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.

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

Tumor-volume simulation during radiotherapy for head-and-neck cancer using a four-level cell population model.

PubMed

Chvetsov, Alexei V; Dong, Lei; Palta, Jantinder R; Amdur, Robert J

2009-10-01

To develop a fast computational radiobiologic model for quantitative analysis of tumor volume during fractionated radiotherapy. The tumor-volume model can be useful for optimizing image-guidance protocols and four-dimensional treatment simulations in proton therapy that is highly sensitive to physiologic changes. The analysis is performed using two approximations: (1) tumor volume is a linear function of total cell number and (2) tumor-cell population is separated into four subpopulations: oxygenated viable cells, oxygenated lethally damaged cells, hypoxic viable cells, and hypoxic lethally damaged cells. An exponential decay model is used for disintegration and removal of oxygenated lethally damaged cells from the tumor. We tested our model on daily volumetric imaging data available for 14 head-and-neck cancer patients treated with an integrated computed tomography/linear accelerator system. A simulation based on the averaged values of radiobiologic parameters was able to describe eight cases during the entire treatment and four cases partially (50% of treatment time) with a maximum 20% error. The largest discrepancies between the model and clinical data were obtained for small tumors, which may be explained by larger errors in the manual tumor volume delineation procedure. Our results indicate that the change in gross tumor volume for head-and-neck cancer can be adequately described by a relatively simple radiobiologic model. In future research, we propose to study the variation of model parameters by fitting to clinical data for a cohort of patients with head-and-neck cancer and other tumors. The potential impact of other processes, like concurrent chemotherapy, on tumor volume should be evaluated.

Trace detection of specific viable bacteria using tetracysteine-tagged bacteriophages.

PubMed

Wu, Lina; Luan, Tian; Yang, Xiaoting; Wang, Shuo; Zheng, Yan; Huang, Tianxun; Zhu, Shaobin; Yan, Xiaomei

2014-01-07

Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.

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.

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.

Optimization of PMA-qPCR for Staphylococcus aureus and determination of viable bacteria in indoor air.

PubMed

Chang, C-W; Lin, M-H

2018-01-01

Staphylococcus aureus may cause infections in humans from mild skin disorders to lethal pneumonia. Rapid and accurate monitoring of viable S. aureus is essential to characterize human exposure. This study evaluated quantitative PCR (qPCR) with propidium monoazide (PMA) to quantify S. aureus. The results showed comparable S. aureus counts between exclusively live cells and mixtures of live/dead cells by qPCR with 1.5 or 2.3 μg/mL PMA (P>.05), illustrating the ability of PMA-qPCR to detect DNA exclusively from viable cells. Moreover, qPCR with 1.5 or 2.3 μg/mL PMA performed optimally with linearity over 10 3 -10 8  CFU/mL (R 2 ≥0.9), whereas qPCR with 10, 23 or 46 μg/mL PMA significantly underestimated viable counts. Staphylococcus aureus and total viable bacteria were further determined with PMA-qPCR (1.5 μg/mL) from 48 samples from a public library and two university dormitories and four from outside. Viable bacteria averaged 1.9×10 4 cells/m 3 , and S. aureus were detected in 22 (42%) samples with a mean of 4.4×10 3 cells/m 3 . The number of S. aureus and viable bacteria were positively correlated (r=.61, P<.005), and percentages of S. aureus relative to viable bacteria averaged 12-44%. The results of field samples suggest that PMA-qPCR can be used to quantify viable S. aureus cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of a solenoid actuated planar valveless micropump with single and multiple inlet-outlet arrangements

NASA Astrophysics Data System (ADS)

Kumar, N.; George, D.; Sajeesh, P.; Manivannan, P. V.; Sen, A. K.

2016-07-01

We report a planar solenoid actuated valveless micropump with multiple inlet-outlet configurations. The self-priming characteristics of the multiple inlet-multiple outlet micropump are studied. The filling dynamics of the micropump chamber during start-up and the effects of fluid viscosity, voltage and frequency on the dynamics are investigated. Numerical simulations for multiple inlet-multiple outlet micropumps are carried out using fluid structure algorithm. With DI water and at 5.0 Vp-p, 20 Hz frequency, the two inlet-two outlet micropump provides a maximum flow rate of 336 μl min-1 and maximum back pressure of 441 Pa. Performance characteristics of the two inlet-two outlet micropump are studied for aqueous fluids of different viscosity. Transport of biological cell lines and diluted blood samples are demonstrated; the flow rate-frequency characteristics are studied. Viability of cells during pumping with multiple inlet multiple outlet configuration is also studied in this work, which shows 100% of cells are viable. Application of the proposed micropump for simultaneous pumping, mixing and distribution of fluids is demonstrated. The proposed integrated, standalone and portable micropump is suitable for drug delivery, lab-on-chip and micro-total-analysis applications.

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.

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 simplified Protocol to Induce Callogenesis in Protoplasts of Date Palm (Phoenix dactylifera L.) Cultivars.

PubMed

Titouh, Khayreddine; Khelifi, Lakhdar; Slaoui, Majda; Boufis, Nazim; Morsli, Abdelkader; Hadj Moussa, Khadidja Titouh; Makhzoum, Abdullah

2015-03-01

In Algeria, date palm is currently confronted to the Bayoud disease. Biotechnological tools such as protoplastsfusion can appear as an alternative to ensure rapid multiplication and improvement of this species. Callogenesis induction in protoplasts isolated from embryogenic callus of three date palm cultivars. Some factors influencing the isolation and culture of protoplasts segregated from the calli of three date palm ( Phoenix dactylifera L.) cultivars (Deglet Nour, Akerbouch and Degla Beida) were studied. Protoplasts of each cultivar were cultured on a semi-solid medium supplemented with various hormonal balances. Maceration with an enzymatic solution containing 1.5% cellulase and 1% macerozyme R10 in the presence of 0.5 M mannitol for more than 16 h with gentle agitation allows isolation of a great number of viable protoplasts. In addition, purification of protoplasts on a cushion of 21 or 25% sucrose was effective in cell debris removal and maximum recovery. The culture of isolated protoplasts on a semi-solidified Murashige and Skoog medium, with 0.3% agarose, 2 mg. L -1 2,4-D and 0.5 mg.L -1 BAP allowed good viable protoplast maintenance as well as cell wall regeneration. After more than two months of culture, cell divisions were still occurring and microcalli became visible to the naked eye, containing a large number of cells. The developed protocol can be useful for application of somatic hybridization to improve date palm cultivars.

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.

Thermal evaluation of laser exposures in an in vitro retinal model by microthermal sensing

NASA Astrophysics Data System (ADS)

Choi, Tae Y.; Denton, Michael L.; Noojin, Gary D.; Estlack, Larry E.; Shrestha, Ramesh; Rockwell, Benjamin A.; Thomas, Robert; Kim, Dongsik

2014-09-01

A temperature detection system using a micropipette thermocouple sensor was developed for use within mammalian cells during laser exposure with an 8.6-μm beam at 532 nm. We have demonstrated the capability of measuring temperatures at a single-cell level in the microscale range by inserting micropipette-based thermal sensors of size ranging from 2 to 4 μm into the membrane of a live retinal pigment epithelium (RPE) cell subjected to a laser beam. We setup the treatment groups of 532-nm laser-irradiated single RPE cell and in situ temperature recordings were made over time. Thermal profiles are given for representative cells experiencing damage resulting from exposures of 0.2 to 2 s. The measured maximum temperature rise for each cell ranges from 39 to 73°C the RPE cells showed a signature of death for all the cases reported herein. In order to check the cell viability, real-time fluorescence microscopy was used to identify the transition of pigmented RPE cells between viable and damaged states due to laser exposure.

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.

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2017-11-01

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC-), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC-) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations-concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate-induced different degrees of transience from VC+ to VC- states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

Distribution of bacterioplankton with active metabolism in waters of the St. Anna Trough, Kara Sea, in autumn 2011

NASA Astrophysics Data System (ADS)

Mosharova, I. V.; Mosharov, S. A.; Ilinskiy, V. V.

2017-01-01

The distribution of bacterioplankton with active electron transport chains, as well as bacteria with intact cell membranes, was investigated for the first time in the region of St. Anna Trough in the Kara Sea. The average number of bacteria with active electron transport chains in the waters of the St. Anna Trough was 15.55 × 103 cells mL-1 (the limits of variation were 1.06-92.17 × 103 cells mL-1). The average number of bacteria with intact membranes was 33.46 × 103 cells mL-1 (the limits of variation were 6.78 to 103.18 × 103 cells mL-1). Almost all bacterioplankton microorganisms in the studied area were potentially viable, and the average share of bacteria with intact membranes was 92.1% of the total number of bacterioplankton (TNB) (the limits of variation were 76.2 to 98.4%). The share of bacteria with active metabolisms was 38.2% of the TNB (the limits of variation were 5.6-93.4%). The shares of the bacteria with active metabolisms were maximum in areas with the most stable environmental conditions (on the shelf and in deep water), whereas on the slope, where the gradients of water temperature and salinity were maximum, these values were lower.

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.

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

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

A method for isolation of rat lymphocyte-rich mononuclear cells from lung tissue useful for determination of nucleoside triphosphate diphosphohydrolase activity.

PubMed

Jaques, Jeandre Augusto Dos S; Peres Rezer, João Felipe; Ruchel, Jader Betsch; Gutierres, Jessié; Bairros, André Valle; Gomes Farias, Iria Luiza; Almeida da Luz, Sonia Cristina; Mello Bertoncheli, Claudia de; Chitolina Schetinger, Maria Rosa; Morsch, Vera Maria; Leal, Daniela Bitencourt Rosa

2011-03-01

Methods for the isolation of peripheral blood mononuclear cells (PBMCs) and human lung mononuclear cells (LMCs) have been proposed previously. This study describes a method that allows the separation of lymphocyte-rich LMCs from rats. Trypan blue was applied to determine cell viability. White blood cell and differential cell counts were also performed. Relationships between nucleoside triphosphate diphosphohydrolase (NTPDase, EC 3.6.1.5) activities expressed in milligrams of protein, millions of cells, and millions of viable cells were examined as linear correlations. The lung tissue yielded 82.46% lymphocytes, 8.6% macrophages, 2.20% monocytes, and 1.27% polymorphonuclear cells (PMNs). In LMCs, a very strong correlation was observed as follows: between NTPDase activity, as determined using ATP or ADP as a substrate, expressed in milligrams of protein and that expressed in millions of cells (r ≥ 0.91), between that expressed in milligrams of protein and that expressed in millions of viable cells (r ≥ 0.91), and between that expressed in millions of cells and that expressed in millions of viable cells (r ≥ 0.98). Based on our results, we affirm that NTPDase activity could be expressed in millions of viable cells, millions of cells, or milligrams of protein. 2010 Elsevier Inc. All rights reserved.

Mathematical estimation of the level of microbial contamination on spacecraft surfaces by volumetric air sampling

NASA Technical Reports Server (NTRS)

Oxborrow, G. S.; Roark, A. L.; Fields, N. D.; Puleo, J. R.

1974-01-01

Microbiological sampling methods presently used for enumeration of microorganisms on spacecraft surfaces require contact with easily damaged components. Estimation of viable particles on surfaces using air sampling methods in conjunction with a mathematical model would be desirable. Parameters necessary for the mathematical model are the effect of angled surfaces on viable particle collection and the number of viable cells per viable particle. Deposition of viable particles on angled surfaces closely followed a cosine function, and the number of viable cells per viable particle was consistent with a Poisson distribution. Other parameters considered by the mathematical model included deposition rate and fractional removal per unit time. A close nonlinear correlation between volumetric air sampling and airborne fallout on surfaces was established with all fallout data points falling within the 95% confidence limits as determined by the mathematical model.

[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.

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.

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.

Soluble Factors Released by Endogenous Viable Cells Enhance the Antioxidant and Chemoattractive Activities of Cryopreserved Amniotic Membrane

PubMed Central

Duan-Arnold, Yi; Gyurdieva, Alexandra; Johnson, Amy; Jacobstein, Douglas A.; Danilkovitch, Alla

2015-01-01

Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment. Progress in tissue preservation has led to commercialization of hAM. The majority of hAM products are devitalized with various degrees of matrix alteration. Data show the importance of hAM matrix preservation, but little is known about the advantages of retaining viable endogenous cells. In this study, we compared the antioxidant and chemoattractive properties of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM) to determine the benefits of cell preservation. Approach: We evaluated the ability of int-hAM and dev-hAM to protect fibroblasts from oxidant-induced cell damage, to suppress oxidants, and to recruit fibroblasts and keratinocytes in vitro. Results: Both the int-hAM–derived conditioned medium (CM) and the int-hAM tissue rescued significantly more fibroblasts from oxidant-induced damage than dev-hAM (844% and 93% more, respectively). The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM. The int-hAM CM enhanced the recruitment of fibroblasts and normal and diseased keratinocytes to a greater extent than dev-hAM (1,555%, 315%, and 151% greater, respectively). Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed. The release of soluble factors that protect fibroblasts from oxidative injury by hAM containing viable cells is a mechanism of hAM antioxidant activity, which is a novel finding of this study. PMID:26029483

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.

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.

Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance

PubMed Central

RAVAL, JAY S.; KOCH, EILEEN; DONNENBERG, ALBERT D.

2014-01-01

Background aims Airborne particulate monitoring is mandated as a component of good manufacturing practice. We present a procedure developed to monitor and interpret airborne particulates in an International Organization for Standardization (ISO) class 7 cleanroom used for the cell processing of Section 351 and Section 361 products. Methods We collected paired viable and non-viable airborne particle data over a period of 1 year in locations chosen to provide a range of air quality. We used receiver operator characteristic (ROC) analysis to determine empirically the relationship between non-viable and viable airborne particle counts. Results Viable and non-viable particles were well-correlated (r 2 = 0.78), with outlier observations at the low end of the scale (non-viable particles without detectable airborne colonies). ROC analysis predicted viable counts ≥0.5/feet 3 (a limit set by the United States Pharmacopeia) at an action limit of ≥32 000 particles (≥0.5 μ)/feet 3 , with 95.6% sensitivity and 50% specificity. This limit was exceeded 2.6 times during 18 months of retrospective daily cleanroom data (an expected false alarm rate of 1.3 times/year). After implementing this action limit, we were alerted in real time to an air-handling failure undetected by our hospital facilities management. Conclusions A rational action limit for non-viable particles was determined based on the correlation with airborne colonies. Reaching or exceeding the action limit of 32 000 non-viable particles/feet 3 triggers suspension of cleanroom cell-processing activities, deep cleaning, investigation of air handling, and a deviation management process. Our full procedure for particle monitoring is available as an online supplement. PMID:22746538

Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.

PubMed

Raval, Jay S; Koch, Eileen; Donnenberg, Albert D

2012-10-01

Airborne particulate monitoring is mandated as a component of good manufacturing practice. We present a procedure developed to monitor and interpret airborne particulates in an International Organization for Standardization (ISO) class 7 cleanroom used for the cell processing of Section 351 and Section 361 products. We collected paired viable and non-viable airborne particle data over a period of 1 year in locations chosen to provide a range of air quality. We used receiver operator characteristic (ROC) analysis to determine empirically the relationship between non-viable and viable airborne particle counts. Viable and non-viable particles were well-correlated (r(2) = 0.78), with outlier observations at the low end of the scale (non-viable particles without detectable airborne colonies). ROC analysis predicted viable counts ≥ 0.5/feet(3) (a limit set by the United States Pharmacopeia) at an action limit of ≥ 32 000 particles (≥ 0.5 µ)/feet(3), with 95.6% sensitivity and 50% specificity. This limit was exceeded 2.6 times during 18 months of retrospective daily cleanroom data (an expected false alarm rate of 1.3 times/year). After implementing this action limit, we were alerted in real time to an air-handling failure undetected by our hospital facilities management. A rational action limit for non-viable particles was determined based on the correlation with airborne colonies. Reaching or exceeding the action limit of 32 000 non-viable particles/feet(3) triggers suspension of cleanroom cell-processing activities, deep cleaning, investigation of air handling, and a deviation management process. Our full procedure for particle monitoring is available as an online supplement.

Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

PubMed

Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

2013-07-01

Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management. Copyright © 2013 Elsevier Inc. All rights reserved.

Oxygen consumption rate and mitochondrial density in human melanoma monolayer cultures and multicellular spheroids.

PubMed

Hystad, M E; Rofstad, E K

1994-05-15

Rate of oxygen consumption per cell has been shown in previous studies to decrease with increasing depth in the viable rim of multicellular spheroids initiated from rodent cells, human colon-carcinoma cells, and human glioma cells, due to progressive accumulation of quiescent cells during spheroid growth. The purpose of our work was to determine oxygen-consumption profiles in human melanoma spheroids. Monolayer cultures of 4 lines (BEX-c, COX-c, SAX-c, and WIX-c) and spheroid cultures of 2 lines (BEX-c and WIX-c) were subjected to investigation. Spheroids were initiated from monolayer cell cultures and grown in spinner flasks. Rate of oxygen consumption was measured with a Clarke-type electrode. Mitochondrial density was determined by stereological analysis of transmission electron micrographs. Thickness of viable rim and cell packing density were assessed by light microscopy of central spheroid sections. Cell-cycle distribution was determined by analysis of DNA histograms measured by flow cytometry. Cell volume was measured by an electronic particle counter. Rate of oxygen consumption per cell differed by a factor of approximately 1.8 between the 4 cell lines and was positively correlated to total volume of mitochondria per cell. Rate of oxygen consumption per cell and total volume of mitochondria per cell were equal for monolayer cell cultures, 600-microns spheroids and 1,200-microns spheroids of the same line. Mitochondrial density and location in the cell did not differ between cells at the spheroid surface, in the middle of the viable rim and adjacent to the central necrosis. Cell-cycle distribution, cell volume, and cell-packing density in the outer and inner halves of the viable rim were not significantly different. Consequently, the rate of oxygen consumption per cell in inner regions of the viable rim was probably equal to that at the spheroid surface, suggesting that oxygen diffusion distances may be shorter in some melanomas than in many other tumor types.

Efficacy and reusability of alginate-immobilized live and heat-inactivated Trichoderma asperellum cells for Cu (II) removal from aqueous solution.

PubMed

Tan, Wei Shang; Ting, Adeline Su Yien

2012-11-01

Cu(II) removal efficacies of alginate-immobilized Trichoderma asperellum using viable and non-viable forms were investigated with respect to time, pH, and initial Cu(II) concentrations. The reusability potential of the biomass was determined based on sorption/desorption tests. Cu(II) biosorption by immobilized heat-inactivated T. asperellum cells was the most efficient, with 134.22mg Cu(II) removed g(-1) adsorbent, compared to immobilized viable cells and plain alginate beads (control) with 105.96 and 94.04mg Cu(II) adsorbed g(-1) adsorbent, respectively. Immobilized non-viable cells achieved equilibrium more rapidly within 4h. For all biosorbents, optimum pH for Cu(II) removal was between pH 4 and 5. Reusability of all biosorbents were similar, with more than 90% Cu(II) desorbed with HCl. These alginate-immobilized cells can be applied to reduce clogging and post-separation process incurred from use of suspended biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

Probabilistic pathway construction.

PubMed

Yousofshahi, Mona; Lee, Kyongbum; Hassoun, Soha

2011-07-01

Expression of novel synthesis pathways in host organisms amenable to genetic manipulations has emerged as an attractive metabolic engineering strategy to overproduce natural products, biofuels, biopolymers and other commercially useful metabolites. We present a pathway construction algorithm for identifying viable synthesis pathways compatible with balanced cell growth. Rather than exhaustive exploration, we investigate probabilistic selection of reactions to construct the pathways. Three different selection schemes are investigated for the selection of reactions: high metabolite connectivity, low connectivity and uniformly random. For all case studies, which involved a diverse set of target metabolites, the uniformly random selection scheme resulted in the highest average maximum yield. When compared to an exhaustive search enumerating all possible reaction routes, our probabilistic algorithm returned nearly identical distributions of yields, while requiring far less computing time (minutes vs. years). The pathways identified by our algorithm have previously been confirmed in the literature as viable, high-yield synthesis routes. Prospectively, our algorithm could facilitate the design of novel, non-native synthesis routes by efficiently exploring the diversity of biochemical transformations in nature. Copyright © 2011 Elsevier Inc. All rights reserved.

Enhanced ethanol production at commercial scale from molasses using high gravity technology by mutant S. cerevisiae.

PubMed

Arshad, Muhammad; Hussain, Tariq; Iqbal, Munawar; Abbas, Mazhar

Very high gravity (VHG) technology was employed on industrial scale to produce ethanol from molasses (fermented) as well as by-products formation estimation. The effect of different Brix° (32, 36 and 40) air-flow rates (0.00, 0.20, 0.40, and 0.60vvm) was studied on ethanol production. The maximum ethanol production was recorded to be 12.2% (v/v) at 40 Brix° with 0.2vvm air-flow rate. At optimum level aeration and 40 Brix° VHG, the residual sugar level was recorded in the range of 12.5-18.5g/L, whereas the viable cell count remained constant up to 50h of fermentation and dry matter production increased with fermentation time. Both water and steam consumption reduced significantly under optimum conditions of Brix° and aeration rate with compromising the ethanol production. Results revealed VHG with continuous air flow is viable technique to reduce the ethanol production cost form molasses at commercial scale. Copyright © 2017. Published by Elsevier Editora Ltda.

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

Synthesis of gold nanostructures with optical properties within the near-infrared window for biomedical applications

NASA Astrophysics Data System (ADS)

Garcia-Soto, Mariano de Jesus

The work reported in this dissertation describes the design and synthesis of different gold nanoshells with strong absorption coefficients at the near-infrared region (NIR) of the spectrum, and includes preliminary studies of their use for the photo-induced heating of pancreatic cancer cells and ex vivo tissues. As the emphasis was on gold nanoshells with maximum extinctions located at 800 nm, the methods explored for their synthesis led us to the preparation of silica-core and hollow gold nanoshells of improved stability, with maximum extinctions at or beyond the targeted within the near-infrared window. The synthesis of silica-core gold nanoshells was investigated first given its relevance as one of the pioneering methods to produce gold nanostructures with strong absorption and scattering coefficients in the visible and the near-infrared regions of the spectrum. By using a classical method of synthesis, we explored the aging of the precursor materials and the effect of using higher concentrations than the customary for the reduction of gold during the shell growth. We found that the aging for one week of the as-prepared or purified precursors, namely, the gold cluster suspensions, and the seeded silica particles, along with higher concentrations of gold in the plating solution, produced fully coated nanoshells of 120 nm in size with smooth surfaces and maximum extinctions around 800 nm. Additional work carried out to reduce the time and steps in the synthesis of silica-core gold nanoshells, led us to improve the seeding step by increasing the ionic strength of the cluster suspension, and also to explore the growth of gold on tin-seeded silica nanoparticles. The synthesis of hollow gold nanoshells (HGS) of with maximum extinctions at the NIR via the galvanic replacement of silver nanoparticles for gold in solution was explored next. A first method explored led us to obtain HGS with maximum extinctions between 650 and 800 nm and sizes between 30 and 80 nm from silver nanoparticles, which were grown by the addition of silver nitrate and a mild reducer. We developed a second method that led us to obtain HGS with maximum extinctions between 750 and 950 nm by adjusting the pH of the precursor solution of the silver particles without much effort or additional steps. The last part of this work consisted in demonstrating the photo-induced heating of two biological systems containing HGS. Photothermal therapy studies of immobilized PANC1 pancreas cancer cells in well-plates were carried out with functionalized HGS. We found that cells exposed to HGS remained viable after incubation. Moreover, the cells incubated with HGS modified with mercaptoundecanoic acid and folic acid turned non-viable after being irradiated with a laser at 800 nm. The other study consisted in the laser-induced heating between 750 and 1000 nm of ex vivo tissues of chicken and pork with nanoshells injected. In comparison with non-injected tissues, it was found that the temperature at the irradiated areas with HGS increased more than 10 °C. Moreover, the extent of the heated area was broader when the laser was used at wavelengths beyond 900 nm, suggesting that the heating was due to the radiation absorbed and transformed into heat primarily by the HGS and at a lesser extent by the water in the tissue.

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.

Serum miRNA Predicts Viable Disease after Chemotherapy in Patients with Testicular Nonseminoma Germ Cell Tumor.

PubMed

Leão, Ricardo; van Agthoven, Ton; Figueiredo, Arnaldo; Jewett, Michael A S; Fadaak, Kamel; Sweet, Joan; Ahmad, Ardalan E; Anson-Cartwright, Lynn; Chung, Peter; Hansen, Aaron; Warde, Padraig; Castelo-Branco, Pedro; O'Malley, Martin; Bedard, Philippe L; Looijenga, Leendert H J; Hamilton, Robert J

2018-02-21

Retroperitoneal lymph node dissection is recommended for residual masses greater than 1 cm after chemotherapy of nonseminomatous germ cell tumors. Currently to our knowledge there is no reliable predictor of post-chemotherapy retroperitoneal lymph node dissection histology. Up to 50% of patients harbor necrosis/fibrosis only so that a potentially morbid surgery has limited therapeutic value. In this study we evaluated the ability of defined serum miRNAs to predict residual viable nonseminomatous germ cell tumors after chemotherapy. Levels of serum miRNA, including miR-371a-3p, miR-373-3p and miR-367-3p, were measured using the ampTSmiR (amplification targeted serum miRNA) test in 82 patients, including 39 in cohort 1 and 43 in cohort 2, who were treated with orchiectomy, chemotherapy and post-chemotherapy retroperitoneal lymph node dissection. miRNA levels were compared to clinical characteristics and serum tumor markers. They correlated with the presence of a viable germ cell tumor vs fibrosis/necrosis and teratoma. ROC analysis was done to determine miRNA discriminative capacity. miRNA levels were significantly associated with disease extent at chemotherapy and they decreased significantly after chemotherapy. Conventional serum tumor maker levels were uninformative after chemotherapy. However, after chemotherapy miRNA levels remained elevated in post-chemotherapy retroperitoneal lymph node dissection specimens of patients harboring viable germ cell tumors. miR-371a-3p demonstrated the highest discriminative capacity for viable germ cell tumors (AUC 0.874, 95% CI 0.774-0.974, p <0.0001). Using an adapted hypothetical cutoff of 3 cm or less for surgical intervention miR-371a-3p correctly stratified all patients with viable residual retroperitoneal germ cell tumors with 100% sensitivity (p = 0.02). To our knowledge our study demonstrates for the first time the potential value of miR-371a-3p to predict viable germ cell tumors in residual masses after chemotherapy. Prospective studies are required to confirm clinical usefulness. Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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.

[The effect of dexamethoxin on the integrity of cytoplasmic membrane in gram-positive and gram-negative microorganisms].

PubMed

Shchetina, V N; Belanov, E F; Starobinets, Z G; Volianskiĭ, Iu L

1990-01-01

Decamethoxin is shown to be able to increase membrane permeability of Pseudomonas aeruginosa, Escherichia coli and Micrococcus lysodeikticus, that is confirmed by a loss of compounds with the absorption maximum at 260 nm by cells. Parallel with this the number of viable individuals has fallen and activity of dehydrogenases has been inhibited. The aspartate and alanine aminotransferase activity was not inhibited by decamethoxin and even increased. Decamethoxin lysed the protoplasts of the tested microorganisms. At high decamethoxin concentrations (over 500 micrograms/ml for P. aeruginosa and over 200 mu/ml--for E. coli) the outflow of components from the cells of gram-negative bacteria ceased, that may be associated with the coagulation changes in the cytoplasm. A loss of the low-molecular components by M. lysodeikticus cells and lysis of protoplasts proceeded less intensely than the same processes in the gram-negative microorganisms, that is explained by a less resistance of M. lysodeikticus to decamethoxin and earlier coagulation of the cytoplasm preventing lysis.

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.

PolyMUMPs MEMS device to measure mechanical stiffness of single cells in aqueous media

NASA Astrophysics Data System (ADS)

Warnat, S.; King, H.; Forbrigger, C.; Hubbard, T.

2015-02-01

A method of experimentally determining the mechanical stiffness of single cells by using differential displacement measurements in a two stage spring system is presented. The spring system consists of a known MEMS reference spring and an unknown cellular stiffness: the ratio of displacements is related to the ratio of stiffness. A polyMUMPs implementation for aqueous media is presented and displacement measurements made from optical microphotographs using a FFT based displacement method with a repeatability of ~20 nm. The approach was first validated on a MEMS two stage spring system of known stiffness. The measured stiffness ratios of control structures (i) MEMS spring systems and (ii) polystyrene microspheres were found to agree with theoretical values. Mechanical tests were then performed on Saccharomyces cerevisiae (Baker’s yeast) in aqueous media. Cells were placed (using a micropipette) inside MEMS measuring structures and compressed between two jaws using an electrostatic actuator and displacements measured. Tested cells showed stiffness values between 5.4 and 8.4 N m-1 with an uncertainty of 11%. In addition, non-viable cells were tested by exposing viable cells to methanol. The resultant mean cell stiffness dropped by factor of 3 × and an explicit discrimination between viable and non-viable cells based on mechanical stiffness was seen.

Enhanced power generation and energy conversion of sewage sludge by CEA-microbial fuel cells.

PubMed

Abourached, Carole; Lesnik, Keaton Larson; Liu, Hong

2014-08-01

The production of methane from sewage sludge through the use of anaerobic digestion has been able to effectively offset energy costs for wastewater treatment. However, significant energy reserves are left unrecovered and effluent standards are not met necessitating secondary processes such as aeration. In the current study a novel cloth-electrode assembly microbial fuel cell (CEA-MFC) was used to generate electricity from sewage sludge. Fermentation pretreatment of the sludge effectively increased the COD of the supernatant and improved reactor performance. Using the CEA-MFC design, a maximum power density of 1200 mW m(-2) was reached after a fermentation pre-treatment time of 96 h. This power density represents a 275% increase over those previously observed in MFC systems. Results indicate continued improvements are possible and MFCs may be a viable modification to existing wastewater treatment infrastructure. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.

PubMed

Fry, L J; Querol, S; Gomez, S G; McArdle, S; Rees, R; Madrigal, J A

2015-08-01

Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion. The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation. A dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7.5 to 10% with detrimental effects observed outside of this range. These results support the use of 7.5-10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw. © 2015 International Society of Blood Transfusion.

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.

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.

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.

Survival of Campylobacter jejuni during Stationary Phase: Evidence for the Absence of a Phenotypic Stationary-Phase Response

PubMed Central

Kelly, Alison F.; Park, Simon F.; Bovill, Richard; Mackey, Bernard M.

2001-01-01

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39°C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50°C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions. PMID:11319108

Development and Validation of a Bioreactor System for Dynamic Loading and Mechanical Characterization of Whole Human Intervertebral Discs in Organ Culture

PubMed Central

Walter, BA; Illien-Junger, S; Nasser, P; Hecht, AC; Iatridis, JC

2014-01-01

Intervertebral disc (IVD) degeneration is a common cause of back pain, and attempts to develop therapies are frustrated by lack of model systems that mimic the human condition. Human IVD organ culture models can address this gap, yet current models are limited since vertebral endplates are removed to maintain cell viability, physiological loading is not applied, and mechanical behaviors are not measured. This study aimed to (i) establish a method for isolating human IVDs from autopsy with intact vertebral endplates, and (ii) develop and validate an organ culture loading system for human or bovine IVDs. Human IVDs with intact endplates were isolated from cadavers within 48 hours of death and cultured for up to 21 days. IVDs remained viable with ~80% cell viability in nucleus and annulus regions. A dynamic loading system was designed and built with the capacity to culture 9 bovine or 6 human IVDs simultaneously while applying simulated physiologic loads (maximum force: 4kN) and measuring IVD mechanical behaviors. The loading system accurately applied dynamic loading regimes (RMS error <2.5N and total harmonic distortion <2.45%), and precisely evaluated mechanical behavior of rubber and bovine IVDs. Bovine IVDs maintained their mechanical behavior and retained >85% viable cells throughout the 3 week culture period. This organ culture loading system can closely mimic physiological conditions and be used to investigate response of living human and bovine IVDs to mechanical and chemical challenges and to screen therapeutic repair techniques. PMID:24725441

A Combined Negative and Positive Enrichment Assay for Cancer Cells Isolation and Purification.

PubMed

Cheng, Boran; Wang, Shuyi; Chen, Yuanyuan; Fang, Yuan; Chen, Fangfang; Wang, Zhenmeng; Xiong, Bin

2016-02-01

Cancer cells that detach from solid tumor and circulate in the peripheral blood (CTCs) have been considered as a new "biomarker" for the detection and characterization of cancers. However, isolating and detecting cancer cells from the cancer patient peripheral blood have been technically challenging, owing to the small sub-population of CTCs (a few to hundreds per milliliter). Here we demonstrate a simple and efficient cancer cells isolation and purification method. A biocompatible and surface roughness controllable TiO2 nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, again, anti-CD45 (a leukocyte common antigen) and anti-EpCAM (epithelial cell adhesion molecule) were then coated onto the surface of the nanofilm for advance depletion of white blood cells (WBCs) and specific isolation of CTCs, respectively. Comparing to the conventional positive enrichment technology, this method exhibited excellent biocompatibility and equally high capture efficiency. Moreover, the maximum number of background cells (WBCs) was removed, and viable and functional cancer cells were isolated with high purity. Utilizing the horizontally packed TiO2 nanofilm improved pure CTC-capture through combining cell-capture-agent and cancer cell-preferred nanoscale topography, which represented a new method capable of obtaining biologically functional CTCs for subsequent molecular analysis. © The Author(s) 2014.

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

Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro.

PubMed

Nishikawa, A; Yoshizato, K

1986-02-01

Epidermal cells were dissociated from tails of the bullfrog tadpole, Rana catesbeiana, and cultured to investigate their response to steroid and thyroid hormones. Charcoal-treated serum (CTS) was used in the growth medium when cells were to be grown in the absence of steroid and thyroid hormones. The cells could be maintained for 2 weeks with a small increase in cell number in medium that contained CTS (CTS medium). Addition of cortisol to CTS medium increased both cellular attachment to the culture dishes and the proliferation of the attached cells with an optimum concentration of 5 X 10(-7) M. The cells remained viable and attached for at least a week. Cortisol stimulated the rate of protein synthesis 1.8-fold but did not alter the rate of DNA synthesis. The cells did not proliferate in the medium containing triiodothyronine (T3) and detached themselves from the dish within 5 days, which occurred in a dose-dependent manner with a maximum effect at 10(-8) M. It drastically decreased the rate of DNA synthesis but did not influence the rate of protein synthesis. These responses of cells to cortisol and T3 may reflect growth and death of tail epidermal cells in vivo at metamorphosis.

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

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

Lui, E.; Moya, Monica L.

This study seeks to validate the reproducibility of previous bioprinting work at Lawrence Livermore National Laboratory (LLNL) on a new Aerotech motion controller system and to modify an existing bioink, fibrin, by adding varying percent volumes of hyaluronic acid (HA). Endothelial and fibroblast cells bioprinted in fibrin gels using the Aerotech system were confirmed to be more than 77 percent viable after one day, and all bioprinted samples retained sterility after one week of culture. To characterize cell behavior in fibrin with HA addition, static co-­culture gels with varying percent volumes of HA were cultured in vitro for one week.more » Resulting confocal microscope images showed increased cell network formation in all concentrations of HA compared to the control (no HA), and rheological tests mimicking static gel compositions displayed positive correlations between gelation time, gel stiffness (G’), and hyaluronic acid concentration. Although the current data is insufficient to quantitatively associate HA concentration with the level of cell vascularization, future work will aim to develop a targeted HA concentration in fibrin for maximum cell network formation, to optimize the printing process parameters for this new bioink composition, and to analyze cell viability in bioprinted fibrin-­HA structures.« 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.

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

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.

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).

Growth and morphology of thermophilic dairy starters in alginate beads.

PubMed

Lamboley, Laurence; St-Gelais, Daniel; Champagne, Claude P; Lamoureux, Maryse

2003-06-01

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.

Solar Cell Fabrication Studies Pertinent to Developing Countries.

NASA Astrophysics Data System (ADS)

Prah, Joseph Henry

That there is a need in the world today, and in the Third World in particular, for developing renewable energy sources is a proposition without question. Toward that end, the harnessing of solar energy has attracted much attention recently. In this thesis, we have addressed the question of Photovoltaics among the many approaches to the problem as being of poignant relevance in the Third World. Based on our studies, which involved the physics of solar cells, various solar cell configurations, the materials for their fabrication and their fabrication sequences, we arrived at the conclusion that silicon homojunction solar cells are best suited to the present needs and environment of, and suitable for development in the Third World, though Cadmium Sulphide-Cuprous Sulphide solar cell could be considered as a viable future candidate. Attendant with the adoption of photovoltaics as electric energy supply, is the problem of technology transfer and development. Towards that goal, we carried out in the laboratory, the fabrication of solar cells using very simple fabrication sequences and materials to demonstrate that tolerable efficiencies are achievable by their use. The view is also presented that for a thriving and viable solar cell industry in the Third World, the sine qua non is an integrated national policies involving all facets of solar cell manufacture and application, namely, material processing and fabrication, basic research, and development and socio -economic acceptance of solar cell appliances. To demonstrate how basic research could benefit solar cell fabrication, we undertook a number of experiments, such as varying our fabrication sequences and materials, finding their radiation tolerance, and carrying out Deep Level Transient Spectroscopy (DLTS) studies, in an attempt to understand some of the fabrication and environmental factors which limit solar cell performance. We thus found that subjecting wafers to preheat treatments does not improve solar cell performance, but rather reduces solar cell radiation tolerance. Also P-type substrate solar cells were found to be more radiation resistant than N-type substrate solar cells. The Deep Level Transient Spectroscopy results showed that carbon and oxygen, as one would expect, are chief contaminants of the silicon wafers that we used in the fabrication of our solar cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

PubMed

Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

2017-03-20

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day -1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10 6 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct viable count as test for toxicity assessment: the effects of four metals on a Salmonella enteritidis strain.

PubMed

Scoglio, M E; Di Pietro, A; Anzalone, C; Calimeri, S; Lo Giudice, D; Trimarchi, G R

2000-01-01

The toxicity of synthetic sewage containing increasing concentrations of arsenic (.125, .25, .5, 1.0 mg L-1), cadmium (.02, .05, .1, .2 mg L-1), lead (.2, .5, 1.0, 2.0 mg L-1) and nickel (.5, 1.0, 2.0, 4.0 mg L-1) has been investigated by determining the total direct count (TDC) and the direct viable count (DVC) of Salmonella enteritidis by means of an immunofluorescence technique (IFA). This has been done in order to evaluate the possibility of using the IFA technique to estimate the toxicity of complex effluents. Arsenic, cadmium and nickel produced a concentration-dependent reduction in the number of viable bacterial cells. This was more clear when the viable bacterial cells were considered than when only the culturable part was used. Lead did not show a concentration-dependent and reproducible effect. At the highest concentrations allowed by the Italian wastewater regulations, lead, cadmium, arsenic and nickel reduced the viable/total bacterial cells ratio to 74.5%, 68.5%, 28.4% and 6.9%, respectively. The toxic effects of the metals were also tested using the standard Microtox assay.

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.

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

PubMed Central

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

2012-01-01

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells. PMID:22158840

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells.

PubMed

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W; Ras, Mat; Allbritton, Nancy L; Sims, Christopher E; Venugopalan, Vasan

2012-06-07

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass-pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s(-1) through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s(-1) and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells.

Effects of water-filtered infrared-A and of heat on cell death, inflammation, antioxidative potential and of free radical formation in viable skin--first results.

PubMed

Piazena, Helmut; Pittermann, Wolfgang; Müller, Werner; Jung, Katinka; Kelleher, Debra K; Herrling, Thomas; Meffert, Peter; Uebelhack, Ralf; Kietzmann, Manfred

2014-09-05

The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy. Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m(-2) which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the Earth's surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent conditions in terms of the resulting skin surface temperatures and exposure time. As compared with untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced to about 50% after convective heating to about 45 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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.

High 400 °C operation temperature blue spectrum concentration solar junction in GaInN/GaN

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

Zhao, Liang; Detchprohm, Theeradetch; Wetzel, Christian

2014-12-15

Transparent wide gap junctions suitable as high temperature, high flux topping cells have been achieved in GaInN/GaN by metal-organic vapor phase epitaxy. In structures of 25 quantum wells (QWs) under AM1.5G illumination, an open circuit voltage of 2.1 V is achieved. Of the photons absorbed in the limited spectral range of <450 nm, 64.2% are converted to electrons collected at the contacts under zero bias. At a fill factor of 45%, they account for a power conversion efficiency of38.6%. Under concentration, the maximum output power density per sun increases from 0.49 mW/cm{sup 2} to 0.51 mW/cm{sup 2} at 40 suns and then falls 0.42 mW/cm{sup 2}more » at 150 suns. Under external heating, a maximum of 0.59 mW/cm{sup 2} is reached at 250 °C. Even at 400 °C, the device is fully operational and exceeds room temperature performance. A defect analysis suggests that significantly higher fill factors and extension into longer wavelength ranges are possible with further development. The results prove GaInN/GaN QW solar junctions a viable and rugged topping cell for concentrator photovoltaics with minimal cooling requirements. By capturing the short range spectrum, they reduce the thermal load to any conventional cells stacked behind.« less

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.

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

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

PubMed

Courtès, Franck; Ebel, Bruno; Guédon, Emmanuel; Marc, Annie

2016-05-01

to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors. Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10(6) cells ml(-1), while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10(7) cells ml(-1). Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l(-1). Considering the LDH content of viable cells measured to be 110 U per 10(9) cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R(2) ≥ 0.95) for the three types of cell populations. The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

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.

Efficacy of UV light treatment for the microbiological decontamination of chicken, associated packaging, and contact surfaces.

PubMed

Haughton, P N; Lyng, J G; Cronin, D A; Morgan, D J; Fanning, S; Whyte, P

2011-04-01

UV light was investigated for the decontamination of raw chicken, associated packaging, and contact surfaces. The UV susceptibilities of a number of Campylobacter isolates (seven Campylobacter jejuni isolates and three Campylobacter coli isolates), Escherichia coli ATCC 25922, and Salmonella enterica serovar Enteritidis ATCC 10376 in liquid media were also investigated. From an initial level of 7 log CFU/ml, no viable Campylobacter cells were detected following exposure to the most intense UV dose (0.192 J/cm(2)) in liquid media (skim milk subjected to ultrahigh-temperature treatment and diluted 1:4 with maximum recovery diluent). Maximum reductions of 4.8 and 6.2 log CFU/ml were achieved for E. coli and serovar Enteritidis, respectively, in liquid media. Considerable differences in susceptibilities were found between the Campylobacter isolates examined, with variations of up to 4 log CFU/ml being observed. UV treatment of raw chicken fillet (0.192 J/cm(2)) reduced C. jejuni, E. coli, serovar Enteritidis, total viable counts, and Enterobacteriaceae by 0.76, 0.98, 1.34, 1.76, and 1.29 log CFU/g, respectively. Following UV treatment of packaging and surface materials, reductions of up to 3.97, 4.50, and 4.20 log CFU/cm(2) were obtained for C. jejuni, E. coli, and serovar Enteritidis, respectively (P < 0.05). Overall, the color of UV-treated chicken was not significantly affected (P ≥ 0.05). The findings of this study indicate that Campylobacter is susceptible to UV technology and that differences in sensitivities exist between investigated isolates. Overall, UV could be used for improving the microbiological quality of raw chicken and for decontaminating associated packaging and surface materials.

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.

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

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.

PubMed

Han, Yi; Liu, Xing-Mao; Liu, Hong; Li, Shi-Chong; Wu, Ben-Chuan; Ye, Ling-Ling; Wang, Qu-Wei; Chen, Zhao-Lie

2006-11-01

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

Growth, metabolic activity, and productivity of immobilized and freely suspended CHO cells in perfusion culture.

PubMed

Hilal-Alnaqbi, Ali; Hu, Alan Y C; Zhang, Zhibing; Al-Rubeai, Mohamed

2013-01-01

Chinese hamster ovary (CHO) cells producing β-galactosidase (β-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10(8) cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10(7) cells/mL medium. The β-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific β-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

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.

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line.

PubMed

Pushparajan, Charlotte; Claus, Juan Daniel; Marshall, Sean David Goldie; Visnovsky, Gabriel

2013-12-01

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctes rhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10(5) cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h(-1)). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10(7) TCID50/ml) than in cultures infected in Sf-900 III (2.0 × 10(7) TCID50/ml) and Sf-900 II (1.4 × 10(7) TCID50/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.

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

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.

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

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01.

PubMed

Divakar, K; Suryia Prabha, M; Nandhinidevi, G; Gautam, P

2017-04-21

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking-Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820 × 10 3 U/L and extracellular protease activity of 172 × 10 3 U/L were obtained at the 16th hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.

Growth yields and fermentation balance of Bacteroides fragilis cultured in glucose-enriched medium.

PubMed

Frantz, J C; McCallum, R E

1979-03-01

Bacteroides fragilis is an obligate anaerobic bacterium classified with the gram-negative, non-sporeforming bacilli and is the Bacteroides species most frequently isolated from human infections. In the present study, experiments were designed to investigate growth characteristics of B. fragilis in a complex medium. In a minimal defined medium, which was employed for comparison purposes, B. fragilis grew with a generation time of 2 h. Growth of the organism in glucose-enriched medium used in the present study was superior. Maximum generation time was 60 min. Total and viable cells (colony-forming units) were 8.9 x 10(9) and 2.1 x 10(9), respectively, at maximum measurable growth. The molar growth yield (Ym) was 51.5. Growth yields were found to reach a maximum 2 to 3 h before maximum growth and to vary with respect to the phase of growth. Estimates of the fermentation products indicated that glucose was the sole energy substrate. Major products included acetic acid, propionic acid, lactic acid, and succinic acid. Other products included ethyl alcohol, pyruvic acid, and fumaric acid. No attempt was made to recover CO2 or formic acid. The OR balances from two experiments were 0.013 and -0.093 and the respective carbon recoveries were 6.268 and 6.241. The results of the present study show that B. fragilis is capable of rapid rates of growth in vitro by using glucose as the sole energy source.

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.

Soil and Vegetation Project. A Detailed Study of Five Overburden Cores and Six Disposal Areas Along the Divide Section Tennessee-Tombigbee Waterway.

DTIC Science & Technology

1983-06-17

a c . 9.. a. 0 GIOV 0’ 0" u4 1% 1’ 0 𔃺 01 0% 01 0" -A < a) 0 C6 CN 0 t 0 -4 C, 1 ol El4 c4- 0 (n 1 0 en 01 (14 0 Q *- ou x --- -14 La.) C)X V) z...clearly related to the initial concentration of viable cells in the inoculant. Determinations of the ability of viable Rhizobia to infect and nodulate...temperatures after 28 days storage have been planted. The extent of nodule formation will be determined. 354 ._ _ ~ Figuire 1. Viable Rhizobia cells

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.

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.

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.

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.

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.

Assessing storage of stability and mercury reduction of freeze-dried Pseudomonas putida within different types of lyoprotectant

NASA Astrophysics Data System (ADS)

Azoddein, Abdul Aziz Mohd; Nuratri, Yana; Azli, Faten Ahada Mohd; Bustary, Ahmad Bazli

2017-12-01

Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose was able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage at 4 °C without vacuum. Polyethylene glycol (PEG) pre-treated freeze dried cells and broth pre-treated freeze dried cells after the freeze-dry process recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introducing freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage of 3 weeks was 17.91 %. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been grown in agar. Result from this study may be beneficial and useful as initial reference before commercialized freeze-dried P. putida.

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.

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.

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.

Examination of an indicative tool for rapidly estimating viable organism abundance in ballast water

NASA Astrophysics Data System (ADS)

Vanden Byllaardt, Julie; Adams, Jennifer K.; Casas-Monroy, Oscar; Bailey, Sarah A.

2018-03-01

Regulatory discharge standards stipulating a maximum allowable number of viable organisms in ballast water have led to a need for rapid, easy and accurate compliance assessment tools and protocols. Some potential tools presume that organisms present in ballast water samples display the same characteristics of life as the native community (e.g. rates of fluorescence). This presumption may not prove true, particularly when ships' ballast tanks present a harsh environment and long transit times, negatively impacting organism health. Here, we test the accuracy of a handheld pulse amplitude modulated (PAM) fluorometer, the Hach BW680, for detecting photosynthetic protists at concentrations above or below the discharge standard (< 10 cells·ml- 1) in comparison to microscopic counts using fluorescein diacetate as a viability probe. Testing was conducted on serial dilutions of freshwater harbour samples in the lab and in situ untreated ballast water samples originating from marine, freshwater and brackish sources utilizing three preprocessing techniques to target organisms in the size range of ≥ 10 and < 50 μm. The BW680 numeric estimates were in agreement with microscopic counts when analyzing freshly collected harbour water at all but the lowest concentrations (< 38 cells·ml- 1). Chi-square tests determined that error is not independent of preprocessing methods: using the filtrate method or unfiltered water, in addition to refining the conversion factor of raw fluorescence to cell size, can decrease the grey area where exceedance of the discharge standard cannot be measured with certainty (at least for the studied populations). When examining in situ ballast water, the BW680 detected significantly fewer viable organisms than microscopy, possibly due to factors such as organism size or ballast water age. Assuming both the BW680 and microscopy with FDA stain were measuring fluorescence and enzymatic activity/membrane integrity correctly, the observed discrepancy between methods may simply reflect that the two methods are measuring different characteristics of life. This is the first study to conduct proof-of-concept testing for a rapid compliance detection tool using freshly collected harbour water concomitantly with in situ ballast water; our results demonstrate that it is important to challenge potential compliance tools with water samples spanning a range of biotic and abiotic conditions.

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

Venkataraman, M.; Natarajan, R.; Raj, Baldev

The reprocessing of spent fuel from Fast Breeder Test Reactor (FBTR) has been successfully demonstrated in the pilot plant, CORAL (COmpact Reprocessing facility for Advanced fuels in Lead shielded cell). Since commissioning in 2003, spent mixed carbide fuel from FBTR of different burnups and varying cooling period, have been reprocessed in this facility. Reprocessing of the spent fuel with a maximum burnup of 100 GWd/t has been successfully carried out so far. The feed backs from these campaigns with progressively increasing specific activities, have been useful in establishing a viable process flowsheet for reprocessing the Prototype Fast Breeder Reactor (PFBR)more » spent fuel. Also, the design of various equipments and processes for the future plants, which are either under design for construction, namely, the Demonstration Fast Reactor Fuel Reprocessing Plant (DFRP) and the Fast reactor fuel Reprocessing Plant (FRP) could be finalized. (authors)« less

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels.

PubMed

Lee, Sung Kuk; Chou, Howard; Ham, Timothy S; Lee, Taek Soon; Keasling, Jay D

2008-12-01

The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

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.

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.

Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus.

PubMed

Ng, Elizabeth W; Yeung, Marie; Tong, Phillip S

2011-01-31

Recognized to confer health benefits to consumers, probiotics such as Lactobacillus acidophilus are commonly incorporated into fermented dairy products worldwide; among which yogurt is a popular delivery vehicle. To materialize most of the putative health benefits associated with probiotics, an adequate amount of viable cells must be delivered at the time of consumption. However, the loss in their viabilities during refrigerated storage has been demonstrated previously. This study focused on the effects of yogurt starter cultures on the survival of five strains of L. acidophilus, with emphases on low pH and acid production. Differential survival behavior between L. acidophilus strains was further analyzed. To this end, viable cell counts of L. acidophilus were determined weekly during 4°C storage in various types of yogurts made with Streptococcus thermophilus alone, L. delbrueckii ssp. bulgaricus alone, both species of the starter cultures, or glucono-delta-lactone (GDL). All yogurt types, except for pasteurized yogurts, were co-fermented with L. acidophilus. Yogurt filtrate was analyzed for the presence of any inhibitory substance and for the amount of hydrogen peroxide. Multiplication of L. acidophilus was not affected by the starter cultures as all strains reached high level on day 0 of the storage period. Throughout the 28-day storage period, cell counts of L. acidophilus PIM703 and SBT2062 remained steady (~6 × 10(7)CFU/g) in yogurts made with both starter cultures, whereas those of ATCC 700396 and NCFM were reduced by a maximum of 3 and 4.6 logs, respectively. When starter cultures were replaced by GDL, all strains survived well, suggesting that a low pH was not a critical factor dictating their survival. In addition, the filtrate collected from yogurts made with starter cultures appeared to have higher inhibitory activities against L. acidophilus than that made with GDL. The presence of viable starter cultures was necessary to adversely affect the survival of some strains, as pasteurized yogurts had no effect on their survival. In particular, the inhibitory effect exerted by L. delbrueckii ssp. bulgaricus on L. acidophilus NCFM was highly pronounced than by S. thermophilus, nevertheless, the same effect was not observed on SBT2062. The inhibition against stationary-phase NCFM cells might be caused by the elevated level of hydrogen peroxide produced by L. delbrueckii ssp. bulgaricus. Delineating factors driving the differences in survival trait among probiotic strains will lead to a more efficacious delivery of health benefits in fermented dairy products through targeted technological interventions. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

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.

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

Performance of serum-free broth media for growth of Renibacterium salmoninarum

USGS Publications Warehouse

Starliper, C.E.; Schill, W.B.; Mathias, J.

1998-01-01

Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth.

Hydrogel Film-Immobilized Lactobacillus brevis RK03 for γ-Aminobutyric Acid Production

PubMed Central

Hsueh, Yi-Huang; Liaw, Wen-Chang; Kuo, Jen-Min; Deng, Chi-Shin

2017-01-01

Hydrogels of 2-hydroxyethyl methacrylate/polyethylene glycol diacrylate (HEMA/PEGDA) have been extensively studied for their use in biomedical and pharmaceutical applications owing to their nontoxic and highly hydrophilic characteristics. Recently, cells immobilized by HEMA/PEGDA hydrogels have also been studied for enhanced production in fermentation. Hydrogel films of HEMA/PEGDA copolymer were generated by Ultraviolet (UV)-initiated photopolymerization. The hydrogel films were used to immobilize viable Lactobacillus brevis RK03 cells for the bioconversion of monosodium glutamate (MSG) to γ-aminobutyric acid (GABA). The mechanical properties and fermentation yields of the L. brevis RK03 cells immobilized on polyacrylate hydrogel films with different monomeric formulations were investigated. Fermentation was carried out in 75 mL de Man, Rogosa and Sharpe (MRS) medium containing various concentrations of MSG. We found that HEMA (93%)/PEGDA (3%) hydrogels (sample H) maximized GABA production. The conversion rate of MSG to GABA reached a maximum value of 98.4% after 240 h. Bioconversion activity gradually declined after 420 h to 83.8% after five cycles of semi-continuous fermentation. Our results suggest that HEMA (93%)/PEGDA (3%) hydrogels have great potential for use in GABA production via semi-continuous fermentation. PMID:29099794

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.

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.

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.

Protective effects of anethole dithiolethione against oxidative stress-induced cytotoxicity in human Jurkat T cells.

PubMed

Khanna, S; Sen, C K; Roy, S; Christen, M O; Packer, L

1998-07-01

The protective effects of anethole dithiolethione (ADT) against H2O2- or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T cells were investigated. Jurkat T cells were pretreated with ADT (10-50 microM) for 18 hr and then challenged with H202 or HNE for up to 4 hr. Cytotoxicity was assessed by measuring: 1) leakage of lactate dehydrogenase from cells to medium; and 2) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. Pretreatment of cells with ADT (10 or 25 microM) for 18 hr significantly protected cells against H202- or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 microM) for 72 hr significantly increased the activities of catalase and glutathione reductase. The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 microM of ADT for 72 hr. A significant increase in cellular GSH was observed in cells that were treated with ADT for 72 hr. Using monobromobimane as a thiol probe, we consistently observed that cells pretreated for 18 hr with ADT (25 or 50 microM) had also increased total thiol content. Exposure of Jurkat T cells to H202 or HNE resulted in a time-dependent decrease in cellular GSH. ADT (10-50 microM, 18 hr) pretreatment circumvented H202-dependent lowering of cellular GSH. In conclusion, ADT proved to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action, suggesting that the drug has a remarkable therapeutic potential.

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.

Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

PubMed

Kamachi, Yasuharu; Omasa, Takeshi

2018-04-01

Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

Online estimation of internal stack temperatures in solid oxide fuel cell power generating units

NASA Astrophysics Data System (ADS)

Dolenc, B.; Vrečko, D.; Juričić, Ɖ.; Pohjoranta, A.; Pianese, C.

2016-12-01

Thermal stress is one of the main factors affecting the degradation rate of solid oxide fuel cell (SOFC) stacks. In order to mitigate the possibility of fatal thermal stress, stack temperatures and the corresponding thermal gradients need to be continuously controlled during operation. Due to the fact that in future commercial applications the use of temperature sensors embedded within the stack is impractical, the use of estimators appears to be a viable option. In this paper we present an efficient and consistent approach to data-driven design of the estimator for maximum and minimum stack temperatures intended (i) to be of high precision, (ii) to be simple to implement on conventional platforms like programmable logic controllers, and (iii) to maintain reliability in spite of degradation processes. By careful application of subspace identification, supported by physical arguments, we derive a simple estimator structure capable of producing estimates with 3% error irrespective of the evolving stack degradation. The degradation drift is handled without any explicit modelling. The approach is experimentally validated on a 10 kW SOFC system.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

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.

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.

Interaction between lactic acid bacteria and yeasts in airag, an alcoholic fermented milk.

PubMed

Sudun; Wulijideligen; Arakawa, Kensuke; Miyamoto, Mari; Miyamoto, Taku

2013-01-01

The interaction between nine lactic acid bacteria (LAB) and five yeast strains isolated from airag of Inner Mongolia Autonomic Region, China was investigated. Three representative LAB and two yeasts showed symbioses were selected and incubated in 10% (w/v) reconstituted skim milk as single and mixed cultures to measure viable count, titratable acidity, ethanol and sugar content every 24 h for 1 week. LAB and yeasts showed high viable counts in the mixed cultures compared to the single cultures. Titratable acidity of the mixed cultures was obviously enhanced compared with that of the single cultures, except for the combinations of Lactobacillus reuteri 940B3 with Saccharomyces cerevisiae 4C and Lactobacillus helveticus 130B4 with Candida kefyr 2Y305. C. kefyr 2Y305 produced large amounts of ethanol (maximum 1.35 g/L), whereas non-lactose-fermenting S. cerevisiae 4C produced large amounts of ethanol only in the mixed cultures. Total glucose and galactose content increased while lactose content decreased in the single cultures of Leuconostoc mesenteroides 6B2081 and Lb. helveticus 130B4. However, both glucose and galactose were completely consumed and lactose was markedly reduced in the mixed cultures with yeasts. The result suggests that yeasts utilize glucose and galactose produced by LAB lactase to promote cell growth. © 2012 The Authors. Animal Science Journal © 2012 Japanese Society of Animal Science.

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.

[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.

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.

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.

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...

Solar energy storage using surfactant micelles

NASA Astrophysics Data System (ADS)

Srivastava, R. C.; Marwadi, P. R.; Latha, P. K.; Bhise, S. B.

1982-09-01

The results of experiments designed to test the soluble reduced form of thionine dye as a suitable solar energy storage agent inside the hydrophobic core of surfactant micelles are discussed. Aqueous solutions of thionine, methylene blue, cetyl pyridinium bromide, sodium lauryl sulphate, iron salts, and iron were employed as samples of anionic, cationic, and nonionic surfactants. The solutions were exposed to light until the dye disappeared, and then added drop-by-drop to surfactant solutions. The resultant solutions were placed in one cell compartment while an aqueous solution with Fe(2+) and Fe(3+) ions were placed in another, with the compartments being furnished with platinum electrodes connected using a saturated KCl-agar bridge. Data was gathered on the short circuit current, maximum power, and internal resistance encountered. Results indicate that dye-surfactant systems are viable candidates for solar energy storage for later conversion to electrical power.

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

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

Kuk Lee, Sung; Chou, Howard; Ham, Timothy S.

2009-12-02

The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology willmore » provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.« less

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.

The biodistribution and pharmacokinetic evaluation of choline-bound gold nanoparticles in a human prostate tumor xenograft model.

PubMed

Razzak, Rene; Zhou, Joe; Yang, XiaoHong; Pervez, Nadim; Bédard, Eric Lr; Moore, Ronald B; Shaw, Andrew; Amanie, John; Roa, Wilson H

2013-06-01

Gold nanoparticles (GNPs) have attracted significant attention in the treatment of cancer due to their potential as novel radiation enhancers, particularly when functionalized with various targeting ligands. The aim of this study was to assess the biodistribution and pharmacokinetic characteristics of a novel choline-bound GNP (choline-GNP) stabilized with polyethelenimine (PEI). Choline bound to 27 nm diameter GNPs was characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Toxicity of choline-GNPs was examined on DU-145 prostate cancer cells using an MTT assay. Using balb/c mice bearing flank DU-145 prostate tumors, choline-GNPs bio-distribution was measured using inductively coupled mass spectroscopy (ICP-MS). Blood, heart, lung, liver, spleen, brain, kidney and tumor gold content were examined at multiple time points over a 24-hour period after tail vein injection. An MTT assay using DU-145 prostate cancer cells yielded a 95% cell viability 72 hours after choline-GNP administration. The tumor GNP area under the concentration-time curve during the first 4 hours (AUC0-4) was 2.2 µg/ml h, representing 13% of the circulating blood GNP concentration over the same time period. The maximum intra-tumor GNP concentration observed was 1.4% of the injected dose per gram of tumor tissue (%ID/g) one hour post injection. GNPs functionalized with choline demonstrates a viable future nanoparticle platform with increased intra-tumor uptake as compared to unconjugated GNPs. Decreased intra-hepatic accumulation appears to be the reason for the improved systemic bioavailability. The next logical translational investigation will incorporate external beam radiation with the observed maximum intra-tumor uptake.

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

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.

[Effect of chloramines disinfection for biofilm formation control on copper and stainless steel pipe materials].

PubMed

Zhou, Ling-ling; Zhang, Yong-ji; Li, Xing; Li, Gui-bai

2008-12-01

Two rotating annular bioreactors (RABs) with copper and stainless steel pipe materials were adopted in the study, the effects of these two pipe materials and chloramines disinfection on biofilms formation in drinking water distribution system were evaluated. The maximum viable bacterial number in biofilm of copper and stainless steel reached 5.5 x 10(3) CFU/cm2 and 2.5 x 10(5) CFU/cm2 at 18th and 21st day without chloramines, and the viable bacterial number at the apparent steady state was 1.0 x 10(3) CFU/cm2 and 1.3 x 10(5) CFU/cm2 respectively. It was obvious that the biomass on copper materials was lower than that of the stainless steel. The maximum viable bacterial on copper and stainless steel under chloramines was 5.0 x 10(2) CFU/cm2 and 5.0 x 10(4) CFU/cm2, which was one order of magnitude lower than that of without chloramines, and its number was 10 CFU/cm2 and 3.5 x 10(4) CFU/cm2 at the steady state. These results illustrated that chloramines had apparent ability in controlling biomass when the biofilm was on steady states, especially for copper material. There was exponential relationship between biomass in biofilm and residue chloramines, which meant less biomass with more chloramines, synergistic effects were observed between chloramines and copper materials on biomass in biofilms inactivation.

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.

Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

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...

Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in Brazil.

PubMed

Campos, C R; Silva, C F; Dias, D R; Basso, L C; Amorim, H V; Schwan, R F

2010-06-01

To evaluate the dominance and persistence of strains of Saccharomyces cerevisiae during the process of sugar cane fermentation for the production of cachaça and to analyse the microbial compounds produced in each fermentative process. Three S. cerevisiae strains were evaluated during seven consecutive 24-h fermentation batches using recycled inocula. The UFLA CA 116 strain had the largest population of viable organisms, and the maximum population was achieved in the fourth batch after 96 h of fermentation. The UFLA CA 1162 and UFLA CA 1183 strains grew more slowly, and the maximum population was reached in the seventh batch. Molecular characterization of isolated yeast cells using PFGE (pulse field gel electrophoresis) revealed that more than 86% of the isolates corresponded to the initially inoculated yeast strain. The concentration of aldehydes, esters, methanol, alcohol and volatile acids in the final-aged beverages were within the legal limits. Cachaça produced by select yeast strains exhibits analytical differences. UFLA CA 1162 and UFLA CA 116 S. cerevisiae isolates can be considered the ideal strains for the artisanal production of cachaça in Brazil. The use of select yeast strains can improve the quality and productivity of cachaça production. Our findings are important for the appropriate monitoring of yeast during sugar cane fermentation. In addition, we demonstrate that UFLA CA 116 and UFLA CA 1162, the ideal yeast strains for cachaça production, are maintained at a high population density. The persistence of these yeast strains in the fermentation of sugar cane juice promotes environmental conditions that prevent or decrease bacterial contamination. Thus, the use of select yeast strains for the production of cachaça is a viable economic alternative to standardize the production of this beverage.

Analysis of gene network robustness based on saturated fixed point attractors

PubMed Central

2014-01-01

The analysis of gene network robustness to noise and mutation is important for fundamental and practical reasons. Robustness refers to the stability of the equilibrium expression state of a gene network to variations of the initial expression state and network topology. Numerical simulation of these variations is commonly used for the assessment of robustness. Since there exists a great number of possible gene network topologies and initial states, even millions of simulations may be still too small to give reliable results. When the initial and equilibrium expression states are restricted to being saturated (i.e., their elements can only take values 1 or −1 corresponding to maximum activation and maximum repression of genes), an analytical gene network robustness assessment is possible. We present this analytical treatment based on determination of the saturated fixed point attractors for sigmoidal function models. The analysis can determine (a) for a given network, which and how many saturated equilibrium states exist and which and how many saturated initial states converge to each of these saturated equilibrium states and (b) for a given saturated equilibrium state or a given pair of saturated equilibrium and initial states, which and how many gene networks, referred to as viable, share this saturated equilibrium state or the pair of saturated equilibrium and initial states. We also show that the viable networks sharing a given saturated equilibrium state must follow certain patterns. These capabilities of the analytical treatment make it possible to properly define and accurately determine robustness to noise and mutation for gene networks. Previous network research conclusions drawn from performing millions of simulations follow directly from the results of our analytical treatment. Furthermore, the analytical results provide criteria for the identification of model validity and suggest modified models of gene network dynamics. The yeast cell-cycle network is used as an illustration of the practical application of this analytical treatment. PMID:24650364

Is self-sufficiency financially viable and ethically justifiable?--a commercial viewpoint.

PubMed

Christie, R B

1994-12-01

Manufacturers of blood products have to maintain the highest possible standards for plasma screening and good manufacturing practices to ensure maximum purity and viral safety. The private sector companies have much experience in implementing and complying with national and international regulations. These requirements involve considerable cost in the areas of (1) plasma collection facilities, (2) research and clinical research, (3) manufacture, and (4) quality control. Total self-sufficiency would mean the loss of many existing resources. An alternative would be a collaboration between the public and private sectors to meet the needs of all patients who require plasma derived products. The current definition of self-sufficiency suggests that it is not financially viable.

Periplasmic Manganese in a Subsurface Bacterium During Anaerobic Growth on Birnessite

NASA Astrophysics Data System (ADS)

Langley, S.; Glasauer, S.; Beveridge, T.

2002-12-01

In subsurface environments, where oxygen is not metabolically available for energy production, bacteria use alternate terminal electron acceptors (TEAs) to respire and grow. Anaerobic TEAs include, but are not limited to, Fe3+ and Mn4+. These metals can be present as mineral phases (e.g., ferrihydrite and hematite in the case of iron; birnessite and pyrolusite in the case of manganese). Bacteria bind strongly to minerals and reduce the metal by a process called dissimilatory metal reduction (DMR). Shewanella putrefaciens strain CN32 is a Gram-negative bacterium capable of DMR. In previous reports, when this organism was grown on birnessite, we observed cytoplasmic granules of a Mn-rich mineral phase, and an unusual deposition of electron-dense material within the periplasm (that region of the cell located between the inner and outer membranes). In an attempt to characterize the periplasmic precipitates, CN32 was inoculated into an anaerobic defined medium (DM), supplemented with 20 mM Mn (birnessite) and incubated in an anaerobic chamber. Reduced and total Mn concentrations were monitored using atomic absorption spectrophotometry, and cell numbers determined by viable counts on trypticase soy agar. TEM, combined with energy dispersive X-ray spectroscopy (EDS), was used to localize and confirm the presence of any Mn-rich depositions. Soluble Mn concentration increased steadily after inoculation, indicating active metabolism and metal reduction by the cells. Viable counts indicated that the cells reached their maximum number on day 9. Stained thin sections from 4-day-old samples examined with TEM showed cells in close association with the mineral. Secondary mineral products derived from birnessite reduction were evident (e.g., manganese phosphate). TEM-EDS also revealed the presence of ~30 nm-thick deposits of electron-dense material in the periplasm of some cells. However, examination of similar sections which had not been previously stained with osmium tetroxide (an oxidizing EM stain), failed to reveal similar periplasmic depositions. We therefore speculate that soluble manganese accumulated within the periplasm of the cells. This pool of soluble manganese was then precipitated upon addition of the oxidizing osmium stain, and appeared as the electron-dense precipitates in stained sections. We believe this is the first report of a pool of soluble manganese accumulating within the periplasm of cells during DMR. At this point, we do not know whether the accumulation is related to the anaerobic metabolism of the cells (i.e., biologically induced), or is simply a passive by-product of their growth on manganese minerals. Further studies are needed to fully investigate this point.

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.

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

Fluorogenic Substrate Detection of Viable Intracellular and Extracellular Pathogenic Protozoa

NASA Astrophysics Data System (ADS)

Jackson, Peter R.; Pappas, Michael G.; Hansen, Brian D.

1985-01-01

Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.

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 ...

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.

Activity of AFN-1252, a novel FabI inhibitor, against Staphylococcus aureus in an in vitro pharmacodynamic model simulating human pharmacokinetics

PubMed Central

Tsuji, Brian T; Harigaya, Yoriko; Lesse, Alan J; Forrest, Alan; Ngo, Dung

2013-01-01

AFN-1252, a potent enoyl-ACP reductase (FabI) inhibitor, is under development for the treatment of Staphylococcus aureus infections. The activity of AFN-1252 against two isolates of S. aureus, MSSA 26213 and MRSA S186, was studied in an in vitro pharmacodynamic model simulating AFN-1252 pharmacokinetics in man. Reductions in bacterial viable count over the first 6 hours were generally 1–2 logs and maximal reductions in viable count were generally achieved at fAUC/MIC ratios of 100–200. Maximum reductions in viable count against MSSA 29213 and MRSA S186 were approximately 4 logs, achieved by 450 mg q12h (fAUC/MIC = 1875) dosing at 28 hours. Staphylococcal resistance to AFN-1252 did not develop throughout the 48-hour experiments. As multidrug resistance continues to increase, these studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections. PMID:23433442

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.

Effects of biotic and abiotic factors on the oxygen content of green sea turtle nests during embryogenesis.

PubMed

Chen, Chiu-Lin; Wang, Chun-Chun; Cheng, I-Jiunn

2010-10-01

Several biotic and abiotic factors can influence nest oxygen content during embryogenesis. Several of these factors were determined during each developmental stage of green sea turtle embryos on Wan-an Island, Penghu Archipelago, Taiwan. We examined oxygen content in 7 nests in 2007 and 11 in 2008. Oxygen in the adjacent sand, total and viable clutch sizes, air, sand and nest temperatures, and sand characters of each nest were also determined. Oxygen content was lower in late stages than in the early and middle stages. It was also lower in the middle layer than in the upper and bottom layers. Nest temperature showed opposite trends, reaching its maximum value in late stages of development. Nest oxygen content was influenced by fraction of viable eggs, total clutch sizes, sand temperatures, maximum nest temperature and maximum change in the nest temperature during incubation. Clutch size during embryogenesis was the most influential factor overall. However, the major influential factors were different for different developmental stages. In the first half of the incubation, the development rate was low, and the change in the nest oxygen content was influenced mainly by the clutch size. During the second half, the rapid embryonic development rate became the dominant factor, and hatchling activities caused even greater oxygen consumption during the last stage of development.

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.

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

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

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...

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

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.

Microbial lipid production by oleaginous Rhodococci cultured in lignocellulosic autohydrolysates

DOE PAGES

Wei, Zhen; Zeng, Guangming; Huang, Fang; ...

2015-07-04

Metabolic synthesis of single cell oils (SCOs) for biodiesel application by heterotrophic oleaginous microorganisms is being hampered by the high cost of culture media. This study investigated the possibility of using loblolly pine and sweetgum autohydrolysates as economic feedstocks for microbial lipid production by oleaginous Rhodococcus opacus ( R. opacus) PD630 and DSM 1069. Results revealed that when the substrates were detoxified by the removal of inhibitors (such as HMF—hydroxymethyl-furfural), the two strains exhibited viable growth patterns after a short adaptation/lag phase. R. opacus PD630 accumulated as much as 28.6 % of its cell dry weight (CDW) in lipids whilemore » growing on detoxified sweetgum autohydrolysate (DSAH) that translates to 0.25 g/l lipid yield. The accumulation of SCOs reached the level of oleagenicity in DSM 1069 cells (28.3 % of CDW) as well, while being cultured on detoxified pine autohydrolysate (DPAH), with the maximum lipid yield of 0.31 g/l. The composition of the obtained microbial oils varied depending on the substrates provided. These results indicate that lignocellulosic autohydrolysates can be used as low-cost fermentation substrates for microbial lipid production by wild-type R. opacus species. Furthermore, the variety of applications for aqueous liquors from lignocellulosic pretreatment has been expanded, allowing for the further optimization of the integrated biorefinery.« less

Overcoming thermal noise in non-volatile spin wave logic

NASA Astrophysics Data System (ADS)

Dutta, Sourav; Nikonov, Dmitri; Manipatruni, Sasikanth; Young, Ian; Naeemi, Azad

Spin waves are propagating disturbances in magnetically ordered materials. To compete as a promising candidate for beyond-CMOS application, the all-magnon based computing system must undergo the essential steps of careful selection of materials and demonstrate robustness with respect to thermal noise/variability. Here, we identify suitable materials and investigate two viable options for translating the theoretical idea of phase-dependent switching of the spin wave detector to a practical realization of a thermally reliable magnonic device by - (a) using the built-in strain in the ME cell, arising from the lattice mismatch and/or thermal expansion coefficient mismatch between the film and the substrate, for compensation of the demagnetization, and (b) using an exchange-spring structure that exhibits a strong exchange-coupling between the ME cell and PMA SWB and provides a modification of the energy landscape of the ME cell magnet. A high switching success and error-free logic functionality can be ensured if the amplitude of the detected spin wave (< θ >) remains higher than a threshold value of around 6°C and the detected phase falls within the window from 280°C through 0 to 20°C or from 100°C to 200°C with a maximum allowable ϕ range of around 100°C.

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.

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.

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.

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.

Listeria monocytogenes serotype 1/2b and 4b isolates from human clinical cases and foods show differences in tolerance to refrigeration and salt stress.

PubMed

Ribeiro, V B; Destro, M T

2014-09-01

Control of Listeria monocytogenes in food processing facilities is a difficult issue because of the ability of this microorganism to form biofilms and adapt to adverse environmental conditions. Survival at high concentrations of sodium chloride and growth at refrigeration temperatures are two other important characteristics of L. monocytogenes isolates. The aim of this study was to compare the growth characteristics under stress conditions at different temperatures of L. monocytogenes serotypes responsible for the majority of clinical cases from different sources. Twenty-two L. monocytogenes isolates, 12 from clinical cases (8 serotype 4b and 4 serotype 1/2a) and 10 from food (6 serotype 4b and 4 serotype 1/2a), and an L. monocytogenes Scott A (serotype 4b) reference strain were analyzed for the ability to grow in brain heart infusion broth plus 1.9 M NaCl (11%) at 4, 10, and 25°C for 73, 42, and 15 days, respectively. The majority of L. monocytogenes strains was viable or even grew at 4°C and under the high osmotic conditions usually used to control pathogens in the food industry. At 10°C, most strains could adapt and grow; however, no significant difference (P > 0.05) was found for lag-phase duration, maximum growth rate, and maximum cell density. At 25°C, all strains were able to grow, and populations increased by up 5 log CFU/ml. Clinical strains had a significantly longer lag phase and lower maximum cell density (P < 0.05) than did food strains. Regarding virulence potential, no significant differences in hemolytic activity were found among serotypes; however, serotype 4b strains were more invasive in Caco-2 cells than were serotype 1/2a strains (P < 0.05). The global tendency of decreasing NaCl concentrations in processed foods for health reasons may facilitate L. monocytogenes survival and growth in these products. Therefore, food companies must consider additional microbial growth barriers to assure product safety.

Extract of Pelargonium sidoides (EPs 7630) improves phagocytosis, oxidative burst, and intracellular killing of human peripheral blood phagocytes in vitro.

PubMed

Conrad, Andreas; Hansmann, Cathrin; Engels, Inge; Daschner, Franz D; Frank, Uwe

2007-01-01

Clinical data show that EPs 7630, an aqueous ethanolic extract from the roots of Pelargonium sidoides, can be used for the treatment of upper respiratory tract infections (URTI). The biological effects of the preparation have not been fully investigated. The objective of this study was to examine the impact of EPs 7630 on the activity of human peripheral blood phagocytes (PBP). A whole blood-based, flow cytometric assay was used to simultaneously assess phagocytosis and oxidative burst. Calcein-AM stained Candida albicans (DSM 1386) were used as target organisms. Oxidative burst was measured by addition of dihydroethidium (DHE). Target organisms and whole blood were co-incubated and analyzed after 0, 2, 4, 6, 10, and 30 min. Intracellular killing of the target organisms was evaluated by determining the number of surviving yeast cells after co-incubation of C. albicans and human whole blood. EPs 7630 was applied in therapeutically relevant concentrations between 0 and 30 microg/ml. Compared with controls EPs 7630 increased the number of phagocytosing PBP during the observed time points between 2 and 10 min in a concentration-dependent manner, with a maximum enhancement of 56% at 2 min (p=0.002). The application of EPs 7630 also led to a significant increase in the number of burst-active PBP for all time points observed beyond 2 min (p<0.001). The maximum augmentation was 120% after application of 30 microg/ml EPs 7630 at 4 min. Using a microbiological assay, intracellular killing was also enhanced by EPs 7630. This was expressed by a significant reduction in the number of surviving target organisms (p<0.001). The maximum reduction in viable yeast cells (-31%) was observed after co-incubation for 120 min with the highest concentration of EPs 7630 (30 microg/ml). In conclusion, the positive effects of EPs 7630 on phagocytosis, oxidative burst, and intracellular killing of yeast cells as test organisms are important components of the compound's biological activity. Our findings constitute a valuable contribution to understanding the clinical effects of EPs 7630.

Preclinical evaluation of parametric image reconstruction of [18F]FMISO PET: correlation with ex vivo immunohistochemistry

NASA Astrophysics Data System (ADS)

Cheng, Xiaoyin; Bayer, Christine; Maftei, Constantin-Alin; Astner, Sabrina T.; Vaupel, Peter; Ziegler, Sibylle I.; Shi, Kuangyu

2014-01-01

Compared to indirect methods, direct parametric image reconstruction (PIR) has the advantage of high quality and low statistical errors. However, it is not yet clear if this improvement in quality is beneficial for physiological quantification. This study aimed to evaluate direct PIR for the quantification of tumor hypoxia using the hypoxic fraction (HF) assessed from immunohistological data as a physiological reference. Sixteen mice with xenografted human squamous cell carcinomas were scanned with dynamic [18F]FMISO PET. Afterward, tumors were sliced and stained with H&E and the hypoxia marker pimonidazole. The hypoxic signal was segmented using k-means clustering and HF was specified as the ratio of the hypoxic area over the viable tumor area. The parametric Patlak slope images were obtained by indirect voxel-wise modeling on reconstructed images using filtered back projection and ordered-subset expectation maximization (OSEM) and by direct PIR (e.g., parametric-OSEM, POSEM). The mean and maximum Patlak slopes of the tumor area were investigated and compared with HF. POSEM resulted in generally higher correlations between slope and HF among the investigated methods. A strategy for the delineation of the hypoxic tumor volume based on thresholding parametric images at half maximum of the slope is recommended based on the results of this study.

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...

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.

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.

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

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.

The Effects of Platelet-Rich Plasma on Bone Marrow Stromal Cell Transplants for Tendon Healing In Vitro

PubMed Central

Morizaki, Yutaka; Zhao, Chunfeng; An, Kai-Nan; Amadio, Peter C.

2010-01-01

Purpose In this study we investigated the effect of platelet-rich plasma (PRP) and bone-marrow derived stromal cell (BMSC)-seeded interposition in an in vitro canine tendon repair model. Methods Bone marrow, peripheral blood, and tendons were harvested from mixed breed dogs. BMSC were cultured and passaged from adherent cells of bone marrow suspension. PRP was purified from peripheral blood using a commercial kit. 192 flexor digitorum profundus tendons were used for the study. Tendons repaired with a simple suture were used as a control group. In treatment groups, a collagen gel patch was interposed at the tendon repair site prior to suture. There were three treatment groups according to the type of collagen patch; a patch with PRP, a patch with BMSC, and a patch with PRP and BMSC. The repaired tendons were evaluated by biomechanical testing and by histological survey after 2 and 4 weeks in tissue culture. To evaluate viability, cells were labeled with PKH26 and surveyed under confocal microscopy after culture. Results The maximum breaking strength and stiffness of the healing tendons with the BMSC-seeded PRP patch was significantly higher than the healing tendons without a patch or with a cell-seeded patch (p<0.02). Viable BMSC were present at both 2 and 4 weeks. Conclusions PRP enhanced the effect of BMSC-seeded collagen gel interposition in this in vitro model. Based on these results we now plan to investigate this effect in vivo. PMID:20951509

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

Regulation of Viable and Optimal Cohorts

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

Aubin, Jean-Pierre, E-mail: aubin.jp@gmail.com

This study deals with the evolution of (scalar) attributes (resources or income in evolutionary demography or economics, position in traffic management, etc.) of a population of “mobiles” (economic agents, vehicles, etc.). The set of mobiles sharing the same attributes is regarded as an instantaneous cohort described by the number of its elements. The union of instantaneous cohorts during a mobile window between two attributes is a cohort. Given a measure defining the number of instantaneous cohorts, the accumulation of the mobile attributes on a evolving mobile window is the measure of the cohort on this temporal mobile window. Imposing accumulationmore » constraints and departure conditions, this study is devoted to the regulation of the evolutions of the attributes which are1.viable in the sense that the accumulations constraints are satisfied at each instant;2.and, among them, optimal, in the sense that both the duration of the temporal mobile window is maximum and that the accumulation on this temporal mobile window is the largest viable one. This value is the “accumulation valuation” function. Viable and optimal evolutions under accumulation constraints are regulated by an “implicit Volterra integro-differential inclusion” built from the accumulation valuation function, solution to an Hamilton–Jacobi–Bellman partial differential equation under constraints which is constructed for this purpose.« less

Canonical Statistical Model for Maximum Expected Immission of Wire Conductor in an Aperture Enclosure

NASA Technical Reports Server (NTRS)

Bremner, Paul G.; Vazquez, Gabriel; Christiano, Daniel J.; Trout, Dawn H.

2016-01-01

Prediction of the maximum expected electromagnetic pick-up of conductors inside a realistic shielding enclosure is an important canonical problem for system-level EMC design of space craft, launch vehicles, aircraft and automobiles. This paper introduces a simple statistical power balance model for prediction of the maximum expected current in a wire conductor inside an aperture enclosure. It calculates both the statistical mean and variance of the immission from the physical design parameters of the problem. Familiar probability density functions can then be used to predict the maximum expected immission for deign purposes. The statistical power balance model requires minimal EMC design information and solves orders of magnitude faster than existing numerical models, making it ultimately viable for scaled-up, full system-level modeling. Both experimental test results and full wave simulation results are used to validate the foundational model.

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution.

PubMed

Centurion, Fernando; Vazquez, Juan M; Calvete, Juan J; Roca, Jordi; Sanz, Libia; Parrilla, Inmaculada; Garcia, Eva M; Martinez, Emilio A

2003-08-01

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

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.

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

Correlation between cardiac remodelling, function, and myocardial contractility in rat hearts 5 weeks after myocardial infarction.

PubMed

Gosselin, H; Qi, X; Rouleau, J L

1998-01-01

Early after infarction, ventricular dysfunction occurs as a result of loss of myocardial tissue. Although papillary muscle studies suggest that reduced myocardial contractility contributes to this ventricular dysfunction, in vivo studies indicate that at rest, cardiac output is normal or near normal, suggesting that contractility of the remaining viable myocardium of the ventricular wall is preserved. However, this has never been verified. To explore this further, 100 rats with various-sized myocardial infarctions had ventricular function assessed by Langendorff preparation or by isolated papillary muscle studies 5 weeks after infarction. Morphologic studies were also done. Rats with large infarctions (54%) had marked ventricular dilatation (dilatation index from 0.23 to 0.75, p < 0.01) and papillary muscle dysfunction (total tension from 6.7 to 3.2 g/mm2, p < 0.01) but only moderate left ventricular dysfunction (maximum developed tension from 206 to 151 mmHg (1 mmHg = 133.3 Pa), p < 0.01), a decrease less than one would expect with an infarct size of 54%. The contractility of the remaining viable myocardium of the ventricle was also moderately depressed (peak systolic midwall stress 91 to 60 mmHg, p < 0.01). Rats with moderate infarctions (32%) had less marked but still moderate ventricular dilatation (dilatation index 0.37, p < 0.001) and moderate papillary muscle dysfunction (total tension 4.2 g/mm2, p < 0.01). However, their decrease in ventricular function was only mild (maximum developed pressure 178 mmHg, p < 0.01) and less than one would expect with an infarct size of 32%. The remaining viable myocardium of the ventricular wall appeared to have normal contractility (peak systolic midwall stress = 86 mmHg, ns). We conclude that in this postinfarction model, in large myocardial infarctions, a loss of contractility of the remaining viable myocardium of the ventricular wall occurs as early as 5 weeks after infarction and that papillary muscle studies slightly overestimate the degree of ventricular dysfunction. In moderate infarctions, the remaining viable myocardium of the ventricular wall has preserved contractility while papillary muscle function is depressed. In this relatively early postinfarction phase, ventricular remodelling appears to help maintain left ventricular function in both moderate and large infarctions.

Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources: a comprehensive study.

PubMed

Boura, Joana S; Santos, Francisco Dos; Gimble, Jeffrey M; Cardoso, Carla M P; Madeira, Catarina; Cabral, Joaquim M S; Silva, Cláudia Lobato da

2013-02-01

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

Dendritic cells for active immunotherapy: optimizing design and manufacture in order to develop commercially and clinically viable products.

PubMed

Nicolette, C A; Healey, D; Tcherepanova, I; Whelton, P; Monesmith, T; Coombs, L; Finke, L H; Whiteside, T; Miesowicz, F

2007-09-27

Dendritic cell (DC) active immunotherapy is potentially efficacious in a broad array of malignant disease settings. However, challenges remain in optimizing DC-based therapy for maximum clinical efficacy within manufacturing processes that permit quality control and scale-up of consistent products. In this review we discuss the critical issues that must be addressed in order to optimize DC-based product design and manufacture, and highlight the DC based platforms currently addressing these issues. Variables in DC-based product design include the type of antigenic payload used, DC maturation steps and activation processes, and functional assays. Issues to consider in development include: (a) minimizing the invasiveness of patient biological material collection; (b) minimizing handling and manipulations of tissue at the clinical site; (c) centralized product manufacturing and standardized processing and capacity for commercial-scale production; (d) rapid product release turnaround time; (e) the ability to manufacture sufficient product from limited starting material; and (f) standardized release criteria for DC phenotype and function. Improvements in the design and manufacture of DC products have resulted in a handful of promising leads currently in clinical development.

The financial viability of an SOFC cogeneration system in single-family dwellings

NASA Astrophysics Data System (ADS)

Alanne, Kari; Saari, Arto; Ugursal, V. Ismet; Good, Joel

In the near future, fuel cell-based residential micro-CHP systems will compete with traditional methods of energy supply. A micro-CHP system may be considered viable if its incremental capital cost compared to its competitors equals to cumulated savings during a given period of time. A simplified model is developed in this study to estimate the operation of a residential solid oxide fuel cell (SOFC) system. A comparative assessment of the SOFC system vis-à-vis heating systems based on gas, oil and electricity is conducted using the simplified model for a single-family house located in Ottawa and Vancouver. The energy consumption of the house is estimated using the HOT2000 building simulation program. A financial analysis is carried out to evaluate the sensitivity of the maximum allowable capital cost with respect to system sizing, acceptable payback period, energy price and the electricity buyback strategy of an energy utility. Based on the financial analysis, small (1-2 kW e) SOFC systems seem to be feasible in the considered case. The present study shows also that an SOFC system is especially an alternative to heating systems based on oil and electrical furnaces.

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.

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.

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.

Protocol for Automated Zooplankton Analysis

DTIC Science & Technology

2010-01-01

on maximum dimension on the smallest axis: organisms > 50 microns (urn) (nominally zooplankton), organisms > 10 um to < 50 um (nominally protists ...viability of protists . Recent work has focused on performing measurements at a variety of geographic locations to demonstrate that these stains...provide a location-independent means to identify viable protists in test samples. NRL recommends staining samples with a combination of two vital stains

The Elicitation Effect of Pathogenic Fungi on Trichodermin Production by Trichoderma brevicompactum

PubMed Central

Shentu, Xu-Ping; Liu, Wei-Ping; Zhan, Xiao-Huan; Yu, Xiao-Ping; Zhang, Chuan-Xi

2013-01-01

The effects of six species of phytopathogenic fungi mycelia as elicitors on trichodermin yield by Trichoderma brevicompactum were investigated. Neither nonviable nor viable mycelia of Botrytis cinerea, Alternaria solani, Colletotrichum lindemuthianum, and Thanatephorus cucumeris demonstrated any elicitation on the accumulation of trichodermin. However, the production of trichodermin was increased by the presence of viable/nonviable Rhizoctonia solani and Fusarium oxysporum mycelia. The strongest elicitation effect was found at the presence of nonviable R. solani. At the presence of nonviable R. solani, the maximum yield of trichodermin (144.55 mg/L) was significantly higher than the Control (67.8 mg/L), and the cultivation time to obtain the maximum yield of trichodermin decreased from 72 h to 60 h. No difference of trichodermin accumulation was observed by changing the concentration of nonviable R. solani from 0.1 to 1.6 g/L. It was observed that the optimum time for adding nonviable R. solani is immediately after inoculation. The diameter of T. brevicompactum mycelial globule after 72 h cultivation with nonviable R. solani elicitor was smaller than that of the Control. PMID:24385883

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.

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

The effect of substrate, season, and agroecological zone on mycoflora and aflatoxin contamination of poultry feed from Khyber Pakhtunkhwa, Pakistan.

PubMed

Alam, Sahib; Shah, Hamid Ullah; Khan, Habibullah; Magan, Naresh

2012-10-01

To study the effects of and interactions among feed types, seasons, and agroecological zones on the total fungal viable count and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) production in poultry feed, an experiment was conducted using three-factorial design. A total of 216 samples of poultry feed ingredients, viz. maize, wheat, rice, cotton seed meal (CSM), and finished products, that is, starter and finisher broilers' rations, were collected from Peshawar, Swat, and D. I. Khan districts of Khyber Pakhtunkhwa, Pakistan, during the winter, spring, summer, and autumn seasons of the year 2007/2008. Analysis of variance showed that there was a complex interaction among all these factors and that this influenced the total fungal viable count and relative concentrations of the aflatoxins produced. Minimum total culturable fungi (6.43 × 10³ CFUs/g) were counted in CSM from D. I. Khan region in winter season while maximum (26.68 × 10³ CFUs/g) in starter ration from Peshawar region in summer. Maximum concentrations of AFB1 (191.65 ng/g), AFB2 (86.85 ng/g), and AFG2 (89.90 ng/g) were examined during the summer season whereas the concentration of AFG1 was maximum (167.82 ng/g) in autumn in finisher ration from Peshawar region. Minimum aflatoxins were produced in the winter season across all the three agroecological zones.

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

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.

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.

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

Air-Abrasive Disinfection of Implant Surfaces in a Simulated Model of Periimplantitis.

PubMed

Quintero, David George; Taylor, Robert Bonnie; Miller, Matthew Braden; Merchant, Keith Roshanali; Pasieta, Scott Anthony

2017-06-01

This in vitro study aimed to evaluate the ability of air-powder abrasion to decontaminate dental implants. Twenty-six implants were inoculated with a Streptococcus sanguinis biofilm media in a novel periimplantitis defect model. Six implants served as controls, and 20 implants were disinfected with either the Cavitron JET Plus or the AIR-FLOW PERIO air-powder abrasion units. Residual bacteria were cultured, and colony forming units (CFUs) were totaled at 24 hours. As expected, negative control implant cultures showed no evidence of viable bacteria. Bacterial growth was observed on all positive control cultures, whereas only 15% of the experimental cultures displayed evidence of viable bacteria. The average CFU per streak for the positive control was 104 compared with a maximum of 10 and 4 CFUs for the Cavitron JET Plus and AIR-FLOW PERIO, respectively. There was a 99.9% reduction in bacteria for both air-powder abrasion instruments. Air-powder abrasion is an effective technique for the decontamination of dental implants, and the Cavitron JET Plus and AIR-FLOW PERIO are equally successful at eliminating viable bacteria from implant surfaces.

Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: The role of glutathione

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

Brechbuhl, Heather M.; Kachadourian, Remy; Min, Elysia

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5–25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression andmore » total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5–30 μM) and DOX (0.025–3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC{sub 50} values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy. -- Graphical abstract: Possible mechanisms by which chrysin enhances doxorubicin-induced toxicity in cancer cells. Highlights: ► Chyrsin sustains a significant depletion of GSH levels in lung cancer cells. ► Chyrsin synergistically potentiates doxorubicin-induced cancer cell cytotoxicity. ► Cancer cell sensitivity correlated with GSH and MRP gene network expression. ► This approach could allow for lower side effects and targeting resistant tumors.« less

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.

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.

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.

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis.

PubMed

Podszun, Susann; Vulto, Paul; Heinz, Helene; Hakenberg, Sydney; Hermann, Carsten; Hankemeier, Thomas; Urban, Gerald A

2012-02-07

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.

Enteric coating of granules containing the probiotic Lactobacillus acidophilus.

PubMed

Pyar, Hassan; Peh, Kok-Khiang

2014-06-01

In the present study, a capsule formulation composed of enteric coated granules of Lactobacillus acidophilus ATCC 4962 was developed using Eudragit L30D-55 as enteric polymer. Optimization of the capsule formulation was achieved with a maximum viable cell count after 2 h of incubation in acid medium and disintegration time of 1 h in buffer pH 6.8. The amount of Eudragit L30D-55 in the capsules correlated with gastric juice resistance. The best protective qualities against artificial gastric juice were observed when capsules were prepared from granules composed of L. acidophilus, corn starch, lactose monohydrate, polyvinylpyrrolidone and coated with 12.5 % (m/V) of Eudragit L30D-55. Capsule formulation of L. acidophilus in edible broth medium suspension serves as a cheap alternative to the expensive freeze-drying procedure for preparing L. acidophilus. In addition, the enteric coating using Eudragit L30D-55 could protect probiotics from the acidic gastric environment and enhance the bioactivity of probiotics along with replacement of pathogenic microbes in human intestine.

Bactericidal Effect of Solar Water Disinfection under Real Sunlight Conditions▿

PubMed Central

Boyle, M.; Sichel, C.; Fernández-Ibáñez, P.; Arias-Quiroz, G. B.; Iriarte-Puña, M.; Mercado, A.; Ubomba-Jaswa, E.; McGuigan, K. G.

2008-01-01

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of Campylobacter jejuni, Yersinia enterocolitica, enteropathogenic Escherichia coli, Staphylococcus epidermidis, and endospores of Bacillus subtilis, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, ∼1,050 W m−2 ± 10 W m−2) was as follows: C. jejuni, 20 min; S. epidermidis, 45 min; enteropathogenic E. coli, 90 min; Y. enterocolitica, 150 min. Following incomplete inactivation of B. subtilis endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process. PMID:18359829

Transplantation for myelodysplastic syndromes: who, when, and which conditioning regimens.

PubMed

Saber, Wael; Horowitz, Mary M

2016-12-02

Allogeneic hematopoietic stem cell transplantation (HCT) is the only curative therapy for myelodysplastic syndrome (MDS). Broad application is hindered by high risks of transplant-related morbidity and mortality, especially in the older age range represented by the MDS population. However, recent advances in strategies to minimize regimen-related toxicity make HCT a viable option for many more patients. Appropriate selection of patients involves consideration of patient factors, including use of geriatric assessment tools and comorbidity scales, that predict risks of regimen-related toxicity as well as disease factors, including genetic markers, which predict survival with both non-HCT and HCT therapy. Optimal timing of HCT for fit patients must consider MDS risk scores and life-years to be gained, with earlier transplantation indicated for patients with intermediate-2 and high-risk disease but judicious delay for lower risk patients. Selection of suitable conditioning regimens must balance risks of toxicity with opportunity for maximum disease control. © 2016 by The American Society of Hematology. All rights reserved.

Antimicrobial activity of hydroxyl radicals generated by hydrogen peroxide photolysis against Streptococcus mutans biofilm.

PubMed

Nakamura, Keisuke; Shirato, Midori; Kanno, Taro; Örtengren, Ulf; Lingström, Peter; Niwano, Yoshimi

2016-10-01

Prevention of dental caries with maximum conservation of intact tooth substance remains a challenge in dentistry. The present study aimed to evaluate the antimicrobial effect of H2O2 photolysis on Streptococcus mutans biofilm, which may be a novel antimicrobial chemotherapy for treating caries. S. mutans biofilm was grown on disk-shaped hydroxyapatite specimens. After 1-24 h of incubation, growth was assessed by confocal laser scanning microscopy and viable bacterial counting. Resistance to antibiotics (amoxicillin and erythromycin) was evaluated by comparing bactericidal effects on the biofilm with those on planktonic bacteria. To evaluate the effect of the antimicrobial technique, the biofilm was immersed in 3% H2O2 and was irradiated with an LED at 365 nm for 1 min. Viable bacterial counts in the biofilm were determined by colony counting. The thickness and surface coverage of S. mutans biofilm increased with time, whereas viable bacterial counts plateaued after 6 h. When 12- and 24-h-old biofilms were treated with the minimum concentration of antibiotics that killed viable planktonic bacteria with 3 log reduction, their viable counts were not significantly decreased, suggesting the biofilm acquired antibiotic resistance by increasing its thickness. By contrast, hydroxyl radicals generated by photolysis of 3% H2O2 effectively killed S. mutans in 24-h-old biofilm, with greater than 5 log reduction. The technique based on H2O2 photolysis is a potentially powerful adjunctive antimicrobial chemotherapy for caries treatment. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Low temperature MBBR nitrification: Microbiome analysis.

PubMed

Young, Bradley; Delatolla, Robert; Kennedy, Kevin; Laflamme, Edith; Stintzi, Alain

2017-03-15

This study aims to investigate post carbon removal moving bed biofilm reactor (MBBR) nitrification through the transition from 20 °C to 1 °C and during through long term operation at 1 °C. Four pilot nitrifying MBBR reactors were operated at various ammonia loading rates to elucidate the temperature effects on ammonia removal rates, cell viability and bacterial communities. The transition from 20 °C to 1 °C and during long term operation at 1 °C were modeled using Arrhenius temperature correction coefficients. Specifically, the steady state removal rates at 1 °C on average were 22.8% of the maximum ammonia removal rate at 20 °C, which corresponds to an Arrhenius temperature correction of 1.086 during steady operation at 1 °C. The microbial communities of the nitrifying MBBR biofilm were shown to be significantly more diverse at 20 °C as compared to 1 °C operation. Although less diverse at 1 °C, 2000 species of bacteria were identified in the nitrifying biofilm during operation at this low temperature. Nitrosomonads were shown to be the dominant ammonia oxidizing bacteria (AOB) and Nitrospira was shown to be the dominant nitrite oxidizing bacteria (NOB) in all the pilot MBBR reactors at all temperatures. The performance of the post carbon removal nitrifying MBBR systems were shown to be enhanced at 1 °C by an increase in the viable embedded biomass as well as thicker biofilm. This effectively increases the number of viable cell present during low temperature operation, which partially compensates for the significant decrease in rate of ammonia removal per nitrifying cell. Operation at the highest loading conditions tested in this study at 1 °C were shown to reduce the ammonia removal rate compared to lower loading conditions at 1 °C. The lower performance at higher loading conditions at 1 °C demonstrated an enrichment in the stress response metagenomics pathways of the system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance

NASA Technical Reports Server (NTRS)

Zachariah, Malcolm M.; Vaishampayan, Parag

2011-01-01

Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).

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.

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.

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.

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.

ONR Far East Scientific Bulletin, Volume 12, Number 4, October-December 1987,

DTIC Science & Technology

1987-12-01

of artificial insemination that have increased the hatching rate of The institute has developed viable prawn eggs in the hatchery from 3 to...this field is the flare observation ’%, % astrophysics, cosmology, and observa- satellite , HINOTORI, meaning firebird, tion or data-reduction...together with the results of the scope at the Okayama Astrophysical Solar Maximum Mission (SMM) satellite , Observatory, magnetic structures inside have

A Between-Squadron Analysis of Cannibalization on the MV-22

DTIC Science & Technology

2015-12-01

queens” or “ wind chimes” among maintainers and often become the lowest priority on a maintenance department’s workload (General Accounting Office, 2001...12b. DISTRIBUTION CODE 13. ABSTRACT (maximum 200 words) The Naval Aviation Maintenance Program recognizes cannibalization as a viable...and maintenance support systems. This thesis analyzed cannibalizations on the MV-22 aircraft platform to examine how the practice varied between

Augmentation of Chemotherapeutic Infusion Effect by TSU-68, an Oral Targeted Antiangiogenic Agent, in a Rabbit VX2 Liver Tumor Model

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

Kim, Hyo-Cheol; Chung, Jin Wook, E-mail: chungjw@snu.ac.kr; Choi, Seung Hong

Purpose: This study was designed to investigate the in vivo effects of combination therapy with TSU-68 and chemotherapeutic infusion in a rabbit VX2 liver tumor model. Methods: This study was approved by the animal care committee at our institute. Three weeks before chemotherapeutic infusion, VX2 carcinoma was implanted into the livers of 32 rabbits. One week after chemotherapeutic infusion, vehicle was administered orally for 3 weeks in the control group (n = 16), and TSU-68 was administered orally at a daily dose of 200 mg/kg for 3 weeks in the treated group (n = 16). Computed tomography (CT) was performedmore » before and 1, 2, 3, and 4 weeks after chemotherapeutic infusion. Tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) on CT scan. The maximum thickness of viable tumor was measured on microscopic sections. Results: According to the RECIST, stable disease was observed in 9 (56%) rabbits and progressive disease in 7 (44%) in the control group, whereas partial response was observed in 1 (6%) rabbit and stable disease in 15 (94%) in the treated group. On pathologic examination, a viable lesion was present in 12 (75%) rabbits in the control group and in 6 (38%) rabbits in the treated group (P = 0.073). The mean maximum thickness of viable tumor in the treated group was significantly smaller than that in the control group (0.74 mm vs. 3.39 mm; P = 0.02). Conclusions: Oral administration of TSU-68 augmented the effect of chemotherapeutic infusion in a rabbit VX2 liver tumor model.« less

A miniaturized, optically accessible bioreactor for systematic 3D tissue engineering research.

PubMed

Laganà, Matteo; Raimondi, Manuela T

2012-02-01

Perfusion bioreactors are widely used in tissue engineering and pharmaceutical research to provide reliable models of tissue growth under controlled conditions. Destructive assays are not able to follow the evolution of the growing tissue on the same construct, so it is necessary to adopt non-destructive analysis. We have developed a miniaturized, optically accessible bioreactor for interstitial perfusion of 3D cell-seeded scaffolds. The scaffold adopted was optically transparent, with highly defined architecture. Computational fluid dynamics (CFD) analysis was useful to predict the flow behavior in the bioreactor scaffold chamber (that was laminar flow, Re = 0.179, with mean velocity equal to 100 microns/s). Moreover, experimental characterization of the bioreactor performance gave that the maximum allowable pressure was 0.06 MPa and allowable flow rate up to 25 ml/min. A method, to estimate quantitatively and non destructively the cell proliferation (from 15 to 43 thousand cells) and tissue growth (from 2% to 43%) during culture time, was introduced and validated. An end point viability test was performed to check the experimental set-up overall suitability for cell culture with successful results. Morphological analysis was performed at the end time point to show the complex tridimensional pattern of the biological tissue growth. Our system, characterized by controlled conditions in a wide range of allowable flow rate and pressure, permits to systematically study the influence of several parameters on engineered tissue growth, using viable staining and a standard fluorescence microscope.

Maltodextrin enhances biofilm elimination by electrochemical scaffold

PubMed Central

Sultana, Sujala T.; Call, Douglas R.; Beyenal, Haluk

2016-01-01

Electrochemical scaffolds (e-scaffolds) continuously generate low concentrations of H2O2 suitable for damaging wound biofilms without damaging host tissue. Nevertheless, retarded diffusion combined with H2O2 degradation can limit the efficacy of this potentially important clinical tool. H2O2 diffusion into biofilms and bacterial cells can be increased by damaging the biofilm structure or by activating membrane transportation channels by exposure to hyperosmotic agents. We hypothesized that e-scaffolds would be more effective against Acinetobacter baumannii and Staphylococcus aureus biofilms in the presence of a hyperosmotic agent. E-scaffolds polarized at −600 mVAg/AgCl were overlaid onto preformed biofilms in media containing various maltodextrin concentrations. E-scaffold alone decreased A. baumannii and S. aureus biofilm cell densities by (3.92 ± 0.15) log and (2.31 ± 0.12) log, respectively. Compared to untreated biofilms, the efficacy of the e-scaffold increased to a maximum (8.27 ± 0.05) log reduction in A. baumannii and (4.71 ± 0.12) log reduction in S. aureus biofilm cell densities upon 10 mM and 30 mM maltodextrin addition, respectively. Overall ~55% decrease in relative biofilm surface coverage was achieved for both species. We conclude that combined treatment with electrochemically generated H2O2 from an e-scaffold and maltodextrin is more effective in decreasing viable biofilm cell density. PMID:27782161

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.

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

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

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.

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.

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.

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.

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

Metal organic frameworks for enzyme immobilization in biofuel cells

NASA Astrophysics Data System (ADS)

Bodell, JaDee

Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following enzymes: nicotinamide adenine dinucleotide (NAD)-dependent alcohol and aldehyde dehydrogenases, and pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases, as well as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase. Tb-meso MOF was shown to immobilize PQQ-dependent enzymes through ? stacking interactions of the heme in the enzyme and the triazine molecules in the ligand of the MOF. However, the PQQ-dependent dehydrogenases did not have enough catalytic activity present to be measured electrochemically. Finally, ZIF-90 was synthesized under aqueous conditions in the presence of FAD-dependent glucose dehydrogenase (GDH) which led to size selective sheltering of FAD-GDH. FAD-GDH had activity an order of magnitude larger than any of the alcohol dehydrogenases, which provided sufficient catalytic activity to measure electrochemically. The FAD-GDH bound within ZIF-90 was used to build a full biofuel cell resulting in an open circuit voltage of 708 +/- 16 mV and a maximum power density of 2.75 +/- 0.40 microW/cm2.

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.

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.

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.

A model for predicting Xanthomonas arboricola pv. pruni growth as a function of temperature

PubMed Central

Llorente, Isidre; Montesinos, Emilio; Moragrega, Concepció

2017-01-01

A two-step modeling approach was used for predicting the effect of temperature on the growth of Xanthomonas arboricola pv. pruni, causal agent of bacterial spot disease of stone fruit. The in vitro growth of seven strains was monitored at temperatures from 5 to 35°C with a Bioscreen C system, and a calibrating equation was generated for converting optical densities to viable counts. In primary modeling, Baranyi, Buchanan, and modified Gompertz equations were fitted to viable count growth curves over the entire temperature range. The modified Gompertz model showed the best fit to the data, and it was selected to estimate the bacterial growth parameters at each temperature. Secondary modeling of maximum specific growth rate as a function of temperature was performed by using the Ratkowsky model and its variations. The modified Ratkowsky model showed the best goodness of fit to maximum specific growth rate estimates, and it was validated successfully for the seven strains at four additional temperatures. The model generated in this work will be used for predicting temperature-based Xanthomonas arboricola pv. pruni growth rate and derived potential daily doublings, and included as the inoculum potential component of a bacterial spot of stone fruit disease forecaster. PMID:28493954

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...

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.

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

Viable myocardium scintigraphy with intravenous nitroglycerine by computed tomography with Tc-99m (MIBI).

PubMed

Ramos Filho, José; Nascimento, Marcos Welber; Silva, Rafael Mariano Gislon da; Camargo, Thiago Negrini de; Almeida, Roberto Simões de; Lima, Eloá Jacinto

2008-09-01

The selection of patients with chronic coronary disease for recanalization is based on the detection of the affected myocardium that is potentially viable. To evaluate the potentially viable ischemic myocardium through single photon emission computed tomography (SPECT) with MIBI after a maximum tolerated dose of I.V. nitroglycerin. We prospectively investigated by SPECT with Tc-99m (MIBI), from April 2004 to November 2005, 40 patients (mean age: 62 +/- 8.9 yrs, 30 men) with coronary obstruction demonstrated angiographically; the myocardium scintigraphy was carried out at rest and after intravenous (I.V.) nitroglycerin, which was started at a dose of 1 microg/kg/min and increased every minute until the systolic blood pressure decreased by 20 mmHg. The decrease in the perfusion of the segments was classified as moderate or severe and compared after the nitroglycerin. The angiographic, hemodynamic and myocardial perfusion variables were analyzed. We analyzed 680 myocardial segments at rest: 538 with a homogenous distribution and 142 with hypoperfusion (54 with moderate and 88 with severe decrease). After the nitroglycerin, there was an increase in the perfusion in 19 (47.5%) of 40 patients and 55 of 142 segments became viable: 33 (61.1%) with moderate and 22 (25%) with severe decrease; both presented a significant increase in the radiotracer distribution (p < 0.001, Chi-square). One of the components with Tc-99m is Tc-99m 2-methoxy-isobutyl-isonitrile (MIBI), which, when used with an optimized dose of I.V. nitroglycerin, can increase the radiotracer uptake in areas with moderate and severe hypoperfusion. The results of the present study suggest the increase in the Tc-99m (MIBI) sensitivity by nitroglycerin for the detection of viable myocardium.

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.

Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium

PubMed Central

Treskes, Philipp; Cowan, Douglas B.; Stamm, Christof; Rubach, Martin; Adelmann, Roland; Wittwer, Thorsten; Wahlers, Thorsten

2015-01-01

Objective The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. Methods Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. Results The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower stimulation frequencies (preconditioned engineered tissue constructs vs maximum embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). Conclusions We have generated skeletal myoblast–based transplantable grafts that electrically couple to myocardium. PMID:22980065

Immune complex-induced human monocyte procoagulant activity. I. a rapid unidirectional lymphocyte-instructed pathway.

PubMed

Schwartz, B S; Edgington, T S

1981-09-01

It has previously been described that soluble antigen:antibody complexes in antigen excess can induce an increase in the procoagulant activity of human peripheral blood mononuclear cells. It has been proposed that this response may explain the presence of fibrin in immune complex-mediated tissue lesions. In the present study we define cellular participants and their roles in the procoagulant response to soluble immune complexes. Monocytes were shown by cell fractionation and by a direct cytologic assay to be the cell of origin of the procoagulant activity; and virtually all monocytes were able to participate in the response. Monocytes, however, required the presence of lymphocytes to respond. The procoagulant response required cell cooperation, and this collaborative interaction between lymphocytes and monocytes appeared to be unidirectional. Lymphocytes once triggered by immune complexes induced monocytes to synthesize the procoagulant product. Intact viable lymphocytes were required to present instructions to monocytes; no soluble mediator could be found to subserve this function. Indeed, all that appeared necessary to induce monocytes to produce procoagulant activity was an encounter with lymphocytes that had previously been in contact with soluble immune complexes. The optimum cellular ratio for this interaction was four lymphocytes per monocyte, about half the ratio in peripheral blood. The procoagulant response was rapid, reaching a maximum within 6 h after exposure to antigen:antibody complexes. The procoagulant activity was consistent with tissue factor because Factors VII and X and prothrombin were required for clotting of fibrinogen. WE propose that this pathway differs from a number of others involving cells of the immune system. Elucidation of the pathway may clarify the role of this lymphocyte-instructed monocyte response in the Shwartzman phenomenon and other thrombohemorrhagic events associated with immune cell function and the formation of immune complexes.

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

[Simultaneous staining with fluorescein diacetate-propidium iodide to determine isolated cochlear outer hair cell viability of guinea pig].

PubMed

Yu, Q; Shi, H; Wang, J

1995-01-01

A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is discribed for use in the determination of isolated cochlear outer hair cell viability. With exciter light, viable cells fluoresce bright green, while nonviable cells are bright red. In cell culture and cytotoxicity studies, double-staining with FDA-PI is a accurate method to discriminate between live and nonviable cells.

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.

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.

Thermostability of Probiotics and Their α -Galactosidases and the Potential for Bean Products.

PubMed

Liu, Xiaoli; Champagne, Claude P; Lee, Byong H; Boye, Joyce I; Casgrain, Michel

2014-01-01

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α -galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α -galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α -galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α -galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α -galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans.

Thermostability of Probiotics and Their α-Galactosidases and the Potential for Bean Products

PubMed Central

Liu, Xiaoli; Champagne, Claude P.; Lee, Byong H.; Boye, Joyce I.; Casgrain, Michel

2014-01-01

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α-galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α-galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α-galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α-galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α-galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans. PMID:24744923

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

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.

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

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.

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.

Effective cancer laser-therapy design through the integration of nanotechnology and computational treatment planning models

NASA Astrophysics Data System (ADS)

Fisher, Jessica W.; Rylander, Marissa Nichole

2008-02-01

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.

Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

PubMed

Pushparajan, Charlotte; Claus, Juan Daniel; Marshall, Sean D G; Visnovsky, Gabriel

2017-12-01

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 10 5 cells ml -1 ) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 10 7 TCID 50 ml -1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm 2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

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.

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

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

Characterization of Heavy Oxide Inorganic Scintillator Crystals for Direct Detection of Fast Neutrons Based on Inelastic Scattering

DTIC Science & Technology

2015-03-01

HEAVY OXIDE INORGANIC SCINTILLATOR CRYSTALS FOR DIRECT DETECTION OF FAST NEUTRONS BASED ON INELASTIC SCATTERING by Philip R. Rusiecki...HEAVY OXIDE INORGANIC SCINTILLATOR CRYSTALS FOR DIRECT DETECTION OF FAST NEUTRONS BASED ON INELASTIC SCATTERING 6. AUTHOR(S) Philip R. Rusiecki 7...ABSTRACT (maximum 200 words) Heavy oxide inorganic scintillators may prove viable in the detection of fast neutrons based on the mechanism of

Mars Rotorcraft: Possibilities, Limitations, and Implications For Human/Robotic Exploration

NASA Technical Reports Server (NTRS)

Young, Larry A.; Aiken, Edwin; Lee, Pascal; Briggs, Geoffrey

2005-01-01

Several research investigations have examined the challenges and opportunities in the use of small robotic rotorcraft for the exploration of Mars. To date, only vehicles smaller than 150 kg have been studied. This paper proposes to examine the question of maximum Mars rotorcraft size, range, and payload/cargo capacity. Implications for the issue of whether or not (from an extreme design standpoint) a manned Mars rotorcraft is viable are also discussed.

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

Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

PubMed Central

Rygaard, Anita Mac; Thøgersen, Mariane Schmidt; Nielsen, Kristian Fog; Gram, Lone

2017-01-01

ABSTRACT Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria. IMPORTANCE Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders. PMID:28213548

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.

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

Instrumentation for optimizing an underground coal-gasification process

NASA Astrophysics Data System (ADS)

Seabaugh, W.; Zielinski, R. E.

1982-06-01

While the United States has a coal resource base of 6.4 trillion tons, only seven percent is presently recoverable by mining. The process of in-situ gasification can recover another twenty-eight percent of the vast resource, however, viable technology must be developed for effective in-situ recovery. The key to this technology is system that can optimize and control the process in real-time. An instrumentation system is described that optimizes the composition of the injection gas, controls the in-situ process and conditions the product gas for maximum utilization. The key elements of this system are Monsanto PRISM Systems, a real-time analytical system, and a real-time data acquisition and control system. This system provides from complete automation of the process but can easily be overridden by manual control. The use of this cost effective system can provide process optimization and is an effective element in developing a viable in-situ technology.

Optimizing Double-Network Hydrogel for Biomedical Soft Robots.

PubMed

Banerjee, Hritwick; Ren, Hongliang

2017-09-01

Double-network hydrogel with standardized chemical parameters demonstrates a reasonable and viable alternative to silicone in soft robotic fabrication due to its biocompatibility, comparable mechanical properties, and customizability through the alterations of key variables. The most viable hydrogel sample in our article shows tensile strain of 851% and maximum tensile strength of 0.273 MPa. The elasticity and strength range of this hydrogel can be customized according to application requirements by simple alterations in the recipe. Furthermore, we incorporated Agar/PAM hydrogel into our highly constrained soft pneumatic actuator (SPA) design and eventually produced SPAs with escalated capabilities, such as larger range of motion, higher force output, and power efficiency. Incorporating SPAs made of Agar/PAM hydrogel resulted in low viscosity, thermos-reversibility, and ultralow elasticity, which we believe can help to combine with the other functions of hydrogel, tailoring a better solution for fabricating biocompatible soft robots.

Controlling the Morphology of BDTT-DPP-Based Small Molecules via End-Group Functionalization for Highly Efficient Single and Tandem Organic Photovoltaic Cells.

PubMed

Kim, Ji-Hoon; Park, Jong Baek; Yang, Hoichang; Jung, In Hwan; Yoon, Sung Cheol; Kim, Dongwook; Hwang, Do-Hoon

2015-11-04

A series of narrow-band gap, π-conjugated small molecules based on diketopyrrolopyrrole (DPP) electron acceptor units coupled with alkylthienyl-substituted-benzodithiophene (BDTT) electron donors were designed and synthesized for use as donor materials in solution-processed organic photovoltaic cells. In particular, by end-group functionalization of the small molecules with fluorine derivatives, the nanoscale morphologies of the photoactive layers of the photovoltaic cells were successfully controlled. The influences of different fluorine-based end-groups on the optoelectronic and morphological properties, carrier mobilities, and the photovoltaic performances of these materials were investigated. A high power conversion efficiency (PCE) of 6.00% under simulated solar light (AM 1.5G) illumination has been achieved for organic photovoltaic cells based on a small-molecule bulk heterojunction system consisting of a trifluoromethylbenzene (CF3) end-group-containing oligomer (BDTT-(DPP)2-CF3) as the donor and [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) as the acceptor. As a result, the introduction of CF3 end-groups has been found to enhance both the short circuit current density (JSC) and fill factor (FF). A tandem photovoltaic device comprising an inverted BDTT-(DPP)2-CF3:PC71BM cell and a poly(3-hexylthiophene) (P3HT):indene-C60-bisadduct (IC60BA)-based cell as the top and bottom cell components, respectively, showed a maximum PCE of 8.30%. These results provide valuable guidelines for the rational design of conjugated small molecules for applications in high-performance organic photovoltaic cells. Furthermore, to the best of our knowledge, this is the first report on the design of fluorine-functionalized BDTT-DPP-based small molecules, which have been shown to be a viable candidate for use in inverted tandem cells.

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.

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

PubMed

Schendel, Stephen A

2017-10-01

Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. 5. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces.

PubMed

Serpaggi, Virginie; Remize, Fabienne; Recorbet, Ghislaine; Gaudot-Dumas, Eliane; Sequeira-Le Grand, Anabelle; Alexandre, Hervé

2012-06-01

Although the viable but not culturable (VBNC) state has been studied in detail in bacteria, it has been suggested that maintenance of viability with loss of culturability also exists in eukaryotic cells, such as in the wine spoilage yeast Brettanomyces. To provide conclusive evidence for the existence of a VBNC state in this yeast, we investigated its capacity to become viable and nonculturable after sulfite stress, and its ability to recover culturability after stressor removal. Sulfite addition induced loss of culturability but maintenance of viability. Increasing the medium pH to decrease the concentration of toxic SO(2) allowed yeast cells to become culturable again, thus demonstrating the occurrence of a VBNC state in Brettanomyces upon SO(2) exposure. Relative to culturable Brettanomyces, VBNC yeast cells were found to display a 22% decrease in size on the basis of laser granulometry. Assays for 4-ethylguaiacol and 4-ethylphenol, volatile phenols produced by Brettanomyces, indicated that spoilage compound production could persist in VBNC cells. These morphological and physiological changes in VBNC Brettanomyces were coupled to extensive protein pattern modifications, as inferred by comparative two-dimensional electrophoresis and mass spectrometric analyses. Upon identification of 53 proteins out of the 168 spots whose abundance was significantly modified in treated cells relative to control, we propose that the SO(2)-induced VBNC state in Brettanomyces is characterized by a reduced glycolytic flux coupled to changes in redox homeostatis/protein turnover-related processes. This study points out the existence of common mechanisms between yeast and bacteria upon entry to the VBNC state. Copyright © 2012 Elsevier Ltd. All rights reserved.

Femtosecond laser cutting of endothelial grafts: comparison of endothelial and epithelial applanation.

PubMed

Bernard, Aurélien; He, Zhiguo; Gauthier, Anne Sophie; Trone, Marie Caroline; Baubeau, Emmanuel; Forest, Fabien; Dumollard, Jean Marc; Peocʼh, Michel; Thuret, Gilles; Gain, Philippe

2015-02-01

Stromal surface quality of endothelial lamellae cut for endothelial keratoplasty with a femtosecond laser (FSL) with epithelial applanation remains disappointing. Applanation of the endothelial side of the cornea, mounted inverted on an artificial chamber, has therefore been proposed to improve cut quality. We compared lamellar quality after FSL cutting using epithelial versus endothelial applanation. Lamellae were cut with an FSL from organ-cultured corneas. After randomization, 7 were cut with epithelial applanation and 7 with endothelial applanation. Lamellae of 50-, 75-, and 100-μm thickness were targeted. Thickness was measured by optical coherence tomography before and immediately after cutting. Viable endothelial cell density was quantified immediately after cutting using triple labeling with Hoechst/ethidium/calcein-AM coupled with image analysis with ImageJ. The stromal surface was evaluated by 9 masked observers using semiquantitative scoring of scanning electronic microscopy images. Histology of 2 samples was also analyzed before lamellar detachment. Precision (difference in target/actual thickness) and thickness regularity [coefficient of variation (CV) of 10 measurements] were significantly better with endothelial applanation (precision: 18 μm; range, 10-30; CV: 11%; range, 8-12) than with epithelial applanation (precision: 84 μm; range, 54-107; P = 0.002; CV: 24%; range, 13-47; P = 0.001). Endothelial applanation provided thinner lamellae. However, viable endothelial cell density was significantly lower after endothelial applanation (1183 cells/mm2; range, 787-1725 versus 1688 cells/mm2; range, 1288-2025; P = 0.018). FSL cutting of endothelial lamellae using endothelial applanation provides thinner more regular grafts with more predictable thickness than with conventional epithelial applanation but strongly reduces the pool of viable endothelial cells.

Gamma Radiation Induces Micronucleated Reticulocytes in 3-D Bone Marrow Bioreactors in Vitro

PubMed Central

Sun, Hongliang; Dertinger, Stephen D.; Hyrien, Ollivier; David Wu, J. H.; Chen, Yuhchyau

2009-01-01

Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3-D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RET). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3-D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th to 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 and 32 hours post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5 – 2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3-D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro. PMID:19786117

Cytotoxicity evaluation of ZnO-eugenol (ZOE) using different ZnO structure on human gingival fibroblast

NASA Astrophysics Data System (ADS)

Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Masudi, Sam'an Malik; Seeni, Azman; Mohamad, Dasmawati; Ann, Ling Chuo; Sirelkhatim, Amna

2017-07-01

Application of ZnO is widely used in many industries, such as in optoelectronic devices, automotive, textile, cosmetics, medical and dentistry. In this study, emphasis was given on ZnO-eugenol (ZOE) that has been used in dental restoration. ZOE contained 80% ZnO and 20% eugenol. ZOE exhibited selective toxicity that could kill bacteria but safe on human cells. The safety of ZOE on humans is critically important. Two types of ZnO with different morphology, namely ZnO-A and ZnO-K were used to make ZOE (ZOE-A and ZOE-K) and the cytotoxicity level on human gingival fibroblast (HGF) cell line were evaluated. Both ZnO were characterized for its morphology and structural using Field Emission Scanning Electron Microscopy (FESEM) and X-ray Diffraction (XRD), respectively. The cytotoxicity level was evaluated using CCK-8 assay where the percentage of viable cells after 72 h were observed. The result showed ZnO-A, containing mostly rod-like shape with a crystallite size of 37.5 nm, had a higher percentage of viable cells after 72 h. Sample ZnO-K, containing irregular shape morphology with bigger crystallite size of 42.2 nm, had a lower percentage of viable cells after 72 h. The HGF cell line was treated with extract dilution of ZOE-A and ZOE-K at 5, 10 and 15%, respectively. At 15% of extracts dilution, 97.3% of the HGF cells survived (for ZOE-A) while the survival percentage of ZOE-K was only 88.1%. This fact was probably due to the larger surface-to-volume ratio of ZnO-A that gave better interlocking bond in ZOE-A. This interlocking bond can prevent the ZnO and eugenol elements leaching out from the ZOE matrix thereby decrease in cytotoxicity effects on HGF.

On the Transport of Viable but Non-Culturable (VBNC) E.coli O157:H7 in Soil and Groundwater

NASA Astrophysics Data System (ADS)

Kartz, C. R.; Kachanoski, G.; Dyck, M. F.

2010-12-01

The influence of the viable but non-culturable (VBNC) state on the expression of specific phenotypic traits of Enterohemorrhagic Escherichia coli O157:H7 as well as its transport behaviour in porous media has been examined in this study. E.coli O157:H7 is a human pathogen capable of entering a viable but non-culturable (VBNC) state following exposure to sublethal stress. In the VBNC state, E.coli O157:H7 is not detectable by standard culture techniques, yet is able to retain its virulence and ability to cause illness in humans. To date there is no in-depth information regarding the transport of VBNC E.coli species in soil or groundwater. Due to the public health risk, it becomes important to examine whether discrepancies exist between the transport behaviors of culturable and VBNC E.coli O157:H7 to help decide if current protocols for detecting this pathogen are accurate. This study identifies and contrasts transport-related properties of the two cell stages including hydrophobicity, extracellular polymeric substance (EPS) composition, and cell widths/lengths. Transport behaviors of the two cellular states are quantified and compared using column transport assays. Our results show that when E.coli O157:H7 cells enter into the VBNC state, there is an accompanied decrease in the hydrophobicity of the cells, shrinking of the cell profile from rod-shaped to coccoid, as well as a significant increase in tightly-bound surface proteins and sugars. Transport assays revealed a notable increase in mass flux when cells were in the VBNC state versus the culturable state. This research will contribute to the current knowledge-base describing E.coli O157:H7 cells in the VBNC state, spark dialogue concerning the accuracy of currently-used identification protocols, as well as add further evidence to the notion that bacteria transport in the subsurface is a truly dynamic process.

Laser-generated Micro-bubbles for Molecular Delivery to Adherent Cells

NASA Astrophysics Data System (ADS)

Genc, Suzanne Lee

We examine the use of optical breakdown in aqueous media as a means to deliver molecules into live adherent cell cultures. This process, called optoinjection (OI), is affected both by the media composition and the cellular exposure to hydrodynamic stresses associated with the cavitation bubble formed by the optical breakdown process. Here we explore the possibility of performing OI using laser microbeams focused at low numerical aperture to provide conditions where OI can be performed at high-throughput. We first investigate the effect of media composition on plasma and cavitation bubble formation. We make the discovery that irradiation of minimal essential media, supports the formation of low-density plasmas (LDP) resulting in the generation of small (2--20 mum radius) cavitation bubbles. This provides gentle specific hydrodynamic perturbations to single or small groups of cells. The addition of supplemental fetal bovine serum to the medium prevents the formation LDPs and the resulting avalanche ionization generates larger (> 100 mum radius) bubbles and more violent hydrodynamic effects. Second, using high-speed photography we provide the first visualization of LDP-generated cavitation bubbles at precise offset locations relative to a boundary on which a cell monolayer can be cultured. These images depict the cellular exposure to different hydrodynamic conditions depending on the normalized offset distance (gamma = s/Rmax) and show how it affects the cellular exposure to shear stresses upon bubble expansion and different distributions of bubble energy upon collapse. Lastly, we examine the effects of pulse energy, parameters, and single vs. multiple laser exposures on the ability to deliver 3-5 kDa dextrans into adherent cells using both small (< 20 mum) and large (100mu m) radius bubbles. For single exposures, we identify several conditions under which OI can be optimized: (a) conditions where cell viability is maximized (˜90%) but optoinjection of viable cells is relatively low (˜30%) and (b) conditions where cell viability is compromised (˜80%) but where the optoinjection of viable cells is higher (˜50%). For multiple exposures in a grid pattern, we generally found reduced optoinjection efficacy but do identify conditions where we achieve injection of viable cells approaching 50%. We correlate these results to the cavitation bubble dynamics.

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.

Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants.

PubMed

Wong, Hui San; Townsend, Kirsty M; Fenwick, Stan G; Maker, Garth; Trengove, Robert D; O'Handley, Ryan M

2010-10-01

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC™ system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.

Basic Techniques in Mammalian Cell Tissue Culture.

PubMed

Phelan, Katy; May, Kristin M

2016-11-01

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. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Growth Phase, Oxygen, Temperature, and Starvation Affect the Development of Viable but Non-culturable State of Vibrio cholerae.

PubMed

Wu, Bin; Liang, Weili; Kan, Biao

2016-01-01

Vibrio cholerae can enter into a viable but non-culturable (VBNC) state in order to survive in unfavorable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW). Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 10(6)-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22 or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 10(8) to 10(6)-10(5) CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB), but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction. Changing a single factor could influence and even block the development of the VBNC state. These findings provide new insight to help design further studies to better understand the mechanisms which trigger the development and regulation of the VBNC state.

Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury

PubMed Central

Matylevitch, Natalia P.; Schuschereba, Steven T.; Mata, Jennifer R.; Gilligan, George R.; Lawlor, David F.; Goodwin, Cleon W.; Bowman, Phillip D.

1998-01-01

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72°C for 1 second. After exposure to temperatures of 58 to 59°C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66°C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72°C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59°C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation. PMID:9708816

Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.

PubMed

Matylevitch, N P; Schuschereba, S T; Mata, J R; Gilligan, G R; Lawlor, D F; Goodwin, C W; Bowman, P D

1998-08-01

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

Breaking the Blood-Brain Barrier With Mannitol to Aid Stem Cell Therapeutics in the Chronic Stroke Brain.

PubMed

Tajiri, Naoki; Lee, Jea Young; Acosta, Sandra; Sanberg, Paul R; Borlongan, Cesar V

2016-01-01

Blood-brain barrier (BBB) permeabilizers, such as mannitol, can facilitate peripherally delivered stem cells to exert therapeutic benefits on the stroke brain. Although this BBB permeation-aided stem cell therapy has been demonstrated in the acute stage of stroke, such BBB permeation in the chronic stage of the disease remains to be examined. Adult Sprague-Dawley rats initially received sham surgery or experimental stroke via the 1-h middle cerebral artery occlusion (MCAo) model. At 1 month after the MCAo surgery, stroke animals were randomly assigned to receive human umbilical cord stem cells only (2 million viable cells), mannitol only (1.1 mol/L mannitol at 4°C), combined human umbilical cord stem cells (200,000 viable cells) and mannitol (1.1 mol/L mannitol at 4°C), and vehicle (phosphate-buffered saline) only. Stroke animals that received human umbilical cord blood cells alone or combined human umbilical cord stem cells and mannitol exhibited significantly improved motor performance and significantly better brain cell survival in the peri-infarct area compared to stroke animals that received vehicle or mannitol alone, with mannitol treatment reducing the stem cell dose necessary to afford functional outcomes. Enhanced neurogenesis in the subventricular zone accompanied the combined treatment of human umbilical cord stem cells and mannitol. We showed that BBB permeation facilitates the therapeutic effects of a low dose of peripherally transplanted stem cells to effectively cause functional improvement and increase neurogenesis in chronic stroke.

The selective progesterone receptor modulator CDB4124 inhibits proliferation and induces apoptosis in uterine leiomyoma cells.

PubMed

Luo, Xia; Yin, Ping; Coon V, John S; Cheng, You-Hong; Wiehle, Ronald D; Bulun, Serdar E

2010-05-15

To evaluate the effects of selective P receptor (PR) 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. Laboratory research. Academic medical center. Premenopausal women (n = 12) undergoing hysterectomy for leiomyoma-related symptoms. Treatment of primary LSM and MSM cells with CDB4124 (10(-8)-10(-6) M) or vehicle for 24, 48, or 72 hours. Western blot for protein expression of proliferating cell nuclear antigen, cleaved polyadenosine 5'-diphosphate-ribose polymerase, Bcl-2, and Krüppel-like transcription factor 11; 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate viable cell numbers; and real-time polymerase chain reaction (PCR) to quantify messenger RNA (mRNA) levels. Treatment with CDB4124 significantly decreased levels of the proliferation marker proliferating cell nuclear antigen, the number of viable LSM cells, and the antiapoptotic protein Bcl-2. On the other hand, treatment with CDB4124 increased levels of the apoptosis marker cleaved polyadenosine 5'-diphosphate-ribose polymerase and the tumor suppressor Krüppel-like transcription factor 11 in a dose- and time-dependent manner in LSM cells. In matched MSM cells, however, CDB4124 did not affect cell proliferation or apoptosis. CDB4124 selectively inhibits proliferation and induces apoptosis in LSM but not in MSM cells. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.

PubMed

Huang, Edwin P; Marquis, Christopher P; Gray, Peter P

2004-11-20

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (c) 2004 Wiley Periodicals, Inc

A 3D human tissue-engineered lung model to study influenza A infection.

PubMed

Bhowmick, Rudra; Derakhshan, Mina; Liang, Yurong; Ritchey, Jerry; Liu, Lin; Gappa-Fahlenkamp, Heather

2018-05-05

Influenza A virus (IAV) claims approximately 250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (2D cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction, would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineering Lung Model (3D-HTLM), we described the 3D culture of primary human small airway epithelial cells (HSAEpCs), and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2.The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

Antibacterial activity of Artemisia asiatica essential oil against some common respiratory infection causing bacterial strains and its mechanism of action in Haemophilus influenzae.

PubMed

Huang, Jiehui; Qian, Chao; Xu, Hongjie; Huang, Yanjie

2018-01-01

The main objective of the current study was to investigate the chemical composition of the essential oil of Artemisia asiatica together with investigating the antibacterial effects it exerts on several common respiratory infection causing bacteria including Haemophilus influenzae. Its mechanism of action was studied using various state-of-the-art assays like scanning electron microscopy, DNA, RNA and protein leakage assays, growth curve assays etc. The essential oil was extracted from the leaves of A. asiatica by supercritical CO 2 fluid extraction technology. Chemical composition of essential oils was analyzed by gas chromatography-mass-spectrometry (GC-MS). The antibacterial activity was evaluated against 6 bacteria by the paper disc diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) values of the essential oil were estimated by agar dilution method. The antibacterial mechanism was evaluated by growth curve, the integrity of cell membrane and scanning electronmicroscope (SEM). Gas chromatographic analysis of the A. asiatica essential oil led to the identification of 16 chemical constituents accounting for 97.2% of the total oil composition. The major components were found to be Piperitone, (z)-davanone, p-cymene and 1, 8-cineole. The essential oil showed maximum growth inhibition against Haemophilus influenzae with a zone of inhibition of 24.5 mm and MIC/MBC values of 1.9/4.5 mg/mL respectively. Bacteria treated with the essential oil led to a rapid decrease in the number of viable cells. On adding the essential oil of A. asiatica to the bacterial culture, the constituents of the bacterial cell got released into the medium and this cell constituent release increased with increasing doses of the essential oil. SEM showed that the bacterial cells treated with the essential oil showed damaged cell wall, deformed cell morphology and shrunken cells. Copyright © 2017. Published by Elsevier Ltd.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria.

PubMed

de Barros, João M S; Lechner, Tabea; Charalampopoulos, Dimitrios; Khutoryanskiy, Vitaliy V; Edwards, Alexander D

2015-09-30

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid. Copyright © 2015 Elsevier B.V. All rights reserved.

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

PubMed

Carlisle, L; Steel, K; Forge, A

1990-11-01

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections.

PubMed

Hagedorn, Mary M; Daly, Jonathan P; Carter, Virginia L; Cole, Kathleen S; Jaafar, Zeehan; Lager, Claire V A; Parenti, Lynne R

2018-04-18

As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.

Revisiting the role of pathological analysis in transarterial chemoembolization-treated hepatocellular carcinoma after transplantation

PubMed Central

Vasuri, Francesco; Malvi, Deborah; Rosini, Francesca; Baldin, Pamela; Fiorentino, Michelangelo; Paccapelo, Alexandro; Ercolani, Giorgio; Pinna, Antonio Daniele; Golfieri, Rita; Morselli-Labate, Antonio Maria; Grigioni, Walter Franco; D’Errico-Grigioni, Antonia

2014-01-01

AIM: To define the histopathological features predictive of post-transplant hepatocellular carcinoma (HCC) recurrence after transarterial chemoembolization, applicable for recipient risk stratification. METHODS: We retrospectively reviewed the specimens of all suspicious nodules (total 275) from 101 consecutive liver transplant recipients which came to our Pathology Unit over a 6-year period. All nodules were sampled and analyzed, and follow-up data were collected. We finally considered 11 histological variables for each patient: total number of nodules, number of viable nodules, size of the major nodule, size of the major viable nodule, occurrence of microscopic vascular invasion, maximum Edmondson's grade, clear cell/sarcomatous changes, and the residual neoplastic volume. Survival data were computed by means of the Kaplan-Meier procedure and analyzed by means of the Cox proportional hazards model. The multivariate linear regression and a k-means cluster analysis were also used in order to compute the standardized histological score. RESULTS: The total number of nodules, the residual neoplastic volume (the total volume of all evaluated nodules minus the necrotic portion) and the microvascular invasion entered the Cox multivariate hazard model with HCC recurrence as dependent variable. The histological score was therefore computed and a cluster analysis sorted recipients into 3 risk groups, with 3.3%, 18.5% and 53.8% respectively of tumor recurrence rates and 1.6%, 11.1% and 38.5% of tumor-related mortality respectively at the end of follow-up. CONCLUSION: The histological score allows a reliable stratification of HCC recurrence risk, especially in those recipients found out to be beyond the Milan criteria after orthotopic liver transplantation (OLT). PMID:25309084

Impact of Saccharomyces cerevisiae strains on traditional sparkling wines production.

PubMed

Di Gianvito, Paola; Perpetuini, Giorgia; Tittarelli, Fabrizia; Schirone, Maria; Arfelli, Giuseppe; Piva, Andrea; Patrignani, Francesca; Lanciotti, Rosalba; Olivastri, Lino; Suzzi, Giovanna; Tofalo, Rosanna

2018-07-01

Sparkling wine fermentation is a challenge for yeasts due to the hostile conditions. A phenotype sought in starters is flocculation, because it reduces riddling time. For this reason, six flocculent Saccharomyces cerevisiae wine strains with different flocculation degree and autolytic activity and two commercial strains were tested for traditional sparkling wine production in a winery. Yeast viability, free aminoacids and high molecular weight nitrogen release and physico-chemical composition of sparkling wines were evaluated. Moreover, strains were tested for their aromatic potential. Obtained data revealed that flocculent yeasts presented oenological performances (in terms of fermentation rate, maximum pressure reached, free aminoacids - AAN and high molecular weight nitrogen - HMWN release) similar to the commercial strains. All considered strains were able to complete fermentation and viable cells of all strains were detected in all sparkling wines produced even after 6 months. F6789 and F10471 strains showed slow fermentation kinetics reaching the maximum of pressure at 180 days. Regarding nitrogen compounds release, FI strain was characterized by the highest amount of AAN and HMWN released, followed by F6789. Strains showed a considerable diversification in terms of number and amount of aroma molecules produced and sparkling wines obtained with autochthonous flocculent strains presented a higher amount of alcohols and esters already after 3 months. Further studies are necessary to select starter strains to improve traditional sparkling wines production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Revisiting the two-layer hypothesis: coexistence of alternative functional rooting strategies in savannas.

PubMed

Holdo, Ricardo M

2013-01-01

The two-layer hypothesis of tree-grass coexistence posits that trees and grasses differ in rooting depth, with grasses exploiting soil moisture in shallow layers while trees have exclusive access to deep water. The lack of clear differences in maximum rooting depth between these two functional groups, however, has caused this model to fall out of favor. The alternative model, the demographic bottleneck hypothesis, suggests that trees and grasses occupy overlapping rooting niches, and that stochastic events such as fires and droughts result in episodic tree mortality at various life stages, thus preventing trees from otherwise displacing grasses, at least in mesic savannas. Two potential problems with this view are: 1) we lack data on functional rooting profiles in trees and grasses, and these profiles are not necessarily reflected by differences in maximum or physical rooting depth, and 2) subtle, difficult-to-detect differences in rooting profiles between the two functional groups may be sufficient to result in coexistence in many situations. To tackle this question, I coupled a plant uptake model with a soil moisture dynamics model to explore the environmental conditions under which functional rooting profiles with equal rooting depth but different depth distributions (i.e., shapes) can coexist when competing for water. I show that, as long as rainfall inputs are stochastic, coexistence based on rooting differences is viable under a wide range of conditions, even when these differences are subtle. The results also indicate that coexistence mechanisms based on rooting niche differentiation are more viable under some climatic and edaphic conditions than others. This suggests that the two-layer model is both viable and stochastic in nature, and that a full understanding of tree-grass coexistence and dynamics may require incorporating fine-scale rooting differences between these functional groups and realistic stochastic climate drivers into future models.

Revisiting the Two-Layer Hypothesis: Coexistence of Alternative Functional Rooting Strategies in Savannas

PubMed Central

Holdo, Ricardo M.

2013-01-01

The two-layer hypothesis of tree-grass coexistence posits that trees and grasses differ in rooting depth, with grasses exploiting soil moisture in shallow layers while trees have exclusive access to deep water. The lack of clear differences in maximum rooting depth between these two functional groups, however, has caused this model to fall out of favor. The alternative model, the demographic bottleneck hypothesis, suggests that trees and grasses occupy overlapping rooting niches, and that stochastic events such as fires and droughts result in episodic tree mortality at various life stages, thus preventing trees from otherwise displacing grasses, at least in mesic savannas. Two potential problems with this view are: 1) we lack data on functional rooting profiles in trees and grasses, and these profiles are not necessarily reflected by differences in maximum or physical rooting depth, and 2) subtle, difficult-to-detect differences in rooting profiles between the two functional groups may be sufficient to result in coexistence in many situations. To tackle this question, I coupled a plant uptake model with a soil moisture dynamics model to explore the environmental conditions under which functional rooting profiles with equal rooting depth but different depth distributions (i.e., shapes) can coexist when competing for water. I show that, as long as rainfall inputs are stochastic, coexistence based on rooting differences is viable under a wide range of conditions, even when these differences are subtle. The results also indicate that coexistence mechanisms based on rooting niche differentiation are more viable under some climatic and edaphic conditions than others. This suggests that the two-layer model is both viable and stochastic in nature, and that a full understanding of tree-grass coexistence and dynamics may require incorporating fine-scale rooting differences between these functional groups and realistic stochastic climate drivers into future models. PMID:23950900

Factors affecting viability of Bifidobacterium bifidum during spray drying.

PubMed

Shokri, Zahra; Fazeli, Mohammad Reza; Ardjmand, Mehdi; Mousavi, Seyyed Mohammad; Gilani, Kambiz

2015-01-25

There is substantial clinical data supporting the role of Bifidobacterium bifidum in human health particularly in benefiting the immune system and suppressing intestinal infections. Compared to the traditional lyophilization, spray-drying is an economical process for preparing large quantities of viable microorganisms. The technique offers high production rates and low operating costs but is not usually used for drying of substances prone to high temperature. The aim of this study was to establish the optimized environmental factors in spray drying of cultured bifidobacteria to obtain a viable and stable powder. The experiments were designed to test variables such as inlet air temperature, air pressure and also maltodextrin content. The combined effect of these variables on survival rateand moisture content of bacterial powder was studied using a central composite design (CCD). Sub-lethal heat-adaptation of a B. bifidum strain which was previously adapted to acid-bile-NaCl led to much more resistance to high outlet temperature during spray drying. The resistant B. bifidum was supplemented with cost friendly permeate, sucrose, yeast extract and different amount of maltodextrin before it was fed into a Buchi B-191 mini spray-dryer. Second-order polynomials were established to identify the relationship between the responses andthe three variables. Results of verification experiments and predicted values from fitted correlations were in close agreement at 95% confidence interval. The optimal values of the variables for maximum survival and minimum moisture content of B. bifidum powder were as follows: inlet air temperature of 111.15°C, air pressure of 4.5 bar and maltodextrin concentration of 6%. Under optimum conditions, the maximum survival of 28.38% was achieved while moisture was maintained at 4.05%. Viable and cost effective spray drying of Bifidobacterium bifidum could be achieved by cultivating heat and acid adapted strain into the culture media containing nutritional protective agents.

Has the Sun Set on Quantum Dot-Sensitized Solar Cells?

DOE PAGES

Wrenn, Toshia L.; McBride, James R.; Smith, Nathanael J.; ...

2015-01-01

This is a reminder, a review and a look toward the future prospects for quantum dot-sensitized solar cells - a reminder of the highly viable, energy-efficient solar cells achievable. This is also a review of ground-breaking devices and their similarities to the near unity photon-to-electron mechanisms of photosynthesis; a look toward architectures that capitalize on the advances observed in previous work.

Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates

NASA Astrophysics Data System (ADS)

Gates, R. D.; Muscatine, L.

1992-09-01

Three maceration methods are described for the isolation of single endoderm cells from marine cnidarians. Two are enzymatic treatments suitable for fleshy anthozoans such as sea anemones and zoanthids. The third employs calcium free sea water and is suitable for stony corals. The viability and morphology of the endoderm cells is described using fluorogenic dyes and scanning and transmission electron microscopy.

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

Identification of Viable Helicobacter pylori in Drinking Water Supplies by Cultural and Molecular Techniques.

PubMed

Santiago, Paula; Moreno, Yolanda; Ferrús, M Antonía

2015-08-01

Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, directly related to peptic ulcer and gastric cancer. It has been suggested that H. pylori can be acquired through different transmission routes, including water. In this study, culture and qPCR were used to detect and identify the presence of H. pylori in drinking water. Furthermore, the combined techniques PMA-qPCR and DVC-FISH were applied for detection of viable cells of H. pylori. Among 24 drinking water samples, 16 samples were positive for the presence of H. pylori, but viable cells were only detected in six samples. Characteristic colonies, covered by a mass of bacterial unspecific growth, were observed on selective agar plates from an only sample, after enrichment. The mixed culture was submitted to DVC-FISH and qPCR analysis, followed by sequencing of the amplicons. Molecular techniques confirmed the growth of H. pylori on the agar plate. Our results demonstrate for the first time that H. pylori can survive and be potentially infective in drinking water, showing that water distribution systems could be a potential route for H. pylori transmission. © 2015 John Wiley & Sons Ltd.

Accumulation of high OPDA level correlates with reduced ROS and elevated GSH benefiting white cell survival in variegated leaves

USDA-ARS?s Scientific Manuscript database

Variegated Epipremnum aureum ‘Marble Queen’ plant has white (VMW) and green (VMG) sectors within the same leaf. The white sector cells containing undifferentiated chloroplasts are viable, but the underlying mechanism for their survival is not clear. Because phytohormones are important for plant grow...

MicroRNAs-449a and -449b exhibit tumor suppressive effects in retinoblastoma

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

Martin, Alissa; Jones, Aunica; Bryar, Paul J.

2013-11-01

Highlights: •We validate miR-449a/b expression in primary human retinoblastomas and cell lines. •Exogenous miRs-449a/b inhibited proliferation in retinoblastoma cell lines. •Exogenous miRs-449a/b increased apoptosis in retinoblastoma cell lines. •miRs-449a/b could serve as viable therapeutic targets for retinoblastoma treatment. -- Abstract: Retinoblastoma is the most common pediatric cancer of the eye. Currently, the chemotherapeutic treatments for retinoblastoma are broad-based drugs such as vincristine, carboplatin, or etoposide. However, therapies targeted directly to aberrant signaling pathways may provide more effective therapy for this disease. The purpose of our study is to illustrate the relationship between the expressions of miRs-449a and -449b to retinoblastomamore » proliferation and apoptosis. We are the first to confirm an inhibitory effect of miR-449a and -449b in retinoblastoma by demonstrating significantly impaired proliferation and increased apoptosis of tumor cells when these miRNAs are overexpressed. This study suggests that these miRNAs could serve as viable therapeutic targets for retinoblastoma treatment.« less

A factor converting viable but nonculturable Vibrio cholerae to a culturable state in eukaryotic cells is a human catalase.

PubMed

Senoh, Mitsutoshi; Hamabata, Takashi; Takeda, Yoshifumi

2015-08-01

In our previous work, we demonstrated that viable but nonculturable (VBNC) Vibrio cholerae O1 and O139 were converted to culturable by coculture with eukaryotic cells. Furthermore, we isolated a factor converting VBNC V. cholerae to culturable (FCVC) from a eukaryotic cell line, HT-29. In this study, we purified FCVC by successive column chromatographies comprising UNO Q-6 anion exchange, Bio-Scale CHT2-1 hydroxyapatite, and Superdex 200 10/300 GL. Homogeneity of the purified FCVC was demonstrated by SDS-PAGE. Nano-LC MS/MS analysis showed that the purified FCVC was a human catalase. An experiment of RNAi knockdown of catalase mRNA from HT-29 cells and treatment of the purified FCVC with a catalase inhibitor, 3-amino-1,2,4-triazole confirmed that the FCVC was a catalase. A possible role of the catalase in converting a VBNC V. cholerae to a culturable state in the human intestine is discussed. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Manipulation of biological cells using a microelectromagnet matrix

NASA Astrophysics Data System (ADS)

Lee, H.; Purdon, A. M.; Westervelt, R. M.

2004-08-01

Noninvasive manipulation of biological cells inside a microfluidic channel was demonstrated using a microelectromagnet matrix. The matrix consists of two layers of straight Au wires, aligned perpendicular to each other, that are covered by insulating layers. By adjusting the current in each independent wire, the microelectromagnet matrix can create versatile magnetic field patterns to control the motion of individual cells in fluid. Single or multiple yeast cells attached to magnetic beads were trapped, continuously moved and rotated, and a viable cell was separated from nonviable cells for cell sorting.

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.

Dual-Stage Crosslinking of a Gel-Phase Bioink Improves Cell Viability and Homogeneity for 3D Bioprinting.

PubMed

Dubbin, Karen; Hori, Yuki; Lewis, Kazuomori K; Heilshorn, Sarah C

2016-10-01

Current bioinks for cell-based 3D bioprinting are not suitable for technology scale-up due to the challenges of cell sedimentation, cell membrane damage, and cell dehydration. A novel bioink hydrogel is presented with dual-stage crosslinking specifically designed to overcome these three major hurdles. This bioink enables the direct patterning of highly viable, multicell type constructs with long-term spatial fidelity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mammalian Cell Tissue Culture.

PubMed

Phelan, Katy; May, Kristin M

2017-07-11

Cultured mammalian cells are used extensively in the field of human genetics. 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. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Long distance biotic dispersal of tropical seagrass seeds by marine mega-herbivores.

PubMed

Tol, Samantha J; Jarvis, Jessie C; York, Paul H; Grech, Alana; Congdon, Bradley C; Coles, Robert G

2017-06-30

Terrestrial plants use an array of animals as vectors for dispersal, however little is known of biotic dispersal of marine angiosperms such as seagrasses. Our study in the Great Barrier Reef confirms for the first time that dugongs (Dugong dugon) and green sea turtles (Chelonia mydas) assist seagrass dispersal. We demonstrate that these marine mega-herbivores consume and pass in faecal matter viable seeds for at least three seagrass species (Zostera muelleri, Halodule uninervis and Halophila decipiens). One to two seagrass seeds per g DW of faecal matter were found during the peak of the seagrass reproductive season (September to December), with viability on excretion of 9.13% ± 4.61% (SE). Using population estimates for these mega-herbivores, and data on digestion time (hrs), average daily movement (km h) and numbers of viable seagrass seeds excreted (per g DW), we calculated potential seagrass seed dispersal distances. Dugongs and green sea turtle populations within this region can disperse >500,000 viable seagrass seeds daily, with a maximum dispersal distance of approximately 650 km. Biotic dispersal of tropical seagrass seeds by dugongs and green sea turtles provides a large-scale mechanism that enhances connectivity among seagrass meadows, and aids in resilience and recovery of these coastal habitats.

Bioaerosol assessment in naturally ventilated historical library building with restricted personnel access.

PubMed

Harkawy, Aleksander; Górny, Rafał L; Ogierman, Leonard; Wlazło, Agnieszka; Ławniczek-Wałczyk, Anna; Niesler, Anna

2011-01-01

The aim of this study was to check the degree and identify the sources of microbial contamination of the Jasna Gora (Bright Hill) monastery library 10 years after disinfection of the incunabula collection. The registered maximum viable indoor microbial concentrations were 1,875 and 7,100 cfu/m³ for stationary and personal measurements, whereas respective total concentrations were 71,000 and 100,000 counts/m3. There was no statistically significant difference between the concentrations of viable microorganisms measured in the stationary using Andersen, GSP, and Button samplers. Moreover, GSP and Button samplers can be interchangeably applied when viable or total microbial levels are stationary or personally measured. The culturable microorganisms constituted 0.5 - 3.9% of the total microflora only. Filamentous fungi were the most prevalent outdoors, whereas Gram-positive cocci and endospore forming Gram-positive rods dominated indoors in the air and settled dust, respectively. Hence, an unrestrained infiltration of ambient air through the draughtiness of the building envelope is probably the main process responsible for indoor fungal pollution, whereas bacterial contaminants have their major sources in the indoor environment. Moreover, even a chemically cleansed library collection, having a restricted personnel access, but under the influence of ambient air, can undergo microbial contamination and becomes an important microbial emission source.

[Prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: detection by cultural and direct microscopic techniques].

PubMed

Muliukin, A L; Demkina, E V; Manucharova, N A; Akimov, V N; Andersen, D; McKay, C; Gal'chenko, V F

2014-01-01

The heterotrophic mesophilic component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. Application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved form subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of preserved fossil soil and permafrost samples collected in the Arctic zone. The differences in response to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.

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

Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway.

PubMed

Hansen, Aviaja A; Herbert, Rodney A; Mikkelsen, Karina; Jensen, Lars Liengård; Kristoffersen, Tommy; Tiedje, James M; Lomstein, Bente Aa; Finster, Kai W

2007-11-01

The viable and non-viable fractions of the bacterial community in a 2347-year-old permafrost soil from Spitsbergen were subjected to a comprehensive investigation using culture-independent and culture-dependent methods. LIVE/DEAD BacLight staining revealed that 26% of the total number of bacterial cells were viable. Quantitatively, aerobic microcolonies, aerobic colony-forming units and culturable anaerobic bacteria comprised a minor fraction of the total number of viable bacteria, which underlines the necessity for alternative cultivation approaches in bacterial cryobiology. Sulfate reduction was detected at temperatures between -2 degrees C and 29 degrees C while methanogenesis was not detected. Bacterial diversity was high with 162 operational taxonomic units observed from 800 16S rDNA clone sequences. The 158 pure cultures isolated from the permafrost soil affiliated with 29 different bacterial genera, the majority of which have not previously been isolated from permafrost habitats. Most of the strains isolated were affiliated to the genera Cellulomonas and Arthrobacter and several of the pure cultures were closely related to bacteria reported from other cryohabitats. Characterization of viable bacterial communities in permafrost soils is important as it will enable identification of functionally important groups together with the as yet undescribed adaptations that bacteria have evolved for surviving subzero temperatures for millennia.

Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors.

PubMed

Chen, Jin; Chaurio, Ricardo A; Maueröder, Christian; Derer, Anja; Rauh, Manfred; Kost, Andriy; Liu, Yi; Mo, Xianming; Hueber, Axel; Bilyy, Rostyslav; Herrmann, Martin; Zhao, Yi; Muñoz, Luis E

2017-01-01

Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo . The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

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.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

PubMed

Lavrov, Andrey I; Kosevich, Igor A

2016-02-01

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions. © 2016 Wiley Periodicals, Inc.

Identifying Stem-like Cells Using Mitochondrial Membrane Potential | Center for Cancer Research

Cancer.gov

Therapies that are based on living cells promise to improve treatments for metastatic cancer and for many degenerative diseases. Lasting treatment of these maladies may require the durable persistence of cells. Long-term engraftment of cells – for months or years – and the generation of large numbers of progeny are characteristics of stem cells. Most approaches to isolate viable hematopoetic stem cells and therapeutically active T cells are based on immunophenotyping using highly multicolored flow cytometry. However, these methods do not directly measure the metabolic features of cells, which are known to be important in predicting cell fate.

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.

Cryopreservation of Human Mucosal Leukocytes.

PubMed

Hughes, Sean M; Shu, Zhiquan; Levy, Claire N; Ferre, April L; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C; Adams Waldorf, Kristina M; Veazey, Ronald S; Germann, Anja; von Briesen, Hagen; McElrath, M Juliana; Dezzutti, Charlene S; Sinclair, Elizabeth; Baker, Chris A R; Shacklett, Barbara L; Gao, Dayong; Hladik, Florian

2016-01-01

Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Artificial neural network associated to UV/Vis spectroscopy for monitoring bioreactions in biopharmaceutical processes.

PubMed

Takahashi, Maria Beatriz; Leme, Jaci; Caricati, Celso Pereira; Tonso, Aldo; Fernández Núñez, Eutimio Gustavo; Rocha, José Celso

2015-06-01

Currently, mammalian cells are the most utilized hosts for biopharmaceutical production. The culture media for these cell lines include commonly in their composition a pH indicator. Spectroscopic techniques are used for biopharmaceutical process monitoring, among them, UV-Vis spectroscopy has found scarce applications. This work aimed to define artificial neural networks architecture and fit its parameters to predict some nutrients and metabolites, as well as viable cell concentration based on UV-Vis spectral data of mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Off-line spectra of supernatant samples taken from batches performed at different dissolved oxygen concentrations in two bioreactor configurations and with two pH control strategies were used to define two artificial neural networks. According to absolute errors, glutamine (0.13 ± 0.14 mM), glutamate (0.02 ± 0.02 mM), glucose (1.11 ± 1.70 mM), lactate (0.84 ± 0.68 mM) and viable cell concentrations (1.89 10(5) ± 1.90 10(5) cell/mL) were suitably predicted. The prediction error averages for monitored variables were lower than those previously reported using different spectroscopic techniques in combination with partial least squares or artificial neural network. The present work allows for UV-VIS sensor development, and decreases cost related to nutrients and metabolite quantifications.

Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

PubMed Central

Panetta, J C; Evans, W E; Cheok, M H

2006-01-01

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance. PMID:16333308

The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

PubMed

Brunner, Matthias; Braun, Philipp; Doppler, Philipp; Posch, Christoph; Behrens, Dirk; Herwig, Christoph; Fricke, Jens

2017-07-01

Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m 3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploratory Development of Transparent Conductor Materials

DTIC Science & Technology

1975-03-01

silicon c,:ll) or b&%ckwa.U (CdS cells ) electrodes. ’Nn oxide and indium oxide are currently the best known transparent condudwtor materials and they are...From an investigation of its fundamental physical properties it was concluded that cadmium stannate is a viable candidate for transparent solar cell ...transparent backwall electrodes in CdS solar cells . A further objective has been the utilization of the high infrared reflectivity of cadmium

Status and Outlook for the U.S. Non-Automotive Fuel Cell Industry: Impacts of Government Policies and Assessment of Future Opportunities

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

Greene, David L.; Duleep, K. G.; Upreti, Girish

Non-Automotive Fuel Cell Industry, Government Policy and Future Opportunities. Fuel cells (FCs)are considered essential future energy technologies by developed and developing economies alike. Several countries, including the United States, Japan, Germany,and South Korea have established publicly funded R&D and market transformation programs to develop viable domestic FC industries for both automotive and nonautomotive applications.

Maladaptive hypertrophy after acute myocardial infarction positive effect of bone marrow-derived stem cell therapy on regional remodeling measured by cardiac MRI.

PubMed

Rolf, Andreas; Assmus, Birgit; Schächinger, Volker; Rixe, Johannes; Möllmann, Susanne; Möllmann, Helge; Dimmeler, Stefanie; Zeiher, Andreas M; Hamm, Christian W; Dill, Thorsten

2011-11-01

In the aftermath of myocardial infarction, increased loading conditions will trigger hypertrophy of viable myocardium. This in turn causes deterioration of regional contractility. Cardiac magnetic resonance imaging (cMRI) allows the exact differentiation of viable and infarcted myocardium and therefore the measurement of regional wall thickness and function. Bone marrow-derived stem cell (BMC) transfer has been shown to improve global function and remodeling. The present study examines the effect of BMC transfer on regional remodeling and function after myocardial infarction by cMRI. Fifty-four patients of the MR substudy of the REPAIR-AMI trial have been studied at baseline and 12-month follow-up. Enddiastolic wall thickness (EDWT) and wall thickening (WT%) have been measured on SSFP cine sequences. Enddiastolic wall thickness decreased in both placebo and BMC groups in viable as well as infarcted segments. The effect was largest in the pre-specified subgroup of patients below the median EF of 48.9% (infarcted segments -1.14 mm Placebo vs. -1.91 mm BMC, p for interaction 0.01, remote segments -0.19 mm Placebo vs. -0.94 mm BMC, p for interaction 0.00001). Corrected for baseline values BMC therapy yielded smaller EDWT at 12 months in infarcted and remote segments (infarcted 7.58 mm Placebo vs. 6.13 mm BMC p = 0.0001, remote 8.76 mm Placebo vs. 7.32 mm BMC, p = 0.0001). This was associated with better contractility within the infarcted segments among BMC patients (WT% 24.17% Placebo vs. 49.31% BMC, p = 0.0001). The WT% was inversely correlated with EDWT (r = -0.37, p = 0.0001). Bone marrow-derived stem cell therapy yields smaller EDWT when compared with placebo patients suggesting a positive effect on maladaptive hypertrophy of viable myocardium. This notion is supported by the enhanced regional contractility within the BMC group which is inversely correlated with EDWT.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Identifying viable regulatory and innovation pathways for regenerative medicine: a case study of cultured red blood cells.

PubMed

Mittra, J; Tait, J; Mastroeni, M; Turner, M L; Mountford, J C; Bruce, K

2015-01-25

The creation of red blood cells for the blood transfusion markets represents a highly innovative application of regenerative medicine with a medium term (5-10 year) prospect for first clinical studies. This article describes a case study analysis of a project to derive red blood cells from human embryonic stem cells, including the systemic challenges arising from (i) the selection of appropriate and viable regulatory protocols and (ii) technological constraints related to stem cell manufacture and scale up to clinical Good Manufacturing Practice (GMP) standard. The method used for case study analysis (Analysis of Life Science Innovation Systems (ALSIS)) is also innovative, demonstrating a new approach to social and natural science collaboration to foresight product development pathways. Issues arising along the development pathway include cell manufacture and scale-up challenges, affected by regulatory demands emerging from the innovation ecosystem (preclinical testing and clinical trials). Our discussion reflects on the efforts being made by regulators to adapt the current pharmaceuticals-based regulatory model to an allogeneic regenerative medicine product and the broader lessons from this case study for successful innovation and translation of regenerative medicine therapies, including the role of methodological and regulatory innovation in future development in the field. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects.

PubMed

Nejadnik, Hossein; Lenkov, Olga; Gassert, Florian; Fretwell, Deborah; Lam, Isaac; Daldrup-Link, Heike E

2016-05-13

Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects

NASA Astrophysics Data System (ADS)

Nejadnik, Hossein; Lenkov, Olga; Gassert, Florian; Fretwell, Deborah; Lam, Isaac; Daldrup-Link, Heike E.

2016-05-01

Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

A summary of data collected by the U.S. Geological Survey at Dasht-e-Nawar, Afghanistan, in support of lithium exploration, June-September 2014

USGS Publications Warehouse

Stillings, Lisa L.; Mack, Thomas J.; Chornack, Michael P.; Kalaly, Siddiq S.; Ahmadi, M. Idrees; Akbar, A. Qasim

2015-01-01

Interpretation of the data from the passive seismic survey suggests that the maximum sediment thickness in the northern lobe of the basin is 107 m, and in the southern lobe of the basin it is 173 m. Although the boreholes did not extend to the basin floor, the low Li concentration observed in pore waters does not suggest the presence of a viable Li brine resource at Dasht-e-Nawar.

Defense Expenditures in Pakistan: A Source of Stimulus for or Competition With the Private Sector

DTIC Science & Technology

1994-01-01

private sector activity, particularly investment, is the only viable option open to the authorities. It follows that for policy purposes the most important issue involves restructuring government expenditures and their financing in a manner that would provide the maximum inducement to private sector capital formation, especially in manufacturing. Operationally, this means finding an optimal balance between the government’s three most important budgetary items: defense, public consumption and infrastructural development. More importantly because

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

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

Patheja, Pooja, E-mail: pooja.patheja8@gmail.com; Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra; Sahu, Khageswar

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the releasemore » of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.« less

Exposure cell number during feeder cell growth-arrest by Mitomycin C is a critical pharmacological aspect in stem cell culture system.

PubMed

Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

2016-01-01

Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10μg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays. Copyright © 2016 Elsevier Inc. All rights reserved.

A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.

PubMed

Zhou, Baoqing; Chen, Bolu; Wu, Xin; Li, Fan; Yu, Pei; Aguilar, Zoraida P; Wei, Hua; Xu, Hengyi

2016-12-01

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×10 2 cfu/mL and 4.4×10 2 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×10 2 cfu/g was obtained in the presence of the Staphylococcus aureus (10 7 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin.

PubMed

Yarani, Reza; Mansouri, Kamran; Mohammadi-Motlagh, Hamid Reza; Bakhtiari, Mitra; Mostafaie, Ali

2013-06-01

Conventional isolation of epidermis from the dermis and disruption of epidermal sheets to liberate the cells, are performed using proteolytic enzymes such as thermolysin or collagenase. Selective population expansion of melanocytes is achieved by suppressing proliferation of keratinocytes and fibroblasts in epidermal cell suspensions, using phorbol esters and cholera toxin. Here, we introduce a new procedure for isolation of epidermal cells, using proteolytic activity of kiwi fruit actinidin, and also an improved growth medium for melanocytes in the presence of leukaemia inhibitory factor (LIF) and forskolin. Dermo-epidermal separation and epidermal sheet cell dispersion were performed using actinidin compared to conventional proteases including collagenase, thermolysin or trypsin. Thereafter, melanocyte culture was performed in two common media and one modified medium to discover optimization for these cells. We found that dermo-epidermal separation and epidermal sheet cell dispersion using kiwi fruit actinidin were considerably better than previously used methods, both from the aspect of less fibroblast and keratinocyte contamination, and of more viable native cells. Also, melanocytes proliferated better in phorbol ester- and cholera toxin-free proliferation medium supplemented with LIF and forskolin. Less contamination and higher numbers of viable cells were actinidin preferential for separation of epidermis and isolation of epidermal cells. Supplementation of LIF and forskolin to new medium increased proliferation potential of melanocytes in comparison to exogenous mitogens. © 2013 Blackwell Publishing Ltd.

Oligoanilines as a suppressor of polysulfide shuttling in lithium–sulfur batteries

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

Chang, Chi-Hao; Chung, Sheng-Heng; Han, Pauline

The migration of small polysulfide (LiPS) chains through the porous polymeric separators seriously jeopardizes the cycle life and energy density of lithium–sulfur (Li–S) batteries. Herein, we present a new concept in which an organic oligoaniline, amine-capped aniline trimer (ACAT), serves as an effective suppressor of the LiPS migration in Li–S cells. The strong interaction between LiPS and ACAT facilitates the formation of bulky ACAT–LiPS complexes (organoLiPS complexes), which are then size-selectively sieved by the porous polymeric separators employed in Li–S cells. Thus, the addition of ACAT significantly ameliorates the electrochemical performances of Li–S batteries due to suppressed LiPS migration. Thismore » new concept offers a viable strategy to achieve practically viable Li–S batteries.« less

Oligoanilines as a suppressor of polysulfide shuttling in lithium–sulfur batteries

DOE PAGES

Chang, Chi-Hao; Chung, Sheng-Heng; Han, Pauline; ...

2017-07-25

The migration of small polysulfide (LiPS) chains through the porous polymeric separators seriously jeopardizes the cycle life and energy density of lithium–sulfur (Li–S) batteries. Herein, we present a new concept in which an organic oligoaniline, amine-capped aniline trimer (ACAT), serves as an effective suppressor of the LiPS migration in Li–S cells. The strong interaction between LiPS and ACAT facilitates the formation of bulky ACAT–LiPS complexes (organoLiPS complexes), which are then size-selectively sieved by the porous polymeric separators employed in Li–S cells. Thus, the addition of ACAT significantly ameliorates the electrochemical performances of Li–S batteries due to suppressed LiPS migration. Thismore » new concept offers a viable strategy to achieve practically viable Li–S batteries.« less

Rapid Detection of Microbial Contamination Using a Microfluidic Device.

PubMed

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

2017-01-01

A portable kinetics fluorometer is developed to detect viable cells which may be contaminating various samples. The portable device acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye and plots it. The slope of the plot depends on the number of colony forming units per milliliter. The device uses resazurin as the indicator dye. Viable cells reduce resazurin to resorufin, which is more fluorescent. Photodiode is used to detect fluorescence change. The photodiode generated current proportional to the intensity of the light that reached it, and an op-amp is used in a transimpedance differential configuration to ensure amplification of the photodiode's signal. A microfluidic chip is designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the resazurin reduction rate. In tests, the E. coli-containing media are injected into the microfluidic chip and the device is able to detect the presence of E. coli in LB media based on the fluorescence change that occurred in the indicator dye. The device provides fast, accurate, and inexpensive means to optical detection of the presence of viable cells and could be used in the field in place of more complex methods, i.e., loop-meditated isothermal amplification of DNA (LAMP) to detect bacteria in pharmaceutical samples (Jimenez et al., J Microbiol Methods 41(3):259-265, 2000) or measuring the intrinsic fluorescence of the bacterial or yeast chromophores (Estes et al., Biosens Bioelectron 18(5):511-519, 2003).

Lab on a fabric: Mass producible and low-cost fabric filters for the high-throughput viable isolation of circulating tumor cells.

PubMed

Bu, Jiyoon; Kang, Yoon-Tae; Lee, Yong-Seok; Kim, Jeongsuk; Cho, Young-Ho; Moon, Byung-In

2017-05-15

Circulating tumor cells (CTCs) play an important role in estimating the presence and the metastatic relapse of tumor. Despite of their importance, isolation of viable CTCs is still struggling, since chemical or mechanical damages are unavoidable when separating less than 1000 of CTCs out of billions of other blood components. Furthermore, the current CTC isolation devices show low productivity, since they are produced after a series of complicated fabrication processes. Here, we present a low-cost and mass-producible fabric filters for the viable CTC isolation and the further molecular assay for profiling cancer-associated markers. The fabric filter, produced by polyester monofilament yarns, can be massively produced at extremely low-cost, by showing productivity of ~22filters/s at ~59filters/USD. By utilizing size-based sorting method, the fabric filter is capable to isolate both epithelial and mesenchymal CTCs, while slots with curved walls are beneficial for preventing the cell rupture by reducing 21.6% of mechanical stress compared to the conventional straight-walled slots. We applied our filter to 11 human blood samples and found that the number of CTCs was closely related to the expression level of Ki-67, which is highly overexpressed in proliferative tumors. The fabric filter might be an appropriate caner-screening tool in developing countries, where people suffer from insufficient healthcare services. Copyright © 2017 Elsevier B.V. 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

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer.

PubMed

Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Dong, Jianjun; Yin, Hua; Yu, Junhong; Chang, Zongming; Wang, Dongfeng

The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30μg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0μg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

An innovative approach to bioremediation of mercury contaminated soils from industrial mining operations.

PubMed

McCarthy, Damien; Edwards, Grant C; Gustin, Mae S; Care, Andrew; Miller, Matthieu B; Sunna, Anwar

2017-10-01

Soils contaminated with mercury (Hg) have proved expensive and logistically difficult to remediate. Research continues into finding suitable environmentally-friendly and efficient ways of achieving this end. Bioremediation is an option, which employs the strategies microorganisms have evolved to deal with Hg. One microbial strategy involves uptake and intracellular volatilisation of mercuric ions, which passively diffuse from the cell and back into the atmosphere. In this work, Pseudomonas veronii cells grown to stationary phase were immobilised in a xanthan gum-based biopolymer via encapsulation. The P. veronii-biopolymer mix was then coated onto natural zeolite granules. Zeolite immobilised cells remained viable for at least 16 weeks stored under ambient room temperature. Furthermore, the immobilised cells were shown to retain both viability and Hg volatilisation functionality after transportation from Australia to the USA, where they were applied to Hg contaminated soil. Maximum flux rates exceeded 10 μg Hg m 2  h -1 from mine tailings (≈7 mg kg -1  Hg with 50% v/v water). This was 4 orders of magnitude above background flux levels. It is envisioned that emitted gaseous elemental mercury (GEM) can be readily captured, and transformed back into metallic Hg, which can then be stored appropriately or recycled. This breaks the Hg cycle, as GEM is no longer translocated back to the atmospheric compartment. The immobilising excipients used in this research overcome many logistical issues with delivery of suitable microbial loads to locations of mercury contamination and presents a facile and inexpensive method of augmenting contaminated sites with selected microbial consortia for bioremediation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of Neurons from Immortalized Dental Pulp Stem Cells for the Study of Neurogenetic Disorders

PubMed Central

Urraca, Nora; Memon, Rawaha; El-Iyachi, Ikbale; Goorha, Sarita; Valdez, Colleen; Tran, Quynh T.; Scroggs, Reese; Miranda-Carboni, Gustavo A.; Donaldson, Martin; Bridges, Dave; Reiter, Lawrence T.

2015-01-01

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSC) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSC that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSC. We immortalized control DPSC using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSC share morphological and electrophysiological properties with non-immortalized DPSC. We also show that differentiation of DPSC into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NSRF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSC can be obtained from teeth stored for ≥72hrs, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSC for the study of disease. PMID:26599327

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders.

PubMed

Urraca, Nora; Memon, Rawaha; El-Iyachi, Ikbale; Goorha, Sarita; Valdez, Colleen; Tran, Quynh T; Scroggs, Reese; Miranda-Carboni, Gustavo A; Donaldson, Martin; Bridges, Dave; Reiter, Lawrence T

2015-11-01

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Fast and Efficient Stochastic Optimization for Analytic Continuation

DOE PAGES

Bao, Feng; Zhang, Guannan; Webster, Clayton G; ...

2016-09-28

In this analytic continuation of imaginary-time quantum Monte Carlo data to extract real-frequency spectra remains a key problem in connecting theory with experiment. Here we present a fast and efficient stochastic optimization method (FESOM) as a more accessible variant of the stochastic optimization method introduced by Mishchenko et al. [Phys. Rev. B 62, 6317 (2000)], and we benchmark the resulting spectra with those obtained by the standard maximum entropy method for three representative test cases, including data taken from studies of the two-dimensional Hubbard model. Genearally, we find that our FESOM approach yields spectra similar to the maximum entropy results.more » In particular, while the maximum entropy method yields superior results when the quality of the data is strong, we find that FESOM is able to resolve fine structure with more detail when the quality of the data is poor. In addition, because of its stochastic nature, the method provides detailed information on the frequency-dependent uncertainty of the resulting spectra, while the maximum entropy method does so only for the spectral weight integrated over a finite frequency region. Therefore, we believe that this variant of the stochastic optimization approach provides a viable alternative to the routinely used maximum entropy method, especially for data of poor quality.« less

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

Propulsion Investigation for Zero and Near-Zero Emissions Aircraft

NASA Technical Reports Server (NTRS)

Snyder, Christopher A.; Berton, Jeffrey J.; Brown, Gerald v.; Dolce, James L.; Dravid, Marayan V.; Eichenberg, Dennis J.; Freeh, Joshua E.; Gallo, Christopher A.; Jones, Scott M.; Kundu, Krishna P.;

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

Laser-induced fusion of human embryonic stem cells with optical tweezers

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

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

Single-cell transcriptome conservation in cryopreserved cells and tissues.

PubMed

Guillaumet-Adkins, Amy; Rodríguez-Esteban, Gustavo; Mereu, Elisabetta; Mendez-Lago, Maria; Jaitin, Diego A; Villanueva, Alberto; Vidal, August; Martinez-Marti, Alex; Felip, Enriqueta; Vivancos, Ana; Keren-Shaul, Hadas; Heath, Simon; Gut, Marta; Amit, Ido; Gut, Ivo; Heyn, Holger

2017-03-01

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

Massachusetts Fuel Cell Bus Project: Demonstrating a Total Transit Solution for Fuel Cell Electric Buses in Boston

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

The Federal Transit Administration's National Fuel Cell Bus Program focuses on developing commercially viable fuel cell bus technologies. Nuvera is leading the Massachusetts Fuel Cell Bus project to demonstrate a complete transit solution for fuel cell electric buses that includes one bus and an on-site hydrogen generation station for the Massachusetts Bay Transportation Authority (MBTA). A team consisting of ElDorado National, BAE Systems, and Ballard Power Systems built the fuel cell electric bus, and Nuvera is providing its PowerTap on-site hydrogen generator to provide fuel for the bus.

Self-Reported Ethnicity and Genetic Ancestry in Relation to Oral Cancer and Pre-Cancer in Puerto Rico

PubMed Central

Erdei, Esther; Sheng, Huiping; Maestas, Erika; Mackey, Amanda; White, Kirsten A.; Li, Lin; Dong, Yan; Taylor, Justin; Berwick, Marianne; Morse, Douglas E.

2011-01-01

Background Hispanics are known to be an extremely diverse and genetically admixed ethnic group. The lack of methodologies to control for ethnicity and the unknown admixture in complex study populations of Hispanics has left a gap in understanding certain cancer disparity issues. Incidence rates for oral and pharyngeal cancer (OPC) in Puerto Rico are among the highest in the Western Hemisphere. We conducted an epidemiological study to examine risk and protective factors, in addition to possible genetic susceptibility components, for oral cancer and precancer in Puerto Rico. Methodology/Principal Findings We recruited 310 Puerto Rico residents who had been diagnosed with either an incident oral squamous cell carcinoma, oral precancer, or benign oral condition. Participants completed an in-person interview and contributed buccal cells for DNA extraction. ABI Biosystem Taqman™ primer sets were used for genotyping 12 ancestry informative markers (AIMs). Ancestral group estimates were generated using maximum likelihood estimation software (LEADMIX), and additional principal component analysis was carried out to detect population substructures. We used unconditional logistic regression to assess the contribution of ancestry to the risk of being diagnosed with either an oral cancer or precancer while controlling for other potential confounders. The maximum likelihood estimates showed that study participants had a group average ancestry contribution of 69.9% European, 24.5% African, and 5.7% detectable Native American. The African and Indigenous American group estimates were significantly higher than anticipated. Neither self-identified ethnicity nor ancestry markers showed any significant associations with oral cancer/precancer risk in our study. Conclusions/Significance The application of ancestry informative markers (AIMs), specifically designed for Hispanics, suggests no hidden population substructure is present based on our sampling and provides a viable approach for the evaluation and control of ancestry in future studies involving Hispanic populations. PMID:21897864

Synthesis and characterization of nanosilver-silicone hydrogel composites for inhibition of bacteria growth.

PubMed

Helaly, F M; El-Sawy, S M; Hashem, A I; Khattab, A A; Mourad, R M

2017-02-01

Nanosilver-silicone hydrogel (NAgSiH) composites for contact lenses were synthesized to asses the antimicrobial effects. Silicone hydrogel (SiH) films were synthesized followed by impregnation in silver nitrate solutions (10, 20, 30, 40, 60, 80ppm) and in-situ chemical reduction of silver ions using sodium borohydride (NaBH 4 ). The silver nano particles (AgNPS) were identified by UV-vis absorption spectroscopy, Energy-dispersive X-ray spectroscopy (EDX) mapping and EDX spectrum. Physico-mechanical and chemical properties of NAgSIH films were studied. The antimicrobial effect of the hydrogels against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus was evaluated. The numbers of viable bacterial cells on NAgSiH surface or in solution compared to control SiH were examined. The NAgSiH films were successfully synthesized. FTIR results indicated that AgNPS had no effect on the bulk structure of the prepared SiH films. From TGA analysis, NAgSiH(R80) and SiH(R0) films had the same maximum decomposition temperature (404°C). UV-vis absorption spectroscopy and EDX mapping and spectrum emphasized that AgNPS were in spherical shape. The maximum absorption wavelength of NAgSiH films were around 400nm. The light transmittance decreased as the concentration of AgNPS increased, but still greater than 90% at wavelength around 555nm. The Young's modulus increased gradually from 1.06MPa of SiH(R0) to highest value 1.38MPa of NAgSiH(R80). AgNPS incorporated into SiH films reduced the bacterial cell growth and prevented colonization. Groups NAgSiH(R60,R80) demonstrated an excellent reduction in bacterial viability in solution and on the SiH surface. NAgSiH composites were successfully synthesized and possessed an excellent antimicrobial effects. Copyright © 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

PubMed

Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

2010-09-01

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

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.

Development of a Quantitative Competitive PCR Assay for Detection and Quantification of Escherichia coli O157:H7 Cells

PubMed Central

Li, Wenli; Drake, Mary Anne

2001-01-01

A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify Escherichia coli O157:H7 cells. From 103 to 108 CFU of E. coli O157:H7 cells/ml was quantified in broth or skim milk, and cell densities predicted by QC-PCR were highly related to viable cell counts (r2 = 0.99 and 0.93, respectively). QC-PCR has potential for quantitative detection of pathogenic bacteria in foods. PMID:11425755

Poly(ethylene glycol) hydrogel microstructures encapsulating living cells

NASA Technical Reports Server (NTRS)

Koh, Won-Gun; Revzin, Alexander; Pishko, Michael V.

2002-01-01

We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.

An investigation into the potential of low head hydro power in Northern Ireland for the production of electricity

NASA Astrophysics Data System (ADS)

Redpath, David; Ward, Michael J.

2017-07-01

The maximum exploitable potential for low head hydroelectric sites (gross head≤10 m) in Northern Ireland (NI) was determined as 12.07 MW using a simple payback analysis for 304 potential sites investigated to derive a classification scheme in terms of economic viability. A techno-economic analysis with validated numerical models from previous research estimated the capital investment required for the development of a hydroelectric plant, using the low head Michell-Banki cross flow turbine, for the 304 sites investigated. The number of potentially viable sites in NI for low head hydro ranged from 198 to 286 with an estimated installed capacity ranging from 11.95 to 12.05 MW. Sites with a limited installed capacity were not economically viable unless increased government support in the form of longer term (25-50 years) low interest loans as well as the current (Renewables Obligations Certificates) Renewables Obligation Certificates scheme is provided and sustained.

Potential of border tax adjustments to deter free riding in international climate agreements

NASA Astrophysics Data System (ADS)

Burcu Irfanoglu, Zeynep; Sesmero, Juan P.; Golub, Alla

2015-02-01

The objective of this study is to conduct assessment of the hypothesis that trade sanctions in the form of border tax adjustments (BTAs) used by the United States against China, constitute a viable enforcement mechanism to sustain compliance with a range of emissions taxes in the context of agreements to curb global emissions of greenhouse gases (GHGs). The performance of BTAs is then compared with those of punitive tariffs on the basis of the range of emission taxes that can be successfully enforced by their implementation. Results show that BTAs are a viable enforcement mechanism for international GHG mitigation agreements. However the maximum level of carbon tax that can be enforced varies dramatically with (1) the marginal damage of pollution perceived by Chinese authorities, and (2) the legal limitations that GATT rules may impose on BTAs. Finally, while BTAs seem a promising enforcement mechanism in the context of climate agreements, punitive tariffs seem to be capable of supporting a much stricter environmental target.

Biosorption of reactive dye from textile wastewater by non-viable biomass of Aspergillus niger and Spirogyra sp.

PubMed

Khalaf, Mahmoud A

2008-09-01

The potential of Aspergillus niger fungus and Spirogyra sp., a fresh water green algae, was investigated as a biosorbents for removal of reactive dye (Synazol) from its multi component textile wastewater. The results showed that pre-treatment of fungal and algal biomasses with autoclaving increased the removal of dye than pre-treatment with gamma-irradiation. The effects of operational parameters (pH, temperature, biomass concentration and time) on dye removal were examined. The results obtained revealed that dried autoclaved biomass of A. niger and Spirogyra sp. exhibited maximum dye removal (88% and 85%, respectively) at pH3, temperature 30 degrees C and 8 gl(-1)(w/v) biomass conc. after 18h contact time. The stability and efficiency of both organisms in the long-term repetitive operation were also investigated. The results showed that the non-viable biomasses possessed high stability and efficiency of dye removal over 3 repeated batches.

Developing integrated patient pathways using hybrid simulation

NASA Astrophysics Data System (ADS)

Zulkepli, Jafri; Eldabi, Tillal

2016-10-01

Integrated patient pathways includes several departments, i.e. healthcare which includes emergency care and inpatient ward; intermediate care which patient(s) will stay for a maximum of two weeks and at the same time be assessed by assessment team to find the most suitable care; and social care. The reason behind introducing the intermediate care in western countries was to reduce the rate of patients that stays in the hospital especially for elderly patients. This type of care setting has been considered to be set up in some other countries including Malaysia. Therefore, to assess the advantages of introducing this type of integrated healthcare setting, we suggest develop the model using simulation technique. We argue that single simulation technique is not viable enough to represent this type of patient pathways. Therefore, we suggest develop this model using hybrid techniques, i.e. System Dynamics (SD) and Discrete Event Simulation (DES). Based on hybrid model result, we argued that the result is viable to be as references for decision making process.

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®

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

PubMed

Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

2017-09-01

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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.

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.

Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis.

PubMed

Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A; Soni, Nipunjot; Mandal, Raju K; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y; Govender, Thavendran; Kruger, Hendrik G; Jawed, Arshad

2016-01-01

For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD 600 nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD 600 nm ): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties.

Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis

PubMed Central

Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A.; Soni, Nipunjot; Mandal, Raju K.; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y.; Govender, Thavendran; Kruger, Hendrik G.; Jawed, Arshad

2016-01-01

For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600 nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600 nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties. PMID:27920762

Genetic analysis of oocyte and embryo production traits in Guzerá breed donors and their associations with age at first calving.

PubMed

Perez, B C; Peixoto, M G C D; Bruneli, F T; Ramos, P V B; Balieiro, J C C

2016-04-26

The objective of this study was to estimate variance components for oocyte and embryo production traits in Guzerá breed female donors, and investigate their associations with age at first calving (AFC). The traits analyzed were the number of viable oocytes (NOV), the number of grade I oocytes (NGI), the number of cleaved embryos (NCLV), and viable embryos produced (NEMB), and the percentages of viable oocytes (POV), grade I oocytes (PGI), cleaved embryos (PCLV), and viable embryos (PEMB). Data were obtained from 5173 ovary puncture and in vitro fertilization (IVF) sessions using 1080 Guzerá female donors of different ages, occurred from March 2005 to July 2013. Variables were log-transformed (logeX+1) prior to analysis. (Co)variance components were estimated by restricted maximum likelihood (REML), using one- and two-trait animal models. Permanent environment and IVF sire (father of the embryos) random effects were included. Estimated heritabilities for NOV, NGI, NCLV, NEMB, POV, PGI, PCLV, and PEMB were 0.19, 0.08, 0.16, 0.14, 0.04, 0.03, 0.01, and 0.07, respectively. Repeatabilities for count traits (NOV, NGI, NCLV, and NEMB) varied from 0.14 and 0.32, higher than estimated for percentage traits (POV, PGI, PCLV, and PEMB), which varied from 0.01 to 0.08. Selection for NOV may be more appropriate in breeding programs than selection for NEMB, because of its strong genetic correlation (0.68) with NEMB and its greater time- and cost-effectiveness. AFC was only weakly associated with the oocyte and embryo production traits, which indicates that there would be no effect on AFC when selecting for these traits.

Aging of Escherichia coli

PubMed Central

Clifton, C. E.

1966-01-01

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905–912. 1966.—The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C14-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C14O2 than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance. PMID:5332874

Enhancement of the neuroprotective activity of Hericium erinaceus mycelium co-cultivated with Allium sativum extract.

PubMed

Kim, Myong Ki; Choi, Woon Yong; Lee, Hyeon Yong

2015-02-01

This study investigated the neuroprotective effects of Hericium erinaceus mycelium enriched with garlic extract (HGE) on rat pheochromocytoma nerve cells (PC12). The survival rates of the PC12 nerve cells and the neurite-bearing cells after the addition of HGE were estimated as 3.5 × 10(3) viable cells/ml and 2.3 × 10(3) viable cells/ml, respectively, which were 50% and 30% higher, respectively, compared with the untreated group. For the in vivo ischemia experiments, after treatment with the HGE extract, the hippocampal CA1 region was more strongly stained (>20%) than the control group, and the HGE extract also promoted higher staining levels than HFB, HM and HGEF, and even the garlic extract. This result indicates that HGE must have neuroprotective effects. Furthermore, HGE greatly decreased p21 gene expression to approximately 70% of the control and decreased p21 gene expression to even lower levels compared with HM, HGEF and the garlic extract. This work suggests that a synergistic effect of the H. erinaceus mycelium and the garlic extract (mainly allicin) exist because the amount of allicin in HGE (5.81 µg/ml) was lower than the garlic extract itself (6.89 µg/ml).

SURVIVAL OF SALMONELLA SPECIES IN RIVER WATER.

EPA Science Inventory

The survival of four Salmonella strains in river water microcosms was monitored using culturing techniques, direct counts, whole cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytrometry. Plate counts of...

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.

MK-2206, an AKT Inhibitor, Promotes Caspase-Independent Cell Death and Inhibits Leiomyoma Growth

PubMed Central

Sefton, Elizabeth C.; Qiang, Wenan; Serna, Vanida; Kurita, Takeshi; Wei, Jian-Jun; Chakravarti, Debabrata

2013-01-01

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs. PMID:24002033

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness▿

PubMed Central

Passerat, Julien; Got, Patrice; Dukan, Sam; Monfort, Patrick

2009-01-01

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death. PMID:19525274

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

PubMed

Patheja, Pooja; Sahu, Khageswar

2017-06-15

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Mammalian Cell Tissue Culture Techniques.

PubMed

Phelan, Katy; May, Kristin M

2016-06-01

Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of 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. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Cryopreservation of Human Mucosal Leukocytes

PubMed Central

Shu, Zhiquan; Levy, Claire N.; Ferre, April L.; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C.; Adams Waldorf, Kristina M.; Veazey, Ronald S.; Germann, Anja; von Briesen, Hagen; McElrath, M. Juliana; Dezzutti, Charlene S.; Sinclair, Elizabeth; Baker, Chris A. R.; Shacklett, Barbara L.; Gao, Dayong; Hladik, Florian

2016-01-01

Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. Methods and Findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes. PMID:27232996

Control of the red tide dinoflagellate Cochlodinium polykrikoides by ozone in seawater.

PubMed

Shin, Minjung; Lee, Hye-Jin; Kim, Min Sik; Park, Noh-Back; Lee, Changha

2017-02-01

The inactivation of C. polykrikoides, a red tide dinoflagellate, by ozonation was investigated in seawater by monitoring numbers of viable and total cells. Parameters affecting the inactivation efficacy of C. polykrikoides such as the ozone dose, initial cell concentration, pH, and temperature were examined. The viable cell number rapidly decreased in the initial stage of the reaction (mostly in 1-2 min), whereas the decrease in total cell number was relatively slow and steady. Increasing ozone dose and decreasing initial cell concentration increased the inactivation efficacy of C. polykrikoides, while increasing pH and temperature decreased the cell inactivation efficacy. The addition of humic acid (a promoter for the ozone decomposition) inhibited the inactivation of C. polykrikoides, whereas bicarbonate ion (an inhibitor for the ozone decomposition) accelerated the C. polykrikoides inactivation. Observations regarding the effects of pH, temperature, humic acid, and bicarbonate ion collectively indicate that the inactivation of C. polykrikoides by ozonation is mainly attributed to oxidative cell damages by molecular ozone, rather than by hydroxyl radical, produced during the ozone decomposition. At high ozone dose (e.g., 5 mg/L), hypobromous acid formed by the reaction of bromide with ozone may partially contribute to cell inactivation. The use of ozone of less than 1 mg/L produced 0.75-2.03 μg/L bromate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity

NASA Technical Reports Server (NTRS)

Evans, H. H.; Horng, M. F.; Evans, T. E.; Jordan, R.; Schwartz, J. L.

2001-01-01

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.

PubMed

Volokhov, Dmitriy V; Graham, Laurie J; Brorson, Kurt A; Chizhikov, Vladimir E

2011-01-01

Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The "gold standard" microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely 'go/no-go' decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates. Published by Elsevier Ltd.

Antimicrobial resistant coliform bacteria in the Gomti river water and determination of their tolerance level.

PubMed

Akhter, Asma; Imran, Mohd; Akhter, Firoz

2014-01-01

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among coliform in the Gomti river water samples was investigated. The coliform populations were isolated on Mac Conky and eosin methylene blue (EMB) agar plates supplemented with antibiotics. The incidence of resistance among the coliform population varied considerably in different drug and water sampling sites. Coliform bacteria showed lower drug resistant viable count in sampling site-III (receiving treated wastewater) as compared to more polluted site-I and site-II. Viable count of coliform population obtained on both medium was recorded higher against erythromycin from sampling site-III. Lower viable count of coliforms was recorded against tetracycline in site-II and III. Similar resistance pattern was obtained in the frequency of E. coli and Enterobacter species from all the three sampling sites. Percentage of antibiotic resistant E. coli was observed higher than Enterobacter spp among the total coliforms against all antibiotics tested without Erythromycin and penicillin in site-I and II respectively. Isolates of E. coli and Enterobacter spp. showed their tolerance level (MIC) in the range of 2-100 against the antibiotics tested. Maximum number of isolates of both genus exhibited their MICs at lower concentration range 2-5µg/ml against ciprofloxacin, tetracyclin and amoxycillin. EMB - Eosin methylene blue, IMViC tests - Indole, Methyl Red, Voges Proskauer and Citrate Utilization Tests, MIC - Minimum inhibitory concentration.

Antimicrobial resistant coliform bacteria in the Gomti river water and determination of their tolerance level

PubMed Central

Akhter, Asma; Imran, Mohd; Akhter, Firoz

2014-01-01

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among coliform in the Gomti river water samples was investigated. The coliform populations were isolated on Mac Conky and eosin methylene blue (EMB) agar plates supplemented with antibiotics. The incidence of resistance among the coliform population varied considerably in different drug and water sampling sites. Coliform bacteria showed lower drug resistant viable count in sampling site-III (receiving treated wastewater) as compared to more polluted site-I and site-II. Viable count of coliform population obtained on both medium was recorded higher against erythromycin from sampling site-III. Lower viable count of coliforms was recorded against tetracycline in site-II and III. Similar resistance pattern was obtained in the frequency of E. coli and Enterobacter species from all the three sampling sites. Percentage of antibiotic resistant E. coli was observed higher than Enterobacter spp among the total coliforms against all antibiotics tested without Erythromycin and penicillin in site-I and II respectively. Isolates of E. coli and Enterobacter spp. showed their tolerance level (MIC) in the range of 2-100 against the antibiotics tested. Maximum number of isolates of both genus exhibited their MICs at lower concentration range 2-5µg/ml against ciprofloxacin, tetracyclin and amoxycillin. Abbreviations EMB - Eosin methylene blue, IMViC tests - Indole, Methyl Red, Voges Proskauer and Citrate Utilization Tests, MIC - Minimum inhibitory concentration. PMID:24966515

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.

Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.

PubMed

Okumura, Naoki; Ueno, Morio; Koizumi, Noriko; Sakamoto, Yuji; Hirata, Kana; Hamuro, Junji; Kinoshita, Shigeru

2009-08-01

The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. MCECs of cynomolgus monkeys were cultured in a medium containing 10 microM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

Model Study To Assess Softwood Hemicellulose Hydrolysates as the Carbon Source for PHB Production in Paraburkholderia sacchari IPT 101.

PubMed

Dietrich, Karolin; Dumont, Marie-Josée; Schwinghamer, Timothy; Orsat, Valérie; Del Rio, Luis F

2018-01-08

Softwood hemicellulose hydrolysates are a cheap source of sugars that can be used as a feedstock to produce polyhydroxybutyrates (PHB), which are biobased and compostable bacterial polyesters. To assess the potential of the hemicellulosic sugars as a carbon source for PHB production, synthetic media containing softwood hemicellulose sugars (glucose, mannose, galactose, xylose, arabinose) and the potentially inhibitory lignocellulose degradation products (acetic acid, 5-hydroxymethylfurfural (HMF), furfural, and vanillin) were fermented with the model strain Paraburkholderia sacchari IPT 101. Relative to pure glucose, individual fermentation for 24 h with 20 g/L mannose or galactose exhibited maximum specific growth rates of 97% and 60%, respectively. On the other hand, with sugar mixtures of glucose, mannose, galactose, xylose, and arabinose, the strain converted all sugars simultaneously to reach a maximum PHB concentration of 5.72 g/L and 80.5% PHB after 51 h. The addition of the inhibitor mixture at the following concentration, sodium acetate (2.11 g/L), HMF (0.67 g/L), furfural (0.66 g/L), and vanillin (0.93 g/L), to the sugar mixture stopped the growth entirely within 24 h. Individually, the inhibitors either had no effect or only reduced growth. Moreover, it was found that a bacterial inoculum with high initial cell density (optical density, OD ≥ 5.6) could overcome the growth inhibition to yield an OD of 13 within 24 h. Therefore, softwood hemicellulose sugars are viable carbon sources for PHB production. Nevertheless, real softwood hemicellulose hydrolysates need detoxification or a high inoculum to overcome inhibitory effects and allow bacterial growth.

A Transient Cell-shielding Method for Viable MSC Delivery Within Hydrophobic Scaffolds Polymerized in situ

DTIC Science & Technology

2015-03-27

cell nutrients and wastes than swollen hydrogels. While hydrophobic biomaterials such as PUR provide a generalizable, biodegradable platform for tissue...Culture of cellularized PUR scaffolds Rat BMSCs were stained with a cytoplasmic dye (VyBrant® CFDA SE Cell Tracer Kit, Life Technologies, per the...growth, adipogenic, or osteo- genic media for up to 21 days and stained with Oil Red O or Alizarin Red S. After staining, dyes were dissolved in

Multi-parameter flow cytometry as a process analytical technology (PAT) approach for the assessment of bacterial ghost production.

PubMed

Langemann, Timo; Mayr, Ulrike Beate; Meitz, Andrea; Lubitz, Werner; Herwig, Christoph

2016-01-01

Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes-called bacterial ghosts (BGs)-that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.

Generation of Viable Cell and Biomaterial Patterns by Laser Transfer

NASA Astrophysics Data System (ADS)

Ringeisen, Bradley

2001-03-01

In order to fabricate and interface biological systems for next generation applications such as biosensors, protein recognition microarrays, and engineered tissues, it is imperative to have a method of accurately and rapidly depositing different active biomaterials in patterns or layered structures. Ideally, the biomaterial structures would also be compatible with many different substrates including technologically relevant platforms such as electronic circuits or various detection devices. We have developed a novel laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), that is able to direct write patterns and three-dimensional structures of numerous biologically active species ranging from proteins and antibodies to living cells. Specifically, we have shown that MAPLE DW is capable of forming mesoscopic patterns of living prokaryotic cells (E. coli bacteria), living mammalian cells (Chinese hamster ovaries), active proteins (biotinylated bovine serum albumin, horse radish peroxidase), and antibodies specific to a variety of classes of cancer related proteins including intracellular and extracellular matrix proteins, signaling proteins, cell cycle proteins, growth factors, and growth factor receptors. In addition, patterns of viable cells and active biomolecules were deposited on different substrates including metals, semiconductors, nutrient agar, and functionalized glass slides. We will present an explanation of the laser-based transfer mechanism as well as results from our recent efforts to fabricate protein recognition microarrays and tissue-based microfluidic networks.

Electronic paramagnetic resonance investigation of the activity of Origanum vulgare L. essential oil on the Listeria monocytogenes membrane.

PubMed

Serio, A; Chiarini, M; Tettamanti, E; Paparella, A

2010-08-01

To evaluate the effect of oregano essential oil on Listeria monocytogenes cytoplasmic membrane. Nitroxide free-radical Electron Paramagnetic Resonance was applied on L. monocytogenes after 30 min exposure to oregano essential oil concentrations ranging from 0 to 1.25%. The impact of essential oil on the number of viable cells was evaluated by plate count. Growth dynamics of survivors in BHI and TSB were evaluated by turbidometry. After exposure to essential oil concentrations up to 0.50%, the membrane fluidity was changed and its order increased. When L. monocytogenes was exposed to higher concentrations, membrane order parameters slightly returned to the values of untreated cells. However, when the cells were exposed to EO in the presence of sodium azide, which impairs energy metabolism, the membrane fluidity was progressively enhanced, even at the lowest EO concentration (0.25%). Microbiological analyses confirmed a progressive reduction of viable count, at increasing essential oil concentrations. Both in BHI and TSB, the Lag phase length increased in treated cells with respect to controls, suggesting a cell damage recovery. The combined approach including microbiological and EPR analyses provided relevant information on membrane modification and cell response to essential oils. EPR approach was demonstrated to be an effective and helpful tool to comprehend the modifications exerted by essential oil on the bacterial membrane.

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.

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.

First study on the formation and resuscitation of viable but nonculturable state and beer spoilage capability of Lactobacillus lindneri.

PubMed

Liu, Junyan; Li, Lin; Li, Bing; Peters, Brian M; Deng, Yang; Xu, Zhenbo; Shirtliff, Mark E

2017-06-01

This study aimed to investigate the spoilage capability of Lactobacillus lindneri during the induction and resuscitation of viable but nonculturable (VBNC) state. L. lindneri strain was identified by sequencing the PCR product (amplifying 16S rRNA gene) using ABI Prism 377 DNA Sequencer. During the VBNC state induction by low temperature storage and beer adaption, total, culturable, and viable cells were assessed by acridine orange direct counting, plate counting, and Live/Dead BacLight bacterial viability kit, respectively. Organic acids and diacetyl concentration were measured by reversed-phase high performance liquid chromatography and head dpace gas chromatography, respectively. VBNC state of L. lindneri was successfully induced by both beer adaption and low temperature storage, and glycerol frozen stock was the optimal way to maintain the VBNC state. Addition of catalase was found to be an effective method for the resuscitation of VBNC L. lindneri cells. Furthermore, spoilage capability remained similar during the induction and resuscitation of VBNC L. lindneri. This is the first report of induction by low temperature storage and resuscitation of VBNC L. lindneri strain, as well as the first identification of spoilage capability of VBNC and resuscitated L. lindneri cells. This study indicated that the potential colonization of L. lindneri strain in brewery environment, formation and resuscitation of VBNC state, as well as maintenance in beer spoilage capability, may be an important risk factor for brewery environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

PubMed

Swift, Benjamin M C; Convery, Thomas W; Rees, Catherine E D

2016-10-02

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Vibrio cholerae Classical Biotype Is Converted to the Viable Non-Culturable State when Cultured with the El Tor Biotype

PubMed Central

Pradhan, Subhra; Mallick, Sanjaya K.; Chowdhury, Rukhsana

2013-01-01

A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains. PMID:23326443