell culture. Our experience is that whereas patients are willing to donate surgical tissue for research, there is a barrier to obtaining consent from next of kin for retrieved tissues to be used for research purposes. © 2013 The Authors. BJU International © 2013 BJU International.

Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

PubMed

Burnat, Mireia; Diestra, Elia; Esteve, Isabel; Solé, Antonio

2010-01-01

In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

Assessment of the commercial viability of selected options for on-orbit servicing (OOS)

NASA Astrophysics Data System (ADS)

Graham, Andrew Robert; Kingston, Jennifer

2015-12-01

The aim of this paper is to determine the commercial viability of on-orbit servicing of communications satellites in geostationary orbit. Previous studies have shown the technical feasibility of servicing as well as the financial viability of some missions, in particular refuelling, therefore this paper analyses these repair missions and life extension missions. A simple parametric model for simulating communications satellite revenue streams is developed and the results are used to determine the maximum possible revenue for a servicing satellite operator. From this, the maximum cost of the servicing satellite as a proportion of the cost of the communications satellite is determined under three profit scenarios representing zero profit, acceptable profit to an operator in a mature industry and acceptable profit to an operator in an emerging industry. The results show that while servicing is financially viable (zero profit scenario), those missions which result in an increase in Comsat life which is a multiple of the mission duration are more likely to be commercially viable. Refuelling is therefore viable in all cases but life extension in most cases is only marginally viable. Repair missions to satellites which are partially operable are also unlikely to deliver sufficient value to justify carrying out servicing. Also the timing of a servicing mission in relation to the expected remaining life expectancy is a major factor in determining the mission's viability.

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

Goven, A.J.; Fitzpatrick, L.C.; Eyambe, G.S.

Acute toxicity in earthworms (Lumbricus terrestris) was assayed immediately after 5-d filter paper exposure to the polychlorinated biphenyl (PCB) Aroclor 1254, using coelomocyte viability, total extruded cell counts (ECC), differential cell counts (DCC), and formation of erythrocyte (ER) and secretory rosettes (SR) with, and phagocytosis of, antigenic rabbit red blood cells (RRBC). Chronic toxicity was assayed using rates by which earthworms replaced viable immunoactive coelomocytes, removed noninvasively immediately after exposure, over an 18-week depuration period. All cytological parameters, except ECC, were acutely affected immediately after exposure, when tissue concentrations were ([anti X] [plus minus] SE) 91.2 [plus minus] 8.19 [mu]gmore » PCB per gram dry mass. Replacement of viable immunoactive coelomocytes occurred within six weeks in unexposed control earthworms. Exposed earthworms showed significant alteration in viability, ECC, DCC, ER, and SR formation, and phagocytosis at 6 and 12 weeks when PCB tissue concentrations were 41 [plus minus] 0.31 and 30.2 [plus minus] 0.88 [mu]g/g dry mass, respectively. Replacement of extruded coelomocytes with normal DCC of viable immunocompetent cells was not observed until week 18, when PCB had decreased to 15.7 [plus minus] 0.83 [mu]g/g dry mass. Low inherent natural variability in coelomocyte viability, ECC, DCC, rosette formation, and phagocytosis, and their sensitivity to sublethal PCB body burdens, indicated that earthworm coelomocytes have potential as nonmammalian biomarkers for assaying acute and chronic sublethal toxicity of xenobiotics.« less

Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants.

PubMed

Overney, Anaïs; Jacques-André-Coquin, Joséphine; Ng, Patricia; Carpentier, Brigitte; Guillier, Laurent; Firmesse, Olivier

2017-03-06

The ability of Listeria monocytogenes to adhere to and persist on surfaces for months or even years may be responsible for its transmission from contaminated surfaces to food products. Hence the necessity to find effective means to prevent the establishment of L. monocytogenes in food processing environments. The aim of this study was to assess, through a fractional experimental design, the environmental factors that could affect the survival of L. monocytogenes cells on surfaces to thereby prevent the persistence of this pathogen in conditions mimicking those encountered in food processing plants: culture with smoked salmon juice or meat exudate, use of two materials with different hygiene status, biofilm of L. monocytogenes in pure-culture or dual-culture with a Pseudomonas fluorescens strain, application of a drying step after cleaning and disinfection (C&D) and comparison of two strains of L. monocytogenes. Bacterial survival was assessed by culture, qPCR to quantify total cells, and propidium monoazide coupled with qPCR to quantify viable cells and highlight viable but non-culturable (VBNC) cells. Our results showed that failure to apply C&D causes cell persistence on surfaces. Moreover, the sanitation procedure leads only to a loss of culturability and appearance of VBNC populations. However, an additional daily drying step after C&D optimises the effectiveness of these procedures to reduce culturable populations. Our results reinforce the importance to use molecular tools to monitor viable pathogens in food processing plants to avoid underestimating the amounts of cells using only methods based on cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of platelet-rich fibrin as an autologous biologic rejuvenating media for avulsed teeth - an in vitro study.

PubMed

Hiremath, Hemalatha; Kulkarni, Sadanand; Sharma, Robin; Hiremath, Vishwanath; Motiwala, Tejas

2014-12-01

The prognosis of replanted avulsed tooth depends on the existence of viable cells in the periodontal ligament and also on those cells which are able to proliferate on the damaged areas of the root. The purpose of this study was to evaluate the survival of periodontal ligament cells (PDL) when soaked in an autologous biologic rejuvenating media after an extra-oral dry time of 40 min. Thirty teeth were selected with intact crown which were advised for Orthodontic extraction having healthy PDL. They were divided into two experimental and two control groups. The positive and negative controls corresponded to 0-min and 1-h dry time, respectively. The experimental teeth were stored dry for 40 min and then immersed in one of the two media, combination of platelet-rich fibrin and platelet poor plasma (PRF+PPP) and PPP for 45 min. The teeth in each group were treated with dispase II and collagenase for 30 min and later centrifuged for 5 min at 50.17 g. The supernatant was removed with sterile micropipette, the cells labelled with 0.4% trypan blue, and the number of viable PDL cells was counted with a haemocytometer, under a light microscope. anova and Mann-Whitney U-test demonstrated statistically significant differences in the viability of PDL cells among experimental groups. Within the parameters of this study, a combination of platelet-rich fibrin and PPP demonstrated higher number of viable PDL cells and hence could be a good biologic rejuvenating media for avulsed teeth. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Intestinal stem cells remain viable after prolonged tissue storage

PubMed Central

Fuller, Megan K.; Faulk, Denver M.; Sundaram, Nambirajan; Mahe, Maxime M.; Stout, Kara M.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Shroyer, Noah F.; Helmrath, Michael A.; Henning, Susan J.

2013-01-01

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such they have significant therapeutic potential. However, it is unknown whether ISCs can survive tissue storage. We hypothesized that, although the majority of epithelial cells may die, ISCs would remain viable for at least 24 h at 4°C. To explore this hypothesis, jejuni of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate buffered saline (PBS) at 4°C. Delayed isolations of epithelia were performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact through 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retain high integrity through 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Culture of isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80%. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated with human tissue, storage at 4°C may offer a valuable temporal window for harvest of crypts or ISCs for therapeutic application. PMID:23820734

Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU.

PubMed

Thomas, P; Sekhar, A C; Mujawar, M M

2012-08-01

To elucidate the cause of high variations and inconsistencies in bacterial CFU observed within and between different experiments while assessing viable bacterial counts through spread plating (SP). Following the inconsistent results, CFU estimations were undertaken through conventional SP using the spreader, or a modified approach that did not use spreader employing four organisms. The latter approach involving spotting-and-tilt-spreading of inoculum on agar surface [spotting spreading (SS)] yielded higher CFU by 11-120% over the weighted average depending on the organism and diluent. The adverse effect owing to the spreader was the most obvious in Escherichia coli followed by Staphylococcus epidermidis, Enterobacter cloacae and Bacillus pumilus. Plate attributes that determined the surface moisture levels of agar medium and the spreading practice adopted by the personnel formed two other major influencing factors. Plating for shorter periods (<60 s) using fresh 15/20 ml plates caused loss of 3-12% CFU owing to inoculum adhesion to spreader irrespective of glass or polypropylene make. On the other hand, prolonging the plating brought down the CFU significantly. Spreader movement on agar surface subsequent to the exhaustion of free moisture, which was marked by the experiencing of some friction to smooth spreader movement, was detrimental to vegetative cells, while Bacillus spores were less affected. The study brings out that the way SP is carried out exerts significant effects on CFU influenced by plate conditions. Prolonged use of spreader on dry agar surface could be highly detrimental to bacterial cells. A mild use of spreader accounting for spreader-adhering inoculum or the practice of SS not involving the spreader is recommended. This study unravels the effects owing to the spreader on bacterial cells and the CFU and recommends an alternate approach of SS to minimize CFU inconsistencies and to maximize the viable bacterial counts. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Neem (Azadirachta indica A. Juss) Oil to Tackle Enteropathogenic Escherichia coli

PubMed Central

Del Serrone, Paola; Nicoletti, Marcello

2015-01-01

Neem (Azadirachta indica A. Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusion test and microdilution test. By molecular biology characterization, fourteen isolates resulted in diarrheagenic E. coli with sixteen primer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA. The NO showed biological activity against all isolates. The bacterial growth inhibition zone by disc diffusion method (100 µL NO) ranged between 9.50 ± 0.70 and 30.00 ± 1.00 mm. The antibacterial activity was furthermore determined at lower NO concentrations (1 : 10–1 : 10,000). The percent of growth reduction ranged between 23.71 ± 1.00 and 99.70 ± 1.53. The highest bacterial growth reduction was 1 : 10 NO concentration with 50 µL of bacterial suspension (ca. 1 × 106 CFU/mL). There is significant difference between the antibacterial activities against pathogenic and nonpathogenic E. coli, as well as NO and ciprofloxacin activities. Viable cells after the different NO concentration treatments were checked by molecular biology assay using PMA dye. On the basis of the obtained results, NO counteracts E. coli and also influences the virulence of E. coli viable cells after NO treatment. The NO metabolomic composition was obtained using fingerprint HPTLC. PMID:26064900

Neem (Azadirachta indica A. Juss) Oil to Tackle Enteropathogenic Escherichia coli.

PubMed

Del Serrone, Paola; Toniolo, Chiara; Nicoletti, Marcello

2015-01-01

Neem (Azadirachta indica A. Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusion test and microdilution test. By molecular biology characterization, fourteen isolates resulted in diarrheagenic E. coli with sixteen primer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA. The NO showed biological activity against all isolates. The bacterial growth inhibition zone by disc diffusion method (100 µL NO) ranged between 9.50 ± 0.70 and 30.00 ± 1.00 mm. The antibacterial activity was furthermore determined at lower NO concentrations (1 : 10-1 : 10,000). The percent of growth reduction ranged between 23.71 ± 1.00 and 99.70 ± 1.53. The highest bacterial growth reduction was 1 : 10 NO concentration with 50 µL of bacterial suspension (ca. 1 × 10(6) CFU/mL). There is significant difference between the antibacterial activities against pathogenic and nonpathogenic E. coli, as well as NO and ciprofloxacin activities. Viable cells after the different NO concentration treatments were checked by molecular biology assay using PMA dye. On the basis of the obtained results, NO counteracts E. coli and also influences the virulence of E. coli viable cells after NO treatment. The NO metabolomic composition was obtained using fingerprint HPTLC.

Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA protects against infection in the swine model of chancroid.

PubMed

Afonina, Galyna; Leduc, Isabelle; Nepluev, Igor; Jeter, Chrystina; Routh, Patty; Almond, Glen; Orndorff, Paul E; Hobbs, Marcia; Elkins, Christopher

2006-04-01

The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

Design of a beneficial product for newborn calves by combining Lactobacilli, minerals, and vitamins.

PubMed

Maldonado, Natalia Cecilia; Silva de Ruiz, Clara; Nader-Macías, María Elena Fátima

2016-10-02

Diarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell's recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.

Potential prebiotic properties of cashew apple (Anacardium occidentale L.) agro-industrial byproduct on Lactobacillus species.

PubMed

Duarte, Francisca Nayara Dantas; Rodrigues, Jéssica Bezerra; da Costa Lima, Maiara; Lima, Marcos Dos Santos; Pacheco, Maria Teresa Bertoldo; Pintado, Maria Manuela Estevez; de Souza Aquino, Jailane; de Souza, Evandro Leite

2017-08-01

The prebiotic effects of a cashew apple (Anacardium occidentale L.) agro-industrial byproduct powder (CAP) on different potentially probiotic Lactobacillus strains, namely Lactobacillus acidophilus LA-05, Lactobacillus casei L-26 and Lactobacillus paracasei L-10, were assessed using in vitro experimental models. Accordingly, the growth of the Lactobacillus strains when cultivated in a broth containing CAP (20 or 30 g L -1 ), glucose (20 g L -1 ) or fructooligosaccharides (FOS) (20 g L -1 ) was monitored over 48 h; the prebiotic activity scores of CAP were determined; and the changes in pH values, production of organic acids and consumption of sugars in growth media were verified. During the 48-h cultivation, similar viable cell counts were observed for the Lactobacillus strains grown in the different media tested. The CAP presented positive prebiotic activity scores toward all the tested Lactobacillus strains, indicating a desirable selective fermentable activity relative to enteric organisms. The cultivation of the Lactobacillus strains in broth containing glucose, FOS or CAP resulted in high viable cell counts, a decreased pH, the production of organic acids and the consumption of sugars over time, revealing intense bacterial metabolic activity. The CAP exerts potential prebiotic effects on different potentially probiotic Lactobacillus strains and should be an added-value ingredient for the food industry. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

E-Clinic: an innovative approach to complex symptom management for allogeneic blood and stem cell transplant patients.

PubMed

Wright, Janice; Purdy, Brendan; McGonigle, Sharon

2007-01-01

The allogeneic blood and stem cell program (ABSCP) at Princess Margaret Hospital, Toronto, performs 75 transplants annually. Many patients live greater than 100 kilometres from the centre and require frequent visits to the hospital for posttransplant care. The weekly travel to clinic, combined with complex symptom issues and the overwhelming desire to be cared for in their home community, is a major burden to patients and care providers. Our team of oncology health professionals, led by the nurse practitioner on service, sought to determine whether telehealth videoconferencing would be a viable option as a care delivery model to meet the complex needs of our remote patients and care partners. We introduced telehealth into the ambulatory clinic as a pilot project in early 2005. Patients were selected based upon symptoms, therapeutic plan and geographical remoteness. Patient progress was monitored with a goal of transitioning patients from posttransplant hospital-based care to partnered self-care in their home communities. The purpose of this article is to illustrate the ABSCP telehealth program development using a patient case study, and to detail the clinical process improvements and overall program successes that have led to the integration of telehealth into everyday clinical practice as a viable service delivery option for patient-centred symptom management and treatment compliance with a geographically remote patient population.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

PubMed

Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

2015-03-01

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

Mechanism of killing of streptococcus mutans by light-activated drugs

NASA Astrophysics Data System (ADS)

Burns, Tracy; Wilson, Michael; Pearson, G. J.

1996-01-01

Recent studies have shown that cariogenic bacteria can be killed when exposed to low power laser light in the presence of a photosensitizing agent. The purpose of this study was to determine the mechanism by which the cariogenic bacterium Streptococcus mutans can be killed by toluidine blue O and helium neon laser light. To determine whether membrane damage occurred, suspensions of sensitized S. mutans were exposed to a 7.3 mW HeNe laser for 30 mins and samples removed every 5 mins. Survivors were enumerated by viable counting on tryptone soya agar plates and cell free filtrates were assayed for phosphate and (beta) -galactosidase. Lipid peroxidation was assessed by assaying for malondialdehyde, a by- product of lipid peroxidation. The role of oxygen and reactive oxygen species was studied by exposing sensitized bacteria to laser light (1) under different atmospheric conditions, (2) in the presence of deuterium oxide, and (3) in the presence of inhibitors of reactive oxygen species. Following exposure of sensitizede S. mutans to 13.2 J of HeNe laser light, 2.6 nmoles of phosphate and 228 nmoles of (beta) -galactosidase were detected in the cell free filtrates. Ten micrometers oles of malondialdehyde were also detected. When the sensitized bacteria were exposed to laser light under anaerobic conditions there was no significant decrease in the viable count compared to a 60% kill in the presence of oxygen. In the presence of D2O there was a 15-fold increase in the numbers of bacteria killed. O.1 M methionine and 0.5 M sodium azide each afforded 98% protection from lethal photosensitization. These results imply that lethal photosensitization results from membrane damage due to lipid peroxidation and that reactive oxygen species are mediators of this process.

Microbial community dynamics during fermentation of doenjang-meju, traditional Korean fermented soybean.

PubMed

Jung, Ji Young; Lee, Se Hee; Jeon, Che Ok

2014-08-18

Bacterial and fungal community dynamics, along with viable plate counts and water content, were investigated in the exterior and interior regions of doenjang-meju, traditional Korean fermented soybean, during its fermentation process. Measurement of viable cells showed that the meju molding equipment might be an important source of bacterial cells (mostly Bacillus) during doenjang-meju fermentation, whereas fungi might be mostly derived from the fermentation environment including incubation shelves, air, and rice straws. Community analysis using rRNA-targeted pyrosequencing revealed that Bacillus among bacteria and Mucor among fungi were predominant in both the exterior and interior regions of doenjang-meju during the early fermentation period. Bacteria such as Ignatzschineria, Myroides, Enterococcus, Corynebacterium, and Clostridium and fungi such as Geotrichum, Scopulariopsis, Monascus, Fusarium, and eventually Aspergillus were mainly detected as the fermentation progressed. Bacillus, an aerobic bacterial group, was predominant in the exterior regions during the entire fermentation period, while anaerobic, facultative anaerobic, and microaerobic bacteria including Enterococcus, Lactobacillus, Clostridium, Myroides, and Ignatzschineria were much more abundant in the interior regions. Principal component analysis (PCA) also indicated that the bacterial communities in the exterior and interior regions were clearly differentiated, suggesting that aeration might be an important factor in determining the bacterial communities during doenjang-meju fermentation. However, PCA showed that fungal communities were not separated in the exterior and interior regions and Pearson's correlation coefficients showed that the major fungal taxa had significantly positive (Mucor and Geotrichum) or negative (Aspergillus) correlations with the water content during doenjang-meju fermentation, indicating that water content might be a significant factor in determining the fungal communities during doenjang-meju fermentation. Copyright © 2014 Elsevier B.V. All rights reserved.

Galacto-oligosaccharides and lactulose as protectants against desiccation of Lactobacillus delbrueckii subsp. bulcaricus.

PubMed

Santos, Mauricio I; Araujo-Andrade, Cuauhtémoc; Esparza-Ibarra, Edgar; Tymczyszyn, Elizabeth; Gómez-Zavaglia, Andrea

2014-01-01

Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was dehydrated on desiccators containing silica gel in the presence of 20% w/w of two types of galacto-oligosaccharides (GOS Biotempo and GOS Cup Oligo H-70®) and lactulose, until no changes in water desorption were detected. After rehydration, bacterial growth was monitored at 37°C by determining: (a) the absorbance at 600 nm and (b) the near infrared spectra (NIR). Principal component analysis (PCA) was then performed on the NIR spectra of samples dehydrated in all conditions. A multiparametric flow cytometry assay was carried out using carboxyfluorescein diacetate and propidium iodide probes to determine the relative composition of damaged, viable, and dead bacteria throughout the growth kinetics. The absorbance at 600 nm and the position of the second derivative band at ∼1370 nm were plotted against the time of incubation. The efficiency of the protectants was GOS Biotempo > GOS Cup Oligo H-70®  > lactulose. The better protectant capacity of GOS Biotempo was explained on the basis of the lower contribution of damaged cells immediately after rehydration (t = 0). PCA showed three groups along PC1, corresponding to the lag, exponential and stationary phases of growth, which explained 99% of the total variance. Along PC2, two groups were observed, corresponding to damaged or viable cells. The results obtained support the use of NIR to monitor the recovery of desiccated microorganisms in real time and without the need of chemical reagents. The use of GOS and lactulose as protectants in dehydration/rehydration processes was also supported. © 2014 American Institute of Chemical Engineers.

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat

NASA Technical Reports Server (NTRS)

Smith, J. D.; Staehelin, L. A.; Todd, P.

1999-01-01

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.

A novel quantitative reverse-transcription PCR (qRT-PCR) for the enumeration of total bacteria, using meat micro-flora as a model.

PubMed

Dolan, Anthony; Burgess, Catherine M; Barry, Thomas B; Fanning, Seamus; Duffy, Geraldine

2009-04-01

A sensitive quantitative reverse-transcription PCR (qRT-PCR) method was developed for enumeration of total bacteria. Using two sets of primers separately to target the ribonuclease-P (RNase P) RNA transcripts of gram positive and gram negative bacteria. Standard curves were generated using SYBR Green I kits for the LightCycler 2.0 instrument (Roche Diagnostics) to allow quantification of mixed microflora in liquid media. RNA standards were used and extracted from known cell equivalents and subsequently converted to cDNA for the construction of standard curves. The number of mixed bacteria in culture was determined by qRT-PCR, and the results correlated (r(2)=0.88, rsd=0.466) with the total viable count over the range from approx. Log(10) 3 to approx. Log(10) 7 CFU ml(-1). The rapid nature of this assay (8 h) and its potential as an alternative method to the standard plate count method to predict total viable counts and shelf life are discussed.

Plants as sources of airborne bacteria, including ice nucleation-active bacteria.

PubMed

Lindemann, J; Constantinidou, H A; Barchet, W R; Upper, C D

1982-11-01

Vertical wind shear and concentration gradients of viable, airborne bacteria were used to calculate the upward flux of viable cells above bare soil and canopies of several crops. Concentrations at soil or canopy height varied from 46 colony-forming units per m over young corn and wet soil to 663 colony-forming units per m over dry soil and 6,500 colony-forming units per m over a closed wheat canopy. In simultaneous samples, concentrations of viable bacteria in the air 10 m inside an alfalfa field were fourfold higher than those over a field with dry, bare soil immediately upwind. The upward flux of viable bacteria over alfalfa was three- to fourfold greater than over dry soil. Concentrations of ice nucleation-active bacteria were higher over plants than over soil. Thus, plant canopies may constitute a major source of bacteria, including ice nucleation-active bacteria, in the air.

Correlation of p63 immunohistochemistry with histology and contrast enhanced MRI in characteristic lesions induced by minimally invasive thermal treatments in a dog prostate

NASA Astrophysics Data System (ADS)

Pascal, A.; Butts-Pauly, K.; Plata, J.; Sommer, G.; Daniel, B.; Bouley, D. M.

2017-03-01

Thermal ablation techniques are important tools to treat low grade tumors in the prostate gland. The use of Magnetic Resonance Imaging (MRI) has been an excellent tool to visualize and assess the thermally ablated areas in real time. In this study slides from dog prostates previously treated with cryoablation or High Intensity Focal Ultrasound (HIFU) were immunohistochemically stained with the biomarker p63, in order to determine if this marker would be helpful for differentiatiating between viable, sub lethally damaged and normal glands. Digitized slides were analyzed using Sedeen Viewer software, and compared with corresponding representative H&E slides and MR images. p63 staining in the cryoablated acute duration prostates was negative in the coagulation necrosis zone (region of interest subjected to the coldest temperatures). In acute duration HIFU treated prostates, the central heat-fixed zone (region of interest subjected to the hottest temperatures) still displayed + p63 staining. Cryoablated or HIFU subacute duration treated prostates were very hemorrhagic, but presented the same stain pattern in the treated areas as the acute duration prostates, and in chronic duration prostates, whether treated with cryo or HIFU, glands displayed robust p63 staining most prevalent in the outer edges of the lesion where there was extensive glandular regeneration. In conclusion, this study demonstrates the value of p63 IHC and its usefulness in detecting viable prostate basal cells in normal dog prostates following either cryoablation of HIFU. Our results suggest that the portions of the lesion with complete loss of p63 staining correspond well to the non-enhancing region in cryoablated prostates, as viewed with MRI. However, p63 staining in the heat-fixed zone in acute harvested HIFU treated prostates remains positive, suggesting either inadequate heat to destroy basal cells, or heat-fixation of the p63 antigen and false positive staining. Therefore p63 staining does not appear to be beneficial in determining cell viability in HIFU-treated tissues, and would not aid in predicting if unwanted tumor cells in a similarly treated area could regenerate.

Biochemical and Cellular Assessment of Acetabular Chondral Flaps Identified During Hip Arthroscopy.

PubMed

Hariri, Sanaz; Truntzer, Jeremy; Smith, Robert Lane; Safran, Marc R

2015-06-01

To analyze chondral flaps debrided during hip arthroscopy to determine their biochemical and cellular composition. Thirty-one full-thickness acetabular chondral flaps were collected during hip arthroscopy. Biochemical analysis was undertaken in 21 flaps from 20 patients, and cellular viability was determined in 10 flaps from 10 patients. Biochemical analysis included concentrations of (1) DNA (an indicator of chondrocyte content), (2) hydroxyproline (an indicator of collagen content), and (3) glycosaminoglycan (an indicator of chondrocyte biosynthesis). Higher values for these parameters indicated more healthy tissue. The flaps were examined to determine the percentage of viable chondrocytes. The percentage of acetabular chondral flap specimens that had concentrations within 1 SD of the mean values reported in previous normal cartilage studies was 38% for DNA, 0% for glycosaminoglycan, and 43% for hydroxyproline. The average cellular viability of our acetabular chondral flap specimens was 39% (SD, 14%). Only 2 of the 10 specimens had more than half the cells still viable. There was no correlation between (1) the gross examination of the joint or knowledge of the patient's demographic characteristics and symptoms and (2) biochemical properties and cell viability of the flap, with one exception: a degenerative appearance of the surrounding cartilage correlated with a higher hydroxyproline concentration. Although full-thickness acetabular chondral flaps can appear normal grossly, the biochemical properties and percentage of live chondrocytes in full-thickness chondral flaps encountered in hip arthroscopy show that this tissue is not normal. There has been recent interest in repairing chondral flaps encountered during hip arthroscopy. These data suggest that acetabular chondral flaps are not biochemically and cellularly normal. Although these flaps may still be valuable mechanically and/or as a scaffold in some conductive or inductive capacity, further study is required to assess the clinical benefit of repair. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Fuel cells feasibility

NASA Technical Reports Server (NTRS)

Schonfeld, D.; Charng, T.

1981-01-01

The technical and economic status of fuel cells is assessed with emphasis on their potential benefits to the Deep Space Network. The fuel cell, what it is, how it operates, and what its outputs are, is reviewed. Major technical problems of the fuel cell and its components are highlighted. Due to these problems and economic considerations it is concluded that fuel cells will not become commercially viable until the early 1990s.

The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

PubMed Central

Ascenso, Andreia; Pedrosa, Tiago; Pinho, Sónia; Pinho, Francisco; de Oliveira, José Miguel P. Ferreira; Cabral Marques, Helena; Oliveira, Helena; Simões, Sandra; Santos, Conceição

2016-01-01

Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer. PMID:26664697

Isolate resistance of Blastocystis hominis to metronidazole.

PubMed

Haresh, K; Suresh, K; Khairul Anus, A; Saminathan, S

1999-04-01

Isolates of Blastocystis hominis from infected immigrant workers from Indonesia, Bangladesh and infected individuals from Singapore and Malaysia were assessed for growth pattern and degree of resistance to different concentrations of metronidazole. Viability of the cells was assessed using eosin-brillian cresyl blue which stained viable cells green and nonviable cells red. The Bangladeshi and Singaporean isolates were nonviable even at the lowest concentration of 0.01 mg/ml, whereas 40% of the initial inoculum of parasites from the Indonesian isolate at day one were still viable in cultures with 1.0 mg/ml metronidazole. The study shows that isolates of B. hominis of different geographical origin have different levels of resistance to metronidazole. The search for more effective drugs to eliminate th parasite appears inevitable, especially since surviving parasites from metronidazole cultures show greater ability to multiply in subcultures than controls.

Laboratory simulation of interplanetary ultraviolet radiation (broad spectrum) and its effects on Deinococcus radiodurans

NASA Astrophysics Data System (ADS)

Paulino-Lima, Ivan Gláucio; Pilling, Sérgio; Janot-Pacheco, Eduardo; de Brito, Arnaldo Naves; Barbosa, João Alexandre Ribeiro Gonçalves; Leitão, Alvaro Costa; Lage, Claudia de Alencar Santos

2010-08-01

The radiation-resistant bacterium Deinococcus radiodurans was exposed to a simulated interplanetary UV radiation at the Brazilian Synchrotron Light Laboratory (LNLS). Bacterial samples were irradiated on different substrates to investigate the influence of surface relief on cell survival. The effects of cell multi-layers were also investigated. The ratio of viable microorganisms remained virtually the same (average 2%) for integrated doses from 1.2 to 12 kJ m -2, corresponding to 16 h of irradiation at most. The asymptotic profiles of the curves, clearly connected to a shielding effect provided by multi-layering cells on a cavitary substrate (carbon tape), means that the inactivation rate may not change significantly along extended periods of exposure to radiation. Such high survival rates reinforce the possibility of an interplanetary transfer of viable microbes.

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

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

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

PubMed Central

2012-01-01

This paper reports a newly fabricated nanophyto-modified wound dressing with microbicidal and anti-adherence properties. Nanofluid-based magnetite doped with eugenol or limonene was used to fabricate modified wound dressings. Nanostructure coated materials were characterized by TEM, XRD, and FT-IR. For the quantitative measurement of biofilm-embedded microbial cells, a culture-based method for viable cell count was used. The optimized textile dressing samples proved to be more resistant to staphylococcal and pseudomonal colonization and biofilm formation compared to the uncoated controls. The functionalized surfaces for wound dressing seems to be a very useful tool for the prevention of wound microbial contamination on viable tissues. PMID:23272823

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

PubMed

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

2012-12-31

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

Effect of Lactobacillus plantarum Tennozu-SU2 on Salmonella Typhimurium Infection in Human Enterocyte-Like HT-29-Luc Cells and BALB/c Mice.

PubMed

Hirano, Shino; Yokota, Yasushi; Eda, Mika; Kuda, Takashi; Shikano, Ayane; Takahashi, Hajime; Kimura, Bon

2017-03-01

The probiotic properties and inhibitory effect on Salmonella Typhimurium adhesion on human enterocyte-like HT-29-Luc cells of three Lactobacillus plantarum strains isolated from fermented fish, beach sand and a coastal plant were determined. Compared with the type strain L. plantarum NBRC 15891 T , which was isolated from pickled cabbage, L. plantarum Tennozu-SU2 isolated from the acorn of a coastal tree showed high autoaggregation in de Man, Rogosa and Sharpe (MRS) broth and an antagonistic effect against S. Typhimurium in brain heart infusion (BHI) broth. Furthermore, heat-killed L. plantarum Tennozu-SU2 cells inhibited S. Typhimurium adhesion on HT-29-Luc cells. Both live and heat-killed L. plantarum Tennozu-SU2 cells showed an inhibitory effect on gut colonisation in BALB/c mice, as assessed by viable Salmonella count in faecal samples and by invasion into liver and spleen tissues. The properties shown in this study suggest that L. plantarum Tennozu-SU2 is useful as a starter and probiotic bacteria in functional food material.

Probiotics in the Space Food System: Delivery, Microgravity Effects, and the Potential Benefit to Crew Health

NASA Technical Reports Server (NTRS)

Castro, S. L.; Ott, C. M.; Douglas, G. L.

2014-01-01

As mission distance and duration increase, the need grows for non-invasive disease prevention and immunomodulation, especially given the limited medical response capability expected for these missions and the immune dysregulation documented in crew. Additionally, changes in diet, lifestyle, antibiotic usage, and the environmental stresses during spaceflight may alter crewmembers' intestinal microbiome. The addition of probiotic bacteria to the space food system is expected to confer immunostimulatory benefits on crewmembers, with the potential to counteract the immune dysregulation that has been documented in spaceflight. Based on previous studies that demonstrated unique microbiological responses to the low shear environment of spaceflight, probiotic organisms hold the potential to induce enhanced beneficial responses through mechanisms, such as beneficial interactions with human immune cells and repression of colonization of pathogens on the mucosa. The work presented here will begin to address two research gaps related to providing probiotics in spaceflight: 1) delivery, and 2) the effect of the low shear microgravity environment on probiotic attributes. The probiotic Lactobacillus acidophilus was selected for investigation due to its wide commercial use and documented benefits that include inhibition of virulence related gene expression in pathogens and mucosal stimulation of immune cells. The delivery system for probiotics has not been determined for spaceflight, where the food system is shelf stable and the lack of refrigeration prevents the use of traditional dairy delivery methods. In order to demonstrate the potential of the space food system to deliver viable probiotic bacteria to crewmembers, the probiotic L. acidophilus was packaged in high barrier flight packaging in nonfat dry milk (NFDM) or retained in commercial capsule form. Viable cells were enumerated over 8 months of storage at 22, 4, and -80ºC. The survival of L. acidophilus rehydrated in NFDM, in a PBS control, and directly from the capsule was also evaluated following stress challenge with simulated gastric and intestinal juices to determine the method that would deliver the most viable cells to the intestine, where they would be expected to confer beneficial effects. L. acidophilus was found to be stable to gastric and intestinal juice challenge when delivered in rehydrated NFDM, even after two hours of exposure. In comparison, L. acidophilus was reduced by 1-5 logs when exposed to gastric and intestinal juice directly and when rehydrated in a PBS control. Shelf life data indicated that L. acidophilus would require refrigerated or frozen storage to remain viable at adequate levels over the multi-year storage periods required for spaceflight. This study suggests that the protective effect provided by the dairy matrix, and not merely rehydration prior to consumption, extends probiotic viability and stress tolerance during storage in spaceflight and in simulated digestion conditions more adequately than a capsule. In addition to effective delivery, it is essential to understand the microgravity effects on the stress tolerances and genetic expression of probiotic bacteria to enable optimization of growth, survival, strain selection, and conferred benefits in spaceflight. In our current study, ground-based characterization of the growth, stress response, and transcriptomic response of L. acidophilus will be accomplished using the low shear modeled microgravity (LSMMG) culture environment in the rotating wall vessel (RWV). We hypothesize that L. acidophilus will maintain or improve its growth and stress response following culture in the LSMMG environment, and that transcriptomic analysis will define the associated molecular mechanism(s), resulting in the ability to optimize strain selection.

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

PubMed

Hastings, Jordan; Kenealey, Jason

2017-01-01

Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G 1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G 1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

BET bromodomain proteins are required for glioblastoma cell proliferation.

PubMed

Pastori, Chiara; Daniel, Mark; Penas, Clara; Volmar, Claude-Henry; Johnstone, Andrea L; Brothers, Shaun P; Graham, Regina M; Allen, Bryce; Sarkaria, Jann N; Komotar, Ricardo J; Wahlestedt, Claes; Ayad, Nagi G

2014-04-01

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.

BET bromodomain proteins are required for glioblastoma cell proliferation

PubMed Central

Pastori, Chiara; Daniel, Mark; Penas, Clara; Volmar, Claude-Henry; Johnstone, Andrea L; Brothers, Shaun P; Graham, Regina M; Allen, Bryce; Sarkaria, Jann N; Komotar, Ricardo J; Wahlestedt, Claes; Ayad, Nagi G

2014-01-01

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors. PMID:24496381

A cell-laden microfluidic hydrogel.

PubMed

Ling, Yibo; Rubin, Jamie; Deng, Yuting; Huang, Catherine; Demirci, Utkan; Karp, Jeffrey M; Khademhosseini, Ali

2007-06-01

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.

Enhancement of Immune Activation Activities of Spirulina maxima Grown in Deep-Sea Water

PubMed Central

Choi, Woon Yong; Kang, Do Hyung; Lee, Hyeon Yong

2013-01-01

In this study, the immuno-modulatory and anticancer activities of marine algae, Spirulina maxima grown in deep-sea water (DSW), were investigated. It was found that the extract of S. maxima, cultured in DSW, effectively suppressed the expression of Bcl2 in A549 cells as well as inhibiting various human cancer cells with concentration dependency, which possibly implies that the extracts may play more important roles in controlling cancer cell growth. The secretion of cytokines IL-6 and TNF-α from human B cells was also greatly increased, compared to those of the extract grown in conventional sea-water. The growth of Human Natural Killer (NK) cells in the presence of the extracts from DSW was significantly higher (12.2 × 104 viable cells/mL) when compared to the control (1.1 × 104 viable cells/mL). Based on HPLC analysis, the increase in the biological activities of the extracts from DSW was caused by considerably high amounts of β-carotene and ascorbic acid because the DSW contained high concentrations and good ratios of several key minerals for biosynthesizing β-carotene and ascorbic acid, as well as maintaining high cell growth. PMID:23743830

What Can We Learn from Solid State NMR on the Electrode-Electrolyte Interface?

PubMed

Haber, Shira; Leskes, Michal

2018-06-11

Rechargeable battery cells are composed of two electrodes separated by an ion-conducting electrolyte. While the energy density of the cell is mostly determined by the redox potential of the electrodes and amount of charge they can store, the processes at the electrode-electrolyte interface govern the battery's lifetime and performance. Viable battery cells rely on unimpeded ion transport across this interface, which depends on its composition and structure. These properties are challenging to determine as interfacial phases are thin, disordered, heterogeneous, and can be very reactive. The recent developments and applications of solid state NMR spectroscopy in the study of interfacial phenomena in rechargeable batteries based on lithium and sodium chemistries are reviewed. The different NMR interactions are surveyed and how these are used to shed light on the chemical composition and architecture of interfacial phases as well as directly probe ion transport across them is described. By combining new methods in solid state NMR spectroscopy with other analytical tools, a holistic description of the electrode-electrolyte interface can be obtained. This will enable the design of improved interfaces for developing battery cells with high energy, high power, and longer lifetime. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Inactivation of the Radiation-Resistant Spoilage Bacterium Micrococcus radiodurans

PubMed Central

Duggan, D. E.; Anderson, A. W.; Elliker, P. R.

1963-01-01

A simplified technique permitting the pipetting of raw puréed meats for quantitative bacteriological study is described for use in determining survival of these non-sporing bacteria, which are exceptionally resistant to radiation. Survival curves, using gamma radiation as the sterilizing agent, were determined in raw beef with four strains of Micrococcus radiodurans. Survival curves of the R1 strain in other meat substrates showed that survival was significantly greater in raw beef and raw chicken than in raw fish or in cooked beef. Resistance was lowest in the buffer. Cells grown in broth (an artificial growth medium) and resuspended in beef did not differ in resistance from cells that had been grown and irradiated in beef. Survival rate was statistically independent of the initial cell concentration, even though there appeared to be a correlation between lower death rate and lower initial cell concentrations. The initial viable count of this culture of the domesticated R1 strain in beef was reduced by a factor of about 10-5 by 3.0 megarad, and 4.0 megarad reduced the initial count by a factor of more than 10-9. Data suggest that M. radiodurans R1 is more resistant to radiation than spore-forming spoilage bacteria for which inactivation rates have been published. PMID:14063780

Endothelial necrosis at 1h post-burn predicts progression of tissue injury

PubMed Central

Hirth, Douglas; McClain, Steve A.; Singer, Adam J.; Clark, Richard A.F.

2013-01-01

Burn injury progression has not been well characterized at the cellular level. To define burn injury progression in terms of cell death, histopathologic spatiotemporal relationships of cellular necrosis and apoptosis were investigated in a validated porcine model of vertical burn injury progression. Cell necrosis was identified by High Mobility Group Box 1 protein and apoptosis by Caspase 3a staining of tissue samples taken 1h, 24h and 7 days post-burn. Level of endothelial cell necrosis at 1h was predictive of level of apoptosis at 24h (Pearson's r=0.87) and of level of tissue necrosis at 7 days (Pearson's r=0.87). Furthermore, endothelial cell necrosis was deeper than interstitial cell necrosis at 1h (p<0.001). Endothelial cell necrosis at 1h divided the zone of injury progression (Jackson's zone of stasis) into an upper subzone with necrotic endothelial cells and initially viable adnexal and interstitial cells at 1h that progressed to necrosis by 24h, and a lower zone with initially viable endothelial cells at 1h, but necrosis and apoptosis of all cell types by 24h. Importantly, this spatiotemporal series of events and rapid progression resembles myocardial infarction and stroke, and implicates mechanisms of these injuries, ischemia, ischemia reperfusion, and programmed cell death, in burn progression. PMID:23627744

Estimation of lactic acid bacterial cell number by DNA quantification.

PubMed

Ishii, Masaki; Matsumoto, Yasuhiko; Sekimizu, Kazuhisa

2018-01-01

Lactic acid bacteria are provided by fermented foods, beverages, medicines, and supplements. Because the beneficial effects of medicines and supplements containing functional lactic acid bacteria are related to the bacterial cell number, it is important to establish a simple method for estimating the total number of lactic acid bacterial cells in the products for quality control. Almost all of the lactic acid bacteria in the products are dead, however, making it difficult to estimate the total number of lactic acid bacterial cells in the products using a standard colony-counting method. Here we estimated the total lactic acid bacterial cell number in samples containing dead bacteria by quantifying the DNA. The number of viable Enterococcus faecalis 0831-07 cells decreased to less than 1 × 10 -8 by 15 min of heat treatment at 80°C. The amount of extracted DNA from heat-treated cells was 78% that of non-heated cells. The number of viable Lactobacillus paraplantarum 11-1 cells decreased to 1 × 10 -4 after 4 days culture. The amount of extracted DNA of the long-cultured cells, however, was maintained at 97%. These results suggest that cell number of lactic acid bacteria killed by heat-treatment or long-term culture can be estimated by DNA quantification.

Blood flow, flow reserve, and glucose utilization in viable and nonviable myocardium in patients with ischemic cardiomyopathy

PubMed Central

Zhang, Xiaoli; Schindler, Thomas H.; Prior, John O.; Sayre, James; Dahlbom, Magnus; Huang, Sung-Cheng

2016-01-01

Purpose The aim of the study was to determine whether glucose uptake in viable myocardium of ischemic cardiomyopathy patients depends on rest myocardial blood flow (MBF) and the residual myocardial flow reserve (MFR). Methods Thirty-six patients with ischemic cardiomyopathy (left ventricular ejection fraction 25±10 %) were studied with 13N-ammonia and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Twenty age-matched normals served as controls. Regional MBF was determined at rest and during dipyridamole hyperemia and regional FDG extraction was estimated from regional FDG to 13N-ammonia activity ratios. Results Rest MBF was reduced in viable (0.42±0.18 ml/min per g) and nonviable regions (0.32±0.09 ml/min per g) relative to remote regions (0.68±0.23 ml/min per g, p<0.001) and to normals (0.63±0.13 ml/min per g). Dipyridamole raised MBFs in controls, remote, viable, and nonviable regions. MBFs at rest (p<0.05) and stress (p<0.05) in viable regions were significantly higher than that in nonviable regions, while MFRs did not differ significantly (p>0.05). Compared to MFR in remote myocardium, MFRs in viable regions were similar (1.39±0.56 vs 1.70±0.45, p>0.05) but were significantly lower in nonviable regions (1.23±0.43, p<0.001). Moreover, the FDG and thus glucose extraction was higher in viable than in remote (1.40±0.14 vs 0.90±0.20, p<0.001) and in nonviable regions (1.13±0.21, p<0.001). The extraction of FDG in viable regions was independent of rest MBF but correlated inversely with MFRs (r=−0.424, p<0.05). No correlation between the FDG extraction and MFR was observed in nonviable regions. Conclusion As in the animal model, decreasing MFRs in viable myocardium are associated with increasing glucose extraction that likely reflects a metabolic adaptation of remodeling hibernating myocytes. PMID:23287994

The potential for reintroduction of tumor cells during intraoperative blood salvage: reduction of risk with use of the RC-400 leukocyte depletion filter.

PubMed

Edelman, M J; Potter, P; Mahaffey, K G; Frink, R; Leidich, R B

1996-02-01

Intraoperative autotransfusion of shed blood is widely utilized in surgery. However, several studies have raised concern about the transmission of tumor cells during oncologic procedures. We compared the ability of a leukocyte depletion filter (RC-400; LDF) to a standard red blood cell filter (SBF) to remove tumor cells derived from urologic malignancies. Cells were suspended in media and passed through a SBF or a LDF. The filtrate was evaluated for the presence of viable cells utilizing the trypan blue exclusion method as well as cell culture. In a second experiment, cells were suspended in fresh bovine blood and processed through a cell saver apparatus followed by filtration with either a SBF or a LDF. Aliquots were cultured after admixture with blood, after processing, and after filtration. The LDF was able to remove tumor cells completely, as demonstrated by both counting with the trypan blue exclusion test and by cell culture. In contrast, admixture with blood processing through the cell saver apparatus nor a standard red blood cell filter removed these cells. Tumor cells derived from urologic malignancies are easily removed with a LDF but not with a SBF. Filtration of blood salvaged at the time of uro-oncologic surgery with a LDF but not with a SBF reduces the potential for reinfusion of viable tumor cells.

Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion

PubMed Central

Paim, A.; Braghirolli, D.I.; Cardozo, N.S.M.; Pranke, P.; Tessaro, I.C.

2018-01-01

Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005–3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion. PMID:29590258

Granulocyte-macrophage colony-stimulating factor responses of oral epithelial cells to Candida albicans.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-06-01

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM-CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM-CSF by ELISA. Fixed organisms, germination-deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM-CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM-CSF secretion. GM-CSF responses were contact-dependent, strain-dependent, required yeast viability and were optimal when the yeast germinated into hyphae.

Al adjuvants can be tracked in viable cells by lumogallion staining.

PubMed

Mile, Irene; Svensson, Andreas; Darabi, Anna; Mold, Matthew; Siesjö, Peter; Eriksson, Håkan

2015-07-01

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved. Copyright © 2015 Elsevier B.V. All rights reserved.

Induction of viable but nonculturable Escherichia coli O157:H7 by high pressure CO2 and its characteristics.

PubMed

Zhao, Feng; Bi, Xiufang; Hao, Yanling; Liao, Xiaojun

2013-01-01

The viable but nonculturable (VBNC) state is a survival strategy adopted by many pathogens when exposed to harsh environmental stresses. In this study, we investigated for the first time that whether high pressure CO2 (HPCD), one of the nonthermal pasteurization techniques, can induce Escherichia coli O157:H7 into the VBNC state. By measuring plate counts, viable cell counts and total cell counts, E. coli O157:H7 in 0.85% NaCl solution (pH 7.0) was able to enter the VBNC state by HPCD treatment at 5 MPa and four temperatures (25°C, 31°C, 34°C and 37°C). Meanwhile, with the improvement of treatment temperature, the time required for E. coli O157:H7 to enter VBNC state would shorten. Enzymatic activities in these VBNC cells were lower than those in the exponential-phase cells by using API ZYM kit, which were also reduced with increasing the treatment temperature, but the mechanical resistance of the VBNC cells to sonication was enhanced. These results further confirmed VBNC state was a self-protection mechanism for some bacteria, which minimized cellular energetic requirements and increased the cell resistance. When incubated in tryptic soy broth at 37°C, the VBNC cells induced by HPCD treatment at 25°C, 31°C and 34°C achieved resuscitation, but their resuscitation capabilities decreased with increasing the treatment temperature. Furthermore, electron microscopy revealed changes in the morphology and interior structure of the VBNC cells and the resuscitated cells. These results demonstrated that HPCD could induce E. coli O157:H7 into the VBNC state. Therefore, it is necessary to detect if there exist VBNC microorganisms in HPCD-treated products by molecular-based methods for food safety.

In silico nanodosimetry: new insights into nontargeted biological responses to radiation.

PubMed

Kuncic, Zdenka; Byrne, Hilary L; McNamara, Aimee L; Guatelli, Susanna; Domanova, Westa; Incerti, Sébastien

2012-01-01

The long-held view that radiation-induced biological damage must be initiated in the cell nucleus, either on or near DNA itself, is being confronted by mounting evidence to suggest otherwise. While the efficacy of cell death may be determined by radiation damage to nuclear DNA, a plethora of less deterministic biological responses has been observed when DNA is not targeted. These so-called nontargeted responses cannot be understood in the framework of DNA-centric radiobiological models; what is needed are new physically motivated models that address the damage-sensing signalling pathways triggered by the production of reactive free radicals. To this end, we have conducted a series of in silico experiments aimed at elucidating the underlying physical processes responsible for nontargeted biological responses to radiation. Our simulation studies implement new results on very low-energy electromagnetic interactions in liquid water (applicable down to nanoscales) and we also consider a realistic simulation of extranuclear microbeam irradiation of a cell. Our results support the idea that organelles with important functional roles, such as mitochondria and lysosomes, as well as membranes, are viable targets for ionizations and excitations, and their chemical composition and density are critical to determining the free radical yield and ensuing biological responses.

21 CFR 610.12 - Sterility.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Factor, Platelets, Red Blood Cells, Plasma, Source Plasma, Smallpox Vaccine, Reagent Red Blood Cells, Anti-Human Globulin, and Blood Grouping Reagents. (2) A manufacturer is not required to comply with the... the test is capable of reliably and consistently detecting the presence of viable contaminating...

21 CFR 610.12 - Sterility.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Factor, Platelets, Red Blood Cells, Plasma, Source Plasma, Smallpox Vaccine, Reagent Red Blood Cells, Anti-Human Globulin, and Blood Grouping Reagents. (2) A manufacturer is not required to comply with the... the test is capable of reliably and consistently detecting the presence of viable contaminating...

Strategies for regeneration of heart muscle.

PubMed

Guyette, Jacques P; Cohen, Ira S; Gaudette, Glenn R

2010-01-01

Regenerative medicine has emerged to the forefront of cardiac research, marrying discoveries in both basic science and engineering to develop viable therapeutic approaches for treating the diseased heart. Signifi cant advancements in gene therapy, stem cell biology, and cardiomyoplasty provide new optimism for regenerating damaged myocardium. Exciting new strategies for endogenous and exogenous regeneration have been proposed. However, questions remain as to whether these approaches can provide enough new myocyte mass to sufficiently restore mechanical function to the heart. In this article, we consider the mechanisms of endogenous cardiomyocyte regeneration and exogenous cell differentiation (with respect to myoblasts, stem cells, and induced pluripotent cells being researched for cell therapies). We begin by reviewing some of the cues that are being harnessed in strategies of gene/cell therapy for regenerating myocardium. We also consider some of the technical challenges that remain in determining new myocyte generation, tracking delivered cells in vivo, and correlating new myocyte contractility with cardiac function. Strategies for regenerating the heart are being realized as both animal and clinical trials suggest that these new approaches provide short-term improvement of cardiac function. However, a more complete understanding of the underlying mechanisms and applications is necessary to sustain longer-term therapeutic success.

Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

PubMed

Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

2015-01-01

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

Microbial assessment of cabin air quality on commercial airliners

NASA Technical Reports Server (NTRS)

La Duc, Myron T.; Stuecker, Tara; Bearman, Gregory; Venkateswaran, Kasthuri

2005-01-01

The microbial burdens of 69 cabin air samples collected from commercial airliners were assessed via conventional culture-dependent, and molecular-based microbial enumeration assays. Cabin air samples from each of four separate flights aboard two different carriers were collected via air-impingement. Microbial enumeration techniques targeting DNA, ATP, and endotoxin were employed to estimate total microbial burden. The total viable microbial population ranged from 0 to 3.6 x10 4 cells per 100 liters of air, as assessed by the ATP-assay. When these same samples were plated on R2A minimal medium, anywhere from 2% to 80% of these viable populations were cultivable. Five of the 29 samples examined exhibited higher cultivable counts than ATP derived viable counts, perhaps a consequence of the dormant nature (and thus lower concentration of intracellular ATP) of cells inhabiting these air cabin samples. Ribosomal RNA gene sequence analysis showed these samples to consist of a moderately diverse group of bacteria, including human pathogens. Enumeration of ribosomal genes via quantitative-PCR indicated that population densities ranged from 5 x 10 1 ' to IO 7 cells per 100 liters of air. Each of the aforementioned strategies for assessing overall microbial burden has its strengths and weaknesses; this publication serves as a testament to the power of their use in concert.

Inactivation of Selected Bacterial Pathogens in Dairy Cattle Manure by Mesophilic Anaerobic Digestion (Balloon Type Digester)

PubMed Central

Manyi-Loh, Christy E.; Mamphweli, Sampson N.; Meyer, Edson L.; Okoh, Anthony I.; Makaka, Golden; Simon, Michael

2014-01-01

Anaerobic digestion of animal manure in biogas digesters has shown promise as a technology in reducing the microbial load to safe and recommended levels. We sought to treat dairy manure obtained from the Fort Hare Dairy Farm by investigating the survival rates of bacterial pathogens, through a total viable plate count method, before, during and after mesophilic anaerobic digestion. Different microbiological media were inoculated with different serial dilutions of manure samples that were withdrawn from the biogas digester at 3, 7 and 14 day intervals to determine the viable cells. Data obtained indicated that the pathogens of public health importance were 90%–99% reduced in the order: Campylobacter sp. (18 days) < Escherichia coli sp. (62 days) < Salmonella sp. (133 days) from a viable count of 10.1 × 103, 3.6 × 105, 7.4 × 103 to concentrations below the detection limit (DL = 102 cfu/g manure), respectively. This disparity in survival rates may be influenced by the inherent characteristics of these bacteria, available nutrients as well as the stages of the anaerobic digestion process. In addition, the highest p-value i.e., 0.957 for E. coli showed the statistical significance of its model and the strongest correlation between its reductions with days of digestion. In conclusion, the results demonstrated that the specific bacterial pathogens in manure can be considerably reduced through anaerobic digestion after 133 days. PMID:25026086

Twitter vs. Facebook: Using Social Media to Promote Collaborative Argumentation in an Online Classroom

ERIC Educational Resources Information Center

Owens, Marissa; Nussbaum, E. Michael

2017-01-01

This study aimed to: 1) evaluate Twitter as a viable tool for promoting collaborative argumentation; 2) determine if scripting through sentence openers promotes a greater number of arguments within an online discussion; and 3) compare Twitter to Facebook as viable tools for promoting collaborative argumentation. Participants were 27 undergraduate…

Chemical decontamination with N-acetyl-L-cysteine-sodium hydroxide improves recovery of viable Mycobacterium avium subsp. paratuberculosis organisms from cultured milk.

PubMed

Bradner, L; Robbe-Austerman, S; Beitz, D C; Stabel, J R

2013-07-01

Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10(2) to 10(8) CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected.

The genomic response of a human uterine endometrial adenocarcinoma cell line to 17alpha-ethynyl estradiol.

PubMed

Naciff, Jorge M; Khambatta, Zubin S; Thomason, Ryan G; Carr, Gregory J; Tiesman, Jay P; Singleton, David W; Khan, Sohaib A; Daston, George P

2009-01-01

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p

Clinically viable magnetic poly(lactide-co-glycolide) (PLGA) particles for MRI-based cell tracking

PubMed Central

Granot, Dorit; Nkansah, Michael K.; Bennewitz, Margaret F.; Tang, Kevin S.; Markakis, Eleni A.; Shapiro, Erik M.

2013-01-01

Purpose To design, fabricate, characterize and in vivo assay clinically viable magnetic particles for MRI-based cell tracking. Methods PLGA encapsulated magnetic nano- and microparticles were fabricated. Multiple biologically relevant experiments were performed to assess cell viability, cellular performance and stem cell differentiation. In vivo MRI experiments were performed to separately test cell transplantation and cell migration paradigms, as well as in vivo biodegradation. Results Highly magnetic nano- (~100 nm) and microparticles (~1–2 μm) were fabricated. Magnetic cell labeling in culture occurred rapidly achieving 3–50 pg Fe/cell at 3 hrs for different particles types, and >100 pg Fe/cell after 10 hours, without the requirement of a transfection agent, and with no effect on cell viability. The capability of magnetically labeled mesenchymal or neural stem cells to differentiate down multiple lineages, or for magnetically labeled immune cells to release cytokines following stimulation, was uncompromised. An in vivo biodegradation study revealed that NPs degraded ~80% over the course of 12 weeks. MRI detected as few as 10 magnetically labeled cells, transplanted into the brains of rats. Also, these particles enabled the in vivo monitoring of endogenous neural progenitor cell migration in rat brains over 2 weeks. Conclusion The robust MRI properties and benign safety profile of these particles make them promising candidates for clinical translation for MRI-based cell tracking. PMID:23568825

Importance of DNA repair in tumor suppression

NASA Astrophysics Data System (ADS)

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Tissue engineering of heart valves: in vitro experiences.

PubMed

Sodian, R; Hoerstrup, S P; Sperling, J S; Daebritz, S H; Martin, D P; Schoen, F J; Vacanti, J P; Mayer, J E

2000-07-01

Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Survival of soil bacteria during prolonged desiccation.

NASA Technical Reports Server (NTRS)

Chen, M.; Alexander, M.

1973-01-01

A determination was made of the kinds and numbers of bacteria surviving when two soils were maintained in the laboratory under dry conditions for more than half a year. Certain non-spore-forming bacteria were found to survive in the dry condition for long periods. A higher percentage of drought-tolerant than drought-sensitive bacteria was able to grow at low water activities. When they were grown in media with high salt concentrations, bacteria generally became more tolerant of prolonged drought and they persisted longer. The percent of cells in a bacterial population that remained viable when exposed to drought stress varied with the stage of growth.

Basic techniques in mammalian cell tissue culture.

PubMed

Phelan, Katy; May, Kristin M

2015-03-02

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. Copyright © 2015 John Wiley & Sons, Inc.

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture

PubMed Central

Freund, Nathaniel W.; Croughan, Matthew S.

2018-01-01

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. PMID:29382079

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture.

PubMed

Freund, Nathaniel W; Croughan, Matthew S

2018-01-28

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production-termed Lactate Supplementation and Adaptation (LSA) technology-through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications.

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

PubMed Central

Riazi, Shadi; Dover, Sara E.; Chikindas, Michael L.

2012-01-01

Aims To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in-vitro. Methods and Results Bacillus coagulans ATCC 7050 was grown at 37 °C for 18 hours. The cell free supernatant was concentrated 10-fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal™ ectocervical (VEC-100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 hours. Conclusions Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a non-cytotoxix antimicrobial for vaginal application. Significance and Impact of the Study This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for control of bacterial vaginosis. PMID:22737982

Histological and Finite Element Analysis of Cell Death due to Irreversible Electroporation

PubMed Central

Long, G.; Bakos, G.; Shires, P. K.; Gritter, L.; Crissman, J. W.; Harris, J. L.; Clymer, J. W.

2014-01-01

Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10μs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism. PMID:24000980

Affinity adsorption of cells to surfaces and strategies for cell detachment.

PubMed

Hubble, John

2007-01-01

The use of bio-specific interactions for the separation and recovery of bio-molecules is now widely established and in many cases the technique has successfully crossed the divide between bench and process scale operation. Although the major specificity advantage of affinity-based separations also applies to systems intended for cell fractionation, developments in this area have been slower. Many of the problems encountered result from attempts to take techniques developed for molecular systems and, with only minor modification to the conditions used, apply them for the separation of cells. This approach tends to ignore or at least trivialise the problems, which arise from the heterogeneous nature of a cell suspension and the multivalent nature of the cell/surface interaction. To develop viable separation processes on a larger scale, effective contacting strategies are required in separators that also allow detachment or recovery protocols that overcome the enhanced binding strength generated by multivalent interactions. The effects of interaction valency on interaction strength needs to be assessed and approaches developed to allow effective detachment and recovery of adsorbed cells without compromising cell viability. This article considers the influence of operating conditions on cell attachment and the extent to which multivalent interactions determine the strength of cell binding and subsequent detachment.

Size and competitive mating success in the yeast Saccharomyces cerevisiae.

PubMed

Smith, Carl; Pomiankowski, Andrew; Greig, Duncan

2014-03-01

In unicellular organisms like yeast, mating with the right partner is critical to future fitness because each individual can only mate once. Because cell size is important for viability, mating with a partner of the right size could be a significant advantage. To investigate this idea, we manipulated the size of unmated yeast cells and showed that their viability depended on environmental conditions; large cells do better on rich medium and small cells do better on poor medium. We also found that the fitness of offspring is determined by the size of their parents. Finally, we demonstrated that when a focal cell of one mating type was placed with a large and a small cell of the opposite mating type, it was more likely to mate with the cell that was closer to the optimum size for growth in a given environment. This pattern was not generated by differences in passive mating efficiency of large and small cells across environments but by competitive mating behavior, mate preference, or both. We conclude that the most likely mechanism underlying this interesting behavior is that yeast cells compete for mates by producing pheromone signals advertising their viability, and cells with the opportunity to choose prefer to mate with stronger signalers because such matings produce more viable offspring.

Prebiotic Potential of Xylooligosaccharides Derived from Corn Cobs and Their In Vitro Antioxidant Activity When Combined with Lactobacillus.

PubMed

Yu, Xiuhua; Yin, Jianyuan; Li, Lin; Luan, Chang; Zhang, Jian; Zhao, Chunfang; Li, Shengyu

2015-07-01

In the present work, the in vitro prebiotic activity of xylooligosaccharides (XOS) derived from corn cobs combined with Lactobacillus plantarum, a probiotic microorganism, was determined. These probiotics exhibited different growth characteristics depending on strain specificity. L. plantarum S2 cells were denser and their growth rates were higher when cultured on XOS. Acetate was found to be the major short-chain fatty acid produced as the end-product of fermentation, and its amount varied from 1.50 to 1.78 mg/ml. The antimicrobial activity of XOS combined with L. plantarum S2 was determined against gastrointestinal pathogens. The results showed that XOS proved to be an effective substrate, enhancing antimicrobial activity for L. plantarum S2. In vivo evaluation of the influence of XOS and L. plantarum S2, used both alone and together, on the intestinal microbiota in a mouse model showed that XOS combined with L. plantarum S2 could increase the viable lactobacilli and bifidobacteria in mice feces and decrease the viable Enterococcus, Enterobacter, and Clostridia spp. Furthermore, in the in vitro antioxidant assay, XOS combined with L. plantarum S2 possessed significant 2,2-diphenyl-1- picrylhydrazyl, 2,2'-azino-bis, and superoxide anion radical-scavenging activities, and the combinations showed better antioxidant activity than either XOS or L. plantarum S2 alone.

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

PubMed

Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

2013-12-01

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Constitutive exposure of phosphatidylserine on viable cells

PubMed Central

Segawa, Katsumori; Suzuki, Jun; Nagata, Shigekazu

2011-01-01

Apoptotic cells are quickly recognized and engulfed by phagocytes to prevent the release of noxious materials from dying cells. Phosphatidylserine (PS) exposed on the surface of apoptotic cells is a proposed “eat-me” signal for the phagocytes. Transmembrane protein 16F (TMEM16F), a membrane protein with eight transmembrane segments, has the Ca-dependent phospholipid scramblase activity. Here we show that when lymphoma cells were transformed with a constitutively active form of TMEM16F, they exposed a high level of PS that was comparable to that observed on apoptotic cells. The PS-exposing cells were morphologically normal and grew normally. They efficiently responded to interleukin 3 and underwent apoptosis upon treatment with Fas ligand. The viable PS-exposing cells bound to peritoneal macrophages at 4 °C, but not at 25 °C. Accordingly, these cells were not engulfed by macrophages. When apoptotic cells were injected i.v. into mice, they were phagocytosed by CD11c+CD8+ dendritic cells (DCs) in the spleen, but the PS-exposing living cells were not phagocytosed by these DCs. Furthermore, when PS-exposing lymphoma cells were transplanted s.c. into nude mice, they generated tumors as efficiently as parental lymphoma cells that did not expose PS. These results indicated that PS exposure alone is not sufficient to be recognized by macrophages as an eat-me signal. PMID:22084121

Sorption and precipitation of Mn2+ by viable and autoclaved Shewanella putrefaciens: Effect of contact time

NASA Astrophysics Data System (ADS)

Chubar, Natalia; Visser, Tom; Avramut, Cristina; de Waard, Helen

2013-01-01

The sorption of Mn(II) by viable and inactivated cells of Shewanella putrefaciens, a non-pathogenic, facultative anaerobic, gram-negative bacterium characterised as a Mn(IV) and Fe(III) reducer, was studied under aerobic conditions, as a function of pH, bacterial density and metal loading. During a short contact time (3-24 h), the adsorptive behaviour of live and dead bacteria toward Mn(II) was sufficiently similar, an observation that was reflected in the studies on adsorption kinetics at various metal loadings, effects of pH, bacteria density, isotherms and drifting of pH during adsorption. Continuing the experiment for an additional 2-30 days demonstrated that the Mn(II) sorption by suspensions of viable and autoclaved cells differed significantly from one another. The sorption to dead cells was characterised by a rapid equilibration and was described by an isotherm. In contrast, the sorption (uptake) to live bacteria exhibited a complex time-dependent uptake. This uptake began as adsorption and ion exchange processes followed by bioprecipitation, and it was accompanied by the formation of polymeric sugars (EPS) and the release of dissolved organic substances. FTIR, EXAFS/XANES and XPS demonstrated that manganese(II) phosphate was the main precipitate formed in 125 ml batches, which is the first evidence of the ability of microbes to synthesise manganese phosphates. XPS and XANES spectra did not detect Mn(II) oxidation. Although the release of protein-like compounds by the viable bacteria increased in the presence of Mn2+ (and, by contrast, the release of carbohydrates did not change), electrochemical analyses did not indicate any aqueous complexation of Mn(II) by the organic ligands.

Cell biology, MRI and geometry: insight into a microscopic/macroscopic marriage.

PubMed

de Oliveira, Sérgio Almeida; Gowdak, Luís Henrique W; Buckberg, Gerald; Krieger, José Eduardo

2006-04-01

The concept of cell therapy as an adjunctive therapy to myocardial surgical revascularization for patients with severe coronary artery disease is illustrated by two case reports of ischemic cardiac disease that were unsuitable for revascularization by coronary grafting. The potential interaction of cell therapy, magnetic resonance imaging (MRI) of viability, and left ventricle (LV) restoration is described. Each patient had an ejection fraction below 30%, a relatively conical heart, and MRI gadolinium scan showing predominantly viable muscle. Intramyocardial injections of autologous bone marrow-derived cells (BMC) were performed along with either incomplete coronary artery bypass grafting (CABG) (to mother regions) or with transmyocardial laser revascularization (TMLR). An improvement in contractile function was seen at 6-12-month intervals after the procedure. The implications of possible underlying mechanisms of improvement in both myocardial perfusion and contractility suggest the striking importance of both micro- and macroenvironment for any cell-based therapeutic strategy. These observations imply that the interaction of cell biology, viability by MRI and geometry may be important in the future, as geometry can be restored surgically, and the new architectural form may develop enhanced function if it contains viable tissue and cell-based treatment can be delivered.

Evolution of β-Cell Replacement Therapy in Diabetes Mellitus: Islet Cell Transplantation

PubMed Central

Jahansouz, Cyrus; Jahansouz, Cameron; Kumer, Sean C.; Brayman, Kenneth L.

2011-01-01

Diabetes mellitus remains one of the leading causes of morbidity and mortality worldwide. According to the Centers for Disease Control and Prevention, approximately 23.6 million people in the United States are affected. Of these individuals, 5 to 10% have been diagnosed with Type 1 diabetes mellitus (T1DM), an autoimmune disease. Although it often appears in childhood, T1DM may manifest at any age, leading to significant morbidity and decreased quality of life. Since the 1960s, the surgical treatment for diabetes mellitus has evolved to become a viable alternative to insulin administration, beginning with pancreatic transplantation. While islet cell transplantation has emerged as another potential alternative, its role in the treatment of T1DM remains to be solidified as research continues to establish it as a truly viable alternative for achieving insulin independence. In this paper, the historical evolution, procurement, current status, benefits, risks, and ongoing research of islet cell transplantation are explored. PMID:22013505

MORPHOLOGICAL AND CULTURAL COMPARISON OF MICROORGANISMS IN SURFACE SOIL AND SUBSURFACE SEDIMENTS AT A PRISTINE STUDY SITE IN OKLAHOMA (JOURNAL VERSION)

EPA Science Inventory

Surface-soil and subsurface microfloras at the site of a shallow aquifer in Oklahoma were examined and compared with respect to (1) total and viable cell numbers, (2) colony and cell types that grew on various plating media, (3) cell morphologies seen in flotation films stripped ...

Hybrid Tandem Solar Cells | Photovoltaic Research | NREL

Science.gov Websites

Hybrid Tandem Solar Cells Hybrid Tandem Solar Cells To achieve aggressive cost reductions in photovoltaics (PV) beyond the 6¢/kWh SunShot Initiative 2020 goal, module efficiency must be increased beyond on a silicon platform and that aim to provide viable prototypes for commercialization. PV Research

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

PubMed

Fujikawa, Takahisa; Hirose, Tetsuro; Fujii, Hideaki; Oe, Shoshiro; Yasuchika, Kentaro; Azuma, Hisaya; Yamaoka, Yoshio

2003-08-01

Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Reduction and restoration of culturability of beer-stressed and low-temperature-stressed Lactobacillus acetotolerans strain 2011-8.

PubMed

Deng, Yang; Liu, Junyan; Li, Lin; Fang, Huijing; Tu, Jingxia; Li, Bing; Liu, Jing; Li, Huiping; Xu, Zhenbo

2015-08-03

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria, regardless of beer type, and therefore pose significant problems for the brewing industry. The aim of this study was to investigate the viable, but putatively non-culturable (VPNC) state of the hard-to-culture beer-spoilage species, Lactobacillus acetotolerans. Upon prolonged contact with degassed beer, L. acetotolerans was found to show decreased culturability. After 17 subcultures in beer, 100-μL aliquots of the culture were no longer culturable on MRS agar until 14 days of incubation despite the presence of 10(5) viable cells, indicating that a large population of cells entered into a VPNC state. Furthermore, a significant reduction or even putative loss of culturability, but maintenance of viability, of L. acetotolerans could also be induced by storing the strain at 0 °C for 105 days. Adding catalase at a concentration of 1000 U/plate enabled the VPNC cells, both induced by beer subculture treatment and cold treatment, to regain culturability with a resuscitation time of 4 days and 3 days, respectively. Scanning electron microscopy results demonstrated that cells decreased in size and gradually changed morphology from short rods to coccoids when they entered the VPNC state. It was concluded that the difficulty in culturing the spoilage bacterium from brewery environments could be partly attributed the hard-to-culture or the viable, but non-culturable characteristic of this organism. Copyright © 2015 Elsevier B.V. All rights reserved.

The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil

PubMed Central

2011-01-01

Background Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU). Methods The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells. Results We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable. Conclusions Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression. PMID:21303518

Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system.

PubMed

Wang, Xiaodan; Yamaguchi, Nobuyasu; Someya, Takashi; Nasu, Masao

2007-10-01

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

PubMed Central

Tario, Joseph D.; Conway, Alexis N.; Muirhead, Katharine A.; Wallace, Paul K.

2018-01-01

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells. The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: Assessment of the dye’s spectral profile on the laboratory’s flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems;Evaluating the effect of labeling on cell growth rate;Testing the fidelity with which dye dilution reports cell division;Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; andVerifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling. PMID:29071683

Iron ion irradiation increases promotes adhesion of monocytic cells to arterial vascular endothelium

NASA Astrophysics Data System (ADS)

Kucik, Dennis; Khaled, Saman; Gupta, Kiran; Wu, Xing; Yu, Tao; Chang, Polly; Kabarowski, Janusz

Radiation causes inflammation, and chronic, low-level vascular inflammation is a risk factor for atherosclerosis. Consistent with this, exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Part of the inflammatory response to radiation is a change in the adhesiveness of the endothelial cells that line the blood vessels, triggering inappropriate accumulation of leukocytes, leading to later, damaging effects of inflammation. Although some studies have been done on the effects of gamma irradiation on vascular endothelium, the response of endothelium to heavy ion radiation likely to be encountered in prolonged space flight has not been determined. We investigated how irradiation of aortic endothelial cells with iron ions affects adhesiveness of cultured aortic endothelial cells for monocytic cells and the consequences of this for development of atherosclerosis. Aortic endothelial cells were irradiated with 600 MeV iron ions at Brookhaven National Laboratory and adhesion-related changes were measured. Cells remained viable for at least 72 hours, and were even able to repair acute damage to cell junctions. We found that iron ion irradiation altered expression levels of specific endothelial cell adhesion molecules. Further, these changes had functional consequences. Using a flow chamber adhesion assay to measure adhesion of monocytic cells to endothelial cells under physiological shear stress, we found that adhesivity of vascular endothelium was enhanced in as little as 24 hours after irradiation. Further, the radiation dose dependence was not monotonic, suggesting that it was not simply the result of endothelial cell damage. We also irradiated aortic arches and carotid arteries of Apolipoprotein-E-deficient mice. Histologic analysis of these mice will be conducted to determine whether effects of radiation on endothelial adhesiveness result in consequences for development of atherosclerosis. (Supported by NSBRI: NCC-9-58-162)

Binding mechanism of patulin to heat-treated yeast cell.

PubMed

Guo, C; Yuan, Y; Yue, T; Hatab, S; Wang, Z

2012-12-01

This study aims to assess the removal mechanism of patulin using heat-treated Saccharomyces cerevisiae cells and identify the role of different cell wall components in the binding process. In order to understand the binding mechanism, viable cells, heat-treated cells, cell wall and intracellular extract were performed to assess their ability to remove patulin. Additionally, the effects of chemical and enzymatic treatments of yeast on the binding ability were tested. The results showed that there was no significant difference between viable (53·28%) and heat-treated yeast cells (51·71%) in patulin binding. In addition, the cell wall fraction decreased patulin by 35·05%, and the cell extract nearly failed to bind patulin. Treatments with protease E, methanol, formaldehyde, periodate or urea significantly decreased (P < 0·05) the ability of heat-treated cells to remove patulin. Fourier transform infrared (FTIR) analysis indicated that more functional groups were involved in the binding process of heat-treated cells. Polysaccharides and protein are important components of yeast cell wall involved in patulin removal. In addition, hydrophobic interactions play a major role in binding processes. Heat-treated S. cerevisiae cells could be used to control patulin contamination in the apple juice industry. Also, our results proof that the patulin removal process is based mainly on the adsorption not degradation. © 2012 The Society for Applied Microbiology.

Angiotensin II improves random-flap viability in a rat model.

PubMed

Okuyama, N; Roda, N; Sherman, R; Guerrero, A; Dougherty, W; Nguyen, T; diZerega, G; Rodgers, K

1999-03-01

Angiotensin II (AII) is a naturally occurring peptide that has been shown to be angiogenic, cause the proliferation of several primary cell types (including endothelial cells), accelerate the repair of dermal injuries, and increase production of growth factors and extracellular matrix. The effect of a single administration of AII on the viability and vascularity of a random flap was assessed in a rat model. In the control model, the viability of the distal portion of the flap was reduced consistently by postoperative day 8. Initially, AII was administered in an aqueous vehicle (phosphate-buffered saline [PBS]) and a viscous vehicle (10% carboxymethyl cellulose [CMC]). Administration of 1 mg per milliliter AII in PBS did not affect the viability of random flaps (1.2 x 7 cm) in this animal model. However, a single administration of a higher dose of AII in PBS (10 mg per milliliter) or 1 mg per milliliter AII in the CMC vehicle resulted in 67% of the grafts being fully viable at postsurgical day 12, in contrast to vehicle-treated control flaps, none of which were fully viable at day 12. Furthermore, the portion of the flap that was viable was increased significantly (p < or = 0.05). Subsequently, a study was conducted to assess the dose-response curve for AII in a CMC vehicle in this rat model. As the dose of AII was reduced, the percentage of animals with fully viable flaps and the percentage of the flap that was viable decreased correspondingly. Administration of 0.03 mg per milliliter AII and greater increased significantly (p < or = 0.05) the viability of the flaps. In conclusion, AII appears to be highly efficacious in increasing the percentage of distal flap surface area survival when administered as a single topical dose to the wound bed.

A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy.

PubMed

Gorokhova, Elena; Mattsson, Lisa; Sundström, Annica M

2012-06-01

Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by ~25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80°C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria.

PubMed

Williams, Timothy J; Schneider, Rene P; Willcox, Mark D P

2003-10-01

Gram negative bacterial adhesion to contact lenses can cause adverse responses. During contact lens wear, components of the tear film adsorb to the contact lens. This study aimed to investigate the effect of this conditioning film on the viability of bacteria. Bacteria adhered to contact lenses which were either unworn, worn for daily-, extended- or overnight-wear or coated with lactoferrin or lysozyme. Numbers of viable and total cells were estimated. The number of viable attached cells was found to be significantly lower than the total number of cells on worn (50% for strain Paer1 on daily-wear lenses) or lactoferrin-coated lenses (56% for strain Paer1). Lysozyme-coated lenses no statistically significant effect on adhesion. The conditioning film gained through wear may not inhibit bacterial adhesion, but may act adversely upon those bacteria that succeed in attaching.

Value-added probiotic development by high-solid fermentation of sweet potato with Saccharomyces boulardii.

PubMed

Campbell, Carmen; Nanjundaswamy, Ananda K; Njiti, Victor; Xia, Qun; Chukwuma, Franklin

2017-05-01

Controlled fermentation of Sweet potato ( Ipomoea batatas ) var. Beauregard by yeast, Saccharomyces boulardii (MAY 796) to enhance the nutritional value of sweet potato was investigated. An average 8.00 × 10 10 Colony Forming Units (CFU)/g of viable cells were obtained over 5-day high-solid fermentation. Yeast cell viability did not change significantly over time at 4°C whereas the number of viable yeast cells reduced significantly at room temperature (25°C), which was approximately 40% in 12 months. Overall, the controlled fermentation of sweet potato by MAY 796 enhanced protein, crude fiber, neutral detergent fiber, acid detergent fiber, amino acid, and fatty acid levels. Development of value-added sweet potato has a great potential in animal feed and human nutrition. S. boulardii - fermented sweet potato has great potential as probiotic-enriched animal feed and/or functional food for human nutrition.

Studies of hexafluorophosphate, tetrafluoroborate, and perchlorate electro-intercalation into graphitic carbon

NASA Astrophysics Data System (ADS)

Seel, Jennifer Ann

There has been some interest in using carbon materials as both working electrodes in electrochemical cells and rechargeable batteries [1--6]. This would result in the intercalation of not only of lithium ions into one carbon electrode but the anion component of the lithium salt, such as PF 6-, into the other carbon electrode. The intercalation of the anion component of the salt into carbon electrodes has not been studied extensively and it is not completely understood. The work presented here will expand on this rarely touched subject through electrochemical cycling as well as in-situ and ex-situ X-ray diffraction experiments. The anions that will be studied are: PF6- , BF4- and ClO4 -. It will be shown that anion intercalation occurs for various types of soft carbons and that the process can be greatly affected by the amount of turbostratic disorder present in the carbon material as well as by the specific anion used. It was discovered that using ethyl methyl sulfone, EMS, as the solvent component of the electrolyte resulted in more stable electrochemical cells than ethylene carbonate/diethyl carbonate, a more common solvent, at the high potentials required for anion intercalation. It was also discovered that PF 6 and BF4 formed staged phases during electrochemical cycling whereas ClO4 did not. The amount of disorder present in the carbon electrode did affect the intercalation of the anion. The samples with a greater amount of disorder present had a larger amount of capacity loss between charge and discharge capacities. It was also found that purer and more distinct staged phases occurred in the more ordered carbon samples. The turbostratically disordered carbon layers may rotate to accommodate PF6 and therefore become slightly more ordered. X-ray diffraction evidence suggests that intercalated PF6 molecules may be free-rotating between the carbon layers. However, the orientation of BF4 molecules between the carbon layers could not be determined. There may also be some co-intercalation of the solvent, mainly with ClO 4 and to a lesser extent BF4 and PF6. It is thought that a large amount of solvent co-intercalation occurs with ClO4 and this is the most probable reason why staged phases were not observed. An unfortunate aspect of this study is that dual carbon cells are not at all viable as commercial cells. The energy densities of dual carbon cells are much lower than the currently available lithium-ion cells. For dual carbon cells to become viable new inexpensive salts and solvents that can operate at high potentials and high concentrations must be discovered. With further investigation, combinations of different anions and solvents may result in higher specific capacities that would also make dual carbon cells more viable.

E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

PubMed

Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

1999-05-01

Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

Cell Expansion-Dependent Inflammatory and Metabolic Profile of Human Bone Marrow Mesenchymal Stem Cells.

PubMed

Prieto, Patricia; Fernández-Velasco, María; Fernández-Santos, María E; Sánchez, Pedro L; Terrón, Verónica; Martín-Sanz, Paloma; Fernández-Avilés, Francisco; Boscá, Lisardo

2016-01-01

Stem cell therapy has emerged as a promising new area in regenerative medicine allowing the recovery of viable tissues. Among the many sources of adult stem cells, bone marrow-derived are easy to expand in culture via plastic adherence and their multipotentiality for differentiation make them ideal for clinical applications. Interestingly, several studies have indicated that MSCs expansion in vitro may be limited mainly due to "cell aging" related to the number of cell divisions in culture. We have determined that MSCs exhibit a progressive decline across successive passages in the expression of stem cell markers, in plasticity and in the inflammatory response, presenting low immunogenicity. We have exposed human MSCs after several passages to TLRs ligands and analyzed their inflammatory response. These cells responded to pro-inflammatory stimuli (i.e., NOS-2 expression) and to anti-inflammatory cytokines (i.e., HO1 and Arg1) until two expansions, rapidly declining upon subculture. Moreover, in the first passages, MSCs were capable to release IL1β, IL6, and IL8, as well as to produce active MMPs allowing them to migrate. Interestingly enough, after two passages, anaerobic glycolysis was enhanced releasing high levels of lactate to the extracellular medium. All these results may have important implications for the safety and efficacy of MSCs-based cell therapies.

Rapid Determination of Endospore Viability by Hyperspectral Reflectance Following Surface Decontamination

DTIC Science & Technology

2008-12-01

Alexandria, VA ABSTRACT Bacterial spores , or endospores, such as those of Bacillus anthracis, are an asymmetrical threat. Decontamination... Bacillus subtilis spores by hypochlorite and chlorine dioxide, J. Appl Microbiol., 95(1), 54-67. ...have the ability to distinguish viable from non-viable endospores. In the laboratory, we have exploited the oxidative alteration of the spore coat

Scottish Ethnicity: Self-Determination Within Great Britain.

DTIC Science & Technology

1985-08-27

Scotland’s politicians in Westminster. There was no effective leadership to coalesce the classes into a viable pressure group . 4 6 Another significant...after the SNP nudged them out of that spot early in the 1970’s. For its part, the SNP is still a viable pressure group , but it has lost much of its

Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics.

PubMed

Xiao, Yinghua; van Hijum, Sacha A F T; Abee, Tjakko; Wells-Bennik, Marjon H J

2015-01-01

The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.

Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

PubMed Central

Xiao, Yinghua; van Hijum, Sacha A. F. T.; Abee, Tjakko; Wells-Bennik, Marjon H. J.

2015-01-01

The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies. PMID:25978838

Novel Synthetic Mono-triazole Glycosides Induce G0/G1 Cell-cycle Arrest and Apoptosis in Cholangiocarcinoma Cells.

PubMed

Obchoei, Sumalee; Saeeng, Rungnapha; Wongkham, Chaisiri; Wongkham, Sopit

2016-11-01

The treatment of cholangiocarcinoma (CCA) is still ineffective and the search for a novel treatment is needed. In this study, eight novel mono-triazole glycosides (W1-W8) were synthesized and tested for their anticancer activities in CCA cell lines. The anti-proliferation effect and the underlying mechanisms of the triazole glycosides were explored. Viable cells were determined using the MTT test. Among glycosides tested, W4 and W5 exhibited the most potent anticancer activity in a dose- and time-dependent fashion. Flow cytometry and wstern blot analysis revealed that W4 and W5 induced G 0 /G 1 phase cell-cycle arrest through down-regulation of cyclin D1, cyclin E and induction of cyclin-dependent kinase inhibitors, p27 and p21 protein expression. Annexin V/propidium iodide (PI) staining demonstrated that W4 and W5 also induced apoptotic cells in a dose-dependent manner via caspase signaling cascade. Together, these findings imply that the novel synthetic glycosides might be a promising anticancer agent for CCA. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

New approach for dry formulation techniques for rhizobacteria

NASA Astrophysics Data System (ADS)

Elchin, A. A.; Mashinistova, A. V.; Gorbunova, N. V.; Muratov, V. S.; Kydralieva, K. A.; Jorobekova, Sh. J.

2009-04-01

Two beneficial Pseudomonas isolates selected from rhizosphere of abundant weed - couch-grass Elytrigia repens L. Nevski have been found to have biocontrol activity. An adequate biocontrol effect requires high yield and long stability of the bacterial preparation [1], which could be achieved by an effective and stable formulation. This study was aimed to test various approaches to dry formulation techniques for Pseudomonas- based preparations. To reach this goal, two drying formulation techniques have been tested: the first one, spray drying and the second, low-temperature contact-convective drying in fluidized bed. The optimal temperature parameters for each technique were estimated. Main merits of the selected approach to dry technique are high yield, moderate specific energy expenditures per 1 kg of evaporated moisture, minimal time of contact of the drying product with drying agent. The technological process for dry formulation included the following stages: the obtaining of cell liquids, the low-temperature concentrating and the subsequent drying of a concentrate. The preliminary technological stages consist in cultivation of the rhizobacteria cultures and concentrating the cell liquids. The following requirements for cultivation regime in laboratory conditions were proposed: optimal temperatures are 26-28°С in 3 days, concentration of viable cells in cell liquid makes 1010-1011 cell/g of absolutely dry substance (ADS). For concentrating the cell liquids the method of a vacuum evaporation, which preserves both rhizobacteria cells and the secondary metabolites of cell liquid, has been used. The process of concentrating was conducted at the minimum possible temperature, i.e. not above 30-33°С. In this case the concentration of viable cells has decreased up to 109-1010 cell/g of ADS. For spray drying the laboratory up-dated drier BUCHI 190, intended for the drying of thermolabile products, was used. The temperatures of an in- and outcoming air did not exceed 50°С and 38°С, respectively. To enrich of dry product yield, 20% of sodium humate [2] was used as filling agent. As a result, concentration of viable cells in yield makes 105-106 cell/g of ADS. Low-temperature contact-convective drying in fluidized bed with use of preliminarily dried heat-carrier was evaluated at 25-30°С. Granules of humic acids (d 3 mm) served as inert carrying agent. So, the concentration of viable cells in dry product makes 108-109 cell/g of ADS. The results presented demonstrated that fluidized bed drying technique applied on rhizobacteria-based BCA had higher beneficial effect in terms of high yield as compared to spray drying. Acknowledgement. This research was supported by the grant of ISTC KR-993.2. 1. Levenfors, J.R., et al. Biological control of snow mould (Microdochium nivale) in winter cereals by Pseudomonas brassicacearum MA250. Biocontrol 2007. 2. Orlov, D.S. (1990) Soil Humic Acids and General Theory of Humification, MSU Publisher, Moscow

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

PubMed

Rodríguez-López, Pedro; Puga, Carmen H; Orgaz, Belén; Cabo, Marta L

2017-09-01

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r 2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Effects of hydrostatic pressure and supercritical carbon dioxide on the viability of Botryococcus braunii algae cells.

PubMed

Yildiz-Ozturk, Ece; Ilhan-Ayisigi, Esra; Togtema, Arnoud; Gouveia, Joao; Yesil-Celiktas, Ozlem

2018-05-01

In bio-based industries, Botryococcus braunii is identified as a potential resource for production of hydrocarbons having a wide range of applications in chemical and biopolymer industries. For a sustainable production platform, the algae cultivation should be integrated with downstream processes. Ideally the algae are not harvested, but the product is isolated while cultivation and growth is continued especially if the doubling time is slow. Consequently, hydrocarbons can be extracted while keeping the algae viable. In this study, the effects of pressure on the viability of B. braunii cells were tested hydrostatically and under supercritical CO 2 conditions. Viability was determined by light microscopy, methylene blue uptake and by re-cultivation of the algae after treatments to follow the growth. It was concluded that supercritical CO 2 was lethal to the algae, whereas hydrostatic pressure treatments up to 150 bar have not affected cell viability and recultivation was successful. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Using of Spherocelle sorbents for construction of immobilized probiotics].

PubMed

Bondarenko, V M; Rybal'chenko, O V; Boldyrev, A G; Potokin, I L; Orlova, O G; Dobritsa, V P

2009-01-01

To assess sorption properties of Spherocelle beads consisting of particles of macroporous celiulose with various charges in relation to bacterial cells of manufacturing probiotic strains from different taxonomic groups. The following manufacturing strains: Bifidobacterium bifidum 1, Lactobacillus plantarum 8PA-3 and Escherichia coli M-17, as well as 3 variants of Spherocelles' matrix: neutral, with positive and negative charges, were used. Spherocelle globules DEAE with a positive charge of the matrix were successively used for designing of immobilized probiotic preparations. Efficacy of sorbent is determined by sorption of > or =1000 viable cells as well as bacterial metabolites interacting in conditions of sorbent-regulated pH on each globule with diameter 100-180 microm. It provides, on the one hand, prolonged viability of probiotic bacteria in culture fluid within 6 months and, on the other hand, optimal pharmacokinetics of preparation due to gradual desorption of metabolites from sorbent globules. Sorbent Spherocell DEAE is biocompatible with cells of manufacturing strains of lactobacilli, bifidobacteria and E. coli and recommended for designing of immobilized probiotics.

Status and Prospects of the Global Automotive Fuel Cell Industry and Plans for Deployment of Fuel Cell Vehicles and Hydrogen Refueling Infrastructure

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

Greene, David L; Duleep, Gopal

2013-06-01

Automobile manufacturers leading the development of mass-market fuel cell vehicles (FCVs) were interviewed in Japan, Korea, Germany and the United States. There is general agreement that the performance of FCVs with respect to durability, cold start, packaging, acceleration, refueling time and range has progressed to the point where vehicles that could be brought to market in 2015 will satisfy customer expectations. However, cost and the lack of refueling infrastructure remain significant barriers. Costs have been dramatically reduced over the past decade, yet are still about twice what appears to be needed for sustainable market success. While all four countries havemore » plans for the early deployment of hydrogen refueling infrastructure, the roles of government, industry and the public in creating a viable hydrogen refueling infrastructure remain unresolved. The existence of an adequate refueling infrastructure and supporting government policies are likely to be the critical factors that determine when and where hydrogen FCVs are brought to market.« less

Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age.

PubMed

Devitt, Sean M; Carter, Cynthia M; Dierov, Raia; Weiss, Scott; Gersch, Robert P; Percec, Ivona

2015-01-01

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2-1159 days) from patients of varying ages (26-62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

The Selective Progesterone Receptor Modulator CDB4124 Inhibits Proliferation and Induces Apoptosis in Uterine Leiomyoma Cells

PubMed Central

Luo, Xia; Yin, Ping; Coon V., John S.; Cheng, You-Hong; Wiehle, Ronald D.; Bulun, Serdar E.

2009-01-01

Objective To evaluate the effects of selective progesterone receptor modulator CDB4124 on cell proliferation and apoptosis in cultured human uterine leiomyoma smooth muscle (LSM) cells and control myometrial smooth muscle (MSM) cells in matched uteri. Design Laboratory research. Setting Academic medical center. Patient(s) Premenopausal women (n=12) undergoing hysterectomy for leiomyoma-related symptoms. Intervention(s) Treatment of primary LSM and MSM cells with CDB4124 (10-8-10-6M) or vehicle for 24, 48 or 72 hours. Main Outcome Measure(s) Western blot for protein expression of proliferating cell nuclear antigen (PCNA), cleaved poly-adenosine 5’-diphosphate-ribose polymerase (PARP), Bcl-2 and Krüppel-like transcription factor 11 (KLF11); MTT assay to evaluate viable cell numbers; and real-time polymerase chain reaction to quantify mRNA levels. Result(s) Treatment with CDB4124 significantly decreased levels of the proliferation marker PCNA, the number of viable LSM cells, and the anti-apoptotic protein Bcl-2. On the other hand, treatment with CDB4124 increased levels of the apoptosis marker cleaved PARP and the tumor suppressor KLF11 in a dose- and time-dependent manner in LSM cells. In matched MSM cells, however, CDB4124 did not affect cell proliferation or apoptosis. Conclusion(s) CDB4124 selectively inhibits proliferation and induces apoptosis in LSM but not in MSM cells. PMID:20056218

Assessment of FUN-1 vital dye staining: Yeast with a block in the vacuolar sorting pathway have impaired ability to form CIVS when stained with FUN-1 fluorescent dye.

PubMed

Essary, Brandin D; Marshall, Pamela A

2009-08-01

FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide] is a fluorescent dye used in studies of yeast and other fungi to monitor cell viability in the research lab and to assay for active fungal infection in the clinical setting. When the plasma membrane is intact, fungal cells internalize FUN-1 and the dye is seen as diffuse green cytosolic fluorescence. FUN-1 is then transported to the vacuole in metabolically active wild type cells and subsequently is compacted into fluorescent red cylindrical intravacuolar structures (CIVS) by an unknown transport pathway. This dye is used to determine yeast viability, as only live cells form CIVS. However, in live Saccharomyces cerevisiae with impaired protein sorting to the yeast vacuole, we report decreased to no CIVS formation, depending on the cellular location of the block in the sorting pathway. Cells with a block in vesicle-mediated transport from the Golgi to prevacuolar compartment (PVC) or with a block in recycling from the PVC to the Golgi demonstrate a substantial impairment in CIVS formation. Instead, the FUN-1 dye is seen either in small punctate structures under fluorescence or as diffuse red cytosol under white light. Thus, researchers using FUN-1 should be cognizant of the limitations of this procedure in determining cell viability as there are viable yeast mutants with impaired CIVS formation.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

PubMed Central

Ciocca, Daniel R.; Rozados, Viviana R.; Carrión, F. Darío Cuello; Gervasoni, Silvia I.; Matar, Pablo; Scharovsky, O. Graciela

2003-01-01

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs. PMID:12820652

Construction of a Bacterial Cell that Contains Only the Set of Essential Genes Necessary to Impart Life

DTIC Science & Technology

2014-08-15

characterized genes from Bacillus subtilis , that is presented in a constitutive expression module. If the B. subtilis gene containing M. mycoides mutant is...essential gene MMYC_0361 with the rlmH gene from Bacillus subtilis . Mycoplasma mycoides containing the B. subtilis rlmH was viable. This tells us the...viable than the function of the conserved hypothetical gene is the same as the input B. subtilis gene. Table of Contents: Section

Reactions of singlet oxygen with pine pollen.

NASA Technical Reports Server (NTRS)

Dowty, B.; Laseter, J. L.; Griffin, G. W.; Politzer, I. R.; Walkinshaw, C. H.

1973-01-01

A study was initiated to determine whether viable atmospheric particles such as plant pollens and fungal spores containing unsaturated lipids can interact with singlet oxygen to give oxygenated products that are potentially toxic. The results obtained confirm that surface and near surface components of common viable particulate matter in the atmosphere may be subject to rapid oxidation by singlet oxygen, leading to products which are probably allylic hydroperoxides. In connection with increasing atmospheric pollution, it is important to note that materials toxic to mammalian lung tissue may be oxidatively produced on the surfaces of viable particulate matter.

Arrhenius parameter determination as a function of heating method and cellular microenvironment based on spatial cell viability analysis.

PubMed

Whitney, Jon; Carswell, William; Rylander, Nichole

2013-06-01

Predictions of injury in response to photothermal therapy in vivo are frequently made using Arrhenius parameters obtained from cell monolayers exposed to laser or water bath heating. However, the impact of different heating methods and cellular microenvironments on Arrhenius predictions has not been thoroughly investigated. This study determined the influence of heating method (water bath and laser irradiation) and cellular microenvironment (cell monolayers and tissue phantoms) on Arrhenius parameters and spatial viability. MDA-MB-231 cells seeded in monolayers and sodium alginate phantoms were heated with a water bath for 3-20 min at 46, 50, and 54 °C or laser irradiated (wavelength of 1064 nm and fluences of 40 W/cm(2) or 3.8 W/cm(2) for 0-4 min) in combination with photoabsorptive carbon nanohorns. Spatial viability was measured using digital image analysis of cells stained with calcein AM and propidium iodide and used to determine Arrhenius parameters. The influence of microenvironment and heating method on Arrhenius parameters and capability of parameters derived from more simplistic experimental conditions (e.g. water bath heating of monolayers) to predict more physiologically relevant systems (e.g. laser heating of phantoms) were assessed. Arrhenius predictions of the treated area (<1% viable) under-predicted the measured areas in photothermally treated phantoms by 23 mm(2) using water bath treated cell monolayer parameters, 26 mm(2) using water bath treated phantom parameters, 27 mm(2) using photothermally treated monolayer parameters, and 0.7 mm(2) using photothermally treated phantom parameters. Heating method and cellular microenvironment influenced Arrhenius parameters, with heating method having the greater impact.

cAMP-CRP acts as a key regulator for the viable but non-culturable state in Escherichia coli.

PubMed

Nosho, Kazuki; Fukushima, Hiroko; Asai, Takehiro; Nishio, Masahiro; Takamaru, Reiko; Kobayashi-Kirschvink, Koseki Joseph; Ogawa, Tetsuhiro; Hidaka, Makoto; Masaki, Haruhiko

2018-03-01

A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

Non-destructive technique for determining the viability of soybean (Glycine max) seeds using FT-NIR spectroscopy.

PubMed

Kusumaningrum, Dewi; Lee, Hoonsoo; Lohumi, Santosh; Mo, Changyeun; Kim, Moon S; Cho, Byoung-Kwan

2018-03-01

The viability of seeds is important for determining their quality. A high-quality seed is one that has a high capability of germination that is necessary to ensure high productivity. Hence, developing technology for the detection of seed viability is a high priority in agriculture. Fourier transform near-infrared (FT-NIR) spectroscopy is one of the most popular devices among other vibrational spectroscopies. This study aims to use FT-NIR spectroscopy to determine the viability of soybean seeds. Viable and artificial ageing seeds as non-viable soybeans were used in this research. The FT-NIR spectra of soybean seeds were collected and analysed using a partial least-squares discriminant analysis (PLS-DA) to classify viable and non-viable soybean seeds. Moreover, the variable importance in projection (VIP) method for variable selection combined with the PLS-DA was employed. The most effective wavelengths were selected by the VIP method, which selected 146 optimal variables from the full set of 1557 variables. The results demonstrated that the FT-NIR spectral analysis with the PLS-DA method that uses all variables or the selected variables showed good performance based on the high value of prediction accuracy for soybean viability with an accuracy close to 100%. Hence, FT-NIR techniques with a chemometric analysis have the potential for rapidly measuring soybean seed viability. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Common bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages through suppression of NF-κB pathways.

PubMed

Oseguera-Toledo, Miguel E; de Mejia, Elvira Gonzalez; Dia, Vermont P; Amaya-Llano, Silvia L

2011-08-01

The objectives of this study were to evaluate the antioxidant capacity of protein hydrolysates of the common bean (Phaseolus vulgaris L.) varieties Negro 8025 and Pinto Durango and determine their effect on the markers of inflammation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Cell viability was determined and the percentage of viable cells was calculated and concentrations that allowed >80% cell viability were used to determine the markers of inflammation. Alcalase hydrolysates and pepsin-pancreatin hydrolysates showed the highest antioxidant capacity after 80 and 120min of hydrolysis, respectively. Alcalase hydrolysates of the common bean Pinto Durango at 120min inhibited inflammation, with IC50 values of 34.9±0.3, 13.9±0.3, 5.0±0.1 and 3.7±0.2μM, while var. Negro needed 43.6±0.2, 61.3±0.3, 14.2±0.3 and 48.2±0.1μM for the inhibition of cyclooxygenase-2 expression, prostaglandin E2 production, inducible nitric oxide synthase expression and nitric oxide production, respectively. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit. In conclusion, hydrolysates from the common bean can be used to combat inflammatory and oxidative-associated diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

A Stirred Microchamber for Oxygen Consumption Rate Measurements With Pancreatic Islets

PubMed Central

Papas, Klearchos K.; Pisania, Anna; Wu, Haiyan; Weir, Gordon C.; Colton, Clark K.

2010-01-01

Improvements in pancreatic islet transplantation for treatment of diabetes are hindered by the absence of meaningful islet quality assessment methods. Oxygen consumption rate (OCR) has previously been used to assess the quality of organs and primary tissue for transplantation. In this study, we describe and characterize a stirred microchamber for measuring OCR with small quantities of islets. The device has a titanium body with a chamber volume of about 200 µL and is magnetically stirred and water jacketed for temperature control. Oxygen partial pressure (pO2) is measured by fluorescence quenching with a fiber optic probe, and OCR is determined from the linear decrease of pO2 with time. We demonstrate that measurements can be made rapidly and with high precision. Measurements with βTC3 cells and islets show that OCR is directly proportional to the number of viable cells in mixtures of live and dead cells and correlate linearly with membrane integrity measurements made with cells that have been cultured for 24 h under various stressful conditions. PMID:17497731

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy.

PubMed

Hensley, Michael Taylor; Tang, Junnan; Woodruff, Kathleen; Defrancesco, Teresa; Tou, Sandra; Williams, Christina M; Breen, Mathew; Meurs, Kathryn; Keene, Bruce; Cheng, Ke

2017-08-01

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Maximising electricity production by controlling the biofilm specific growth rate in microbial fuel cells.

PubMed

Ledezma, Pablo; Greenman, John; Ieropoulos, Ioannis

2012-08-01

The aim of this work is to study the relationship between growth rate and electricity production in perfusion-electrode microbial fuel cells (MFCs), across a wide range of flow rates by co-measurement of electrical output and changes in population numbers by viable counts and optical density. The experiments hereby presented demonstrate, for the first time to the authors' knowledge, that the anodic biofilm specific growth rate can be determined and controlled in common with other loose matrix perfusion systems. Feeding with nutrient-limiting conditions at a critical flow rate (50.8 mL h(-1)) resulted in the first experimental determination of maximum specific growth rate μ(max) (19.8 day(-1)) for Shewanella spp. MFC biofilms, which is considerably higher than those predicted or assumed via mathematical modelling. It is also shown that, under carbon-energy limiting conditions there is a strong direct relationship between growth rate and electrical power output, with μ(max) coinciding with maximum electrical power production. Copyright © 2012 Elsevier Ltd. All rights reserved.

An Image Analysis Algorithm for Malaria Parasite Stage Classification and Viability Quantification

PubMed Central

Moon, Seunghyun; Lee, Sukjun; Kim, Heechang; Freitas-Junior, Lucio H.; Kang, Myungjoo; Ayong, Lawrence; Hansen, Michael A. E.

2013-01-01

With more than 40% of the world’s population at risk, 200–300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays. PMID:23626733

In vitro antimicrobial effect of the tissue conditioner containing silver nanoparticles

PubMed Central

2011-01-01

PURPOSE The aim of this study was to identify in vitro antimicrobial activity of the tissue conditioner containing silver nanoparticles on microbial strains, Staphylococcus aureus, Streptococcus mutans and Candida albicans. MATERIALS AND METHODS Experimental disc samples (20.0×3.0 mm) of tissue conditioner (GC Soft-Liner, GC cooperation, Tokyo, Japan) containing 0.1 - 3.0% silver nanoparticles (0%: control) were fabricated. Samples were placed on separate culture plate dish and microbial suspensions (100 µL) of tested strains were inoculated then incubated at 37℃. Microbial growth was verified at 24 hrs and 72 hrs and the antimicrobial effects of samples were evaluated as a percentage of viable cells in withdrawn suspension (100 µL). Data were recorded as the mean of three colony forming unit (CFU) numerations and the borderline of the antimicrobial effect was determined at 0.1% viable cells. RESULTS A 0.1% silver nanoparticles combined to tissue conditioner displayed minimal bactericidal effect against Staphylococcus aureus and Streptococcus mutans strains, a 0.5% for fungal strain. Control group did not show any microbial inhibitory effect and there were no statistical difference between 24 hrs and extended 72 hrs incubation time (P > .05). CONCLUSION Within the limitation of this in vitro study, the results suggest that the tissue conditioner containing silver nanoparticles could be an antimicrobial dental material in denture plaque control. Further mechanical stability and toxicity studies are still required. PMID:21503189

Immunization with the Haemophilus ducreyi Hemoglobin Receptor HgbA Protects against Infection in the Swine Model of Chancroid

PubMed Central

Afonina, Galyna; Leduc, Isabelle; Nepluev, Igor; Jeter, Chrystina; Routh, Patty; Almond, Glen; Orndorff, Paul E.; Hobbs, Marcia; Elkins, Christopher

2006-01-01

The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid. PMID:16552053

Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

PubMed

Wolf, Moritz K F; Closet, Aurélie; Bzowska, Monika; Bielser, Jean-Marc; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

2018-05-21

Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mycobacterium tuberculosis Catalase Inhibits the Formation of Mast Cell Extracellular Traps

PubMed Central

Campillo-Navarro, Marcia; Leyva-Paredes, Kahiry; Donis-Maturano, Luis; Rodríguez-López, Gloria M.; Soria-Castro, Rodolfo; García-Pérez, Blanca Estela; Puebla-Osorio, Nahum; Ullrich, Stephen E.; Luna-Herrera, Julieta; Flores-Romo, Leopoldo; Sumano-López, Héctor; Pérez-Tapia, Sonia M.; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

2018-01-01

Tuberculosis is one of the leading causes of human morbidity and mortality. Mycobacterium tuberculosis (Mtb) employs different strategies to evade and counterattack immune responses persisting for years. Mast cells are crucial during innate immune responses and help clear infections via inflammation or by direct antibacterial activity through extracellular traps (MCETs). Whether Mtb induce MCETs production is unknown. In this study, we report that viable Mtb did not induce DNA release by mast cells, but heat-killed Mtb (HK-Mtb) did. DNA released by mast cells after stimulation with HK-Mtb was complexed with histone and tryptase. MCETs induced with PMA and HK-Mtb were unable to kill live Mtb bacilli. Mast cells stimulated with HK-Mtb induced hydrogen peroxide production, whereas cells stimulated with viable Mtb did not. Moreover, MCETs induction by HK-Mtb was dependent of NADPH oxidase activity, because its blockade resulted in a diminished DNA release by mast cells. Interestingly, catalase-deficient Mtb induced a significant production of hydrogen peroxide and DNA release by mast cells, indicating that catalase produced by Mtb prevents MCETs release by degrading hydrogen peroxide. Our findings show a new strategy employed by Mtb to overcome the immune response through inhibiting MCETs formation, which could be relevant during early stages of infection. PMID:29892297

Mycobacterium tuberculosis Catalase Inhibits the Formation of Mast Cell Extracellular Traps.

PubMed

Campillo-Navarro, Marcia; Leyva-Paredes, Kahiry; Donis-Maturano, Luis; Rodríguez-López, Gloria M; Soria-Castro, Rodolfo; García-Pérez, Blanca Estela; Puebla-Osorio, Nahum; Ullrich, Stephen E; Luna-Herrera, Julieta; Flores-Romo, Leopoldo; Sumano-López, Héctor; Pérez-Tapia, Sonia M; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

2018-01-01

Tuberculosis is one of the leading causes of human morbidity and mortality. Mycobacterium tuberculosis (Mtb) employs different strategies to evade and counterattack immune responses persisting for years. Mast cells are crucial during innate immune responses and help clear infections via inflammation or by direct antibacterial activity through extracellular traps (MCETs). Whether Mtb induce MCETs production is unknown. In this study, we report that viable Mtb did not induce DNA release by mast cells, but heat-killed Mtb (HK-Mtb) did. DNA released by mast cells after stimulation with HK-Mtb was complexed with histone and tryptase. MCETs induced with PMA and HK-Mtb were unable to kill live Mtb bacilli. Mast cells stimulated with HK-Mtb induced hydrogen peroxide production, whereas cells stimulated with viable Mtb did not. Moreover, MCETs induction by HK-Mtb was dependent of NADPH oxidase activity, because its blockade resulted in a diminished DNA release by mast cells. Interestingly, catalase-deficient Mtb induced a significant production of hydrogen peroxide and DNA release by mast cells, indicating that catalase produced by Mtb prevents MCETs release by degrading hydrogen peroxide. Our findings show a new strategy employed by Mtb to overcome the immune response through inhibiting MCETs formation, which could be relevant during early stages of infection.

Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes.

PubMed

Li, J; Kolling, G L; Matthews, K R; Chikindas, M L

2003-01-01

To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state. When cold and CO2 stressed L. monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population. By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included). If there were VBNC state cells present, we should have observed a significant difference between the above two numbers. In fact, there was no significant difference between the results obtained from those two methods. There were no VBNC state cells observed in the stressed cell population. We conclude that cold and CO2 do not induce L. monocytogenes to enter a VBNC state. Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation. Whether they would induce L. monocytogenes into a VBNC state is of a great concern for microbial food safety.