Effects of the antimicrobial peptide protegrine 1 on sperm viability and bacterial load of boar seminal doses.

PubMed

Sancho, S; Briz, M; Yeste, M; Bonet, S; Bussalleu, E

2017-10-01

The presence of bacteria adversely affects boar sperm quality of seminal doses intended for artificial insemination. Currently, the most common measure to prevent bacteriospermia is the addition of antibiotics in semen extenders; however, mounting evidence shows that microbial resistance exists. A promising alternative to replace antibiotics are antimicrobial peptides. In this study, the effects of the antimicrobial peptide protegrine 1 (PG1) on the sperm viability and bacterial load of boar seminal doses were evaluated. Three different concentrations of PG1 (2.5, 25 and 100 μg/ml) were tested over a storing period of 10 days at 17°C. Sperm viability was analysed by fluorescence microscopy (SYBR14/propidium iodide), and bacterial load was assessed by plating 100 μl of each sample in Luria-Bertani medium and incubated at 37°C for 72 hr under aerobic conditions. Protegrine 1 was effective in controlling the bacterial load in all the assessed concentrations (p < .05), reaching the lowest values at the highest concentrations of the antimicrobial peptide. Nevertheless, sperm viability was significantly (p < .05) reduced by all tested concentrations of this peptide, the most cytotoxic effects being observed at the highest PG1 concentrations. Despite these results, the use of PG1 as an alternative to antibiotics cannot be totally discarded, as further studies using the truncated form of this peptide are needed. © 2017 Blackwell Verlag GmbH.

Improvement in the Viability of Cryopreserved Cells by Microencapsulation

NASA Astrophysics Data System (ADS)

Matsumoto, Yoshifumi; Morinaga, Yukihiro; Ujihira, Masanobu; Oka, Kotaro; Tanishita, Kazuo

The advantages of microencapsulated cells over those of suspended cells were evaluated for improving viability in cryopreservation. Rat pheochromocytoma (PC12) cells were selected as the test biological cells and then microencapsulated in alginate-polylysine-alginate membranes. These microencapsulated PC12 cells were frozen by differential scanning calorimetry (DSC) at various cooling rates, from 0.5 to 10°C/min. Their latent heat was measured during freezing from 4 to -80°C. The post-thaw viability was evaluated by dopamine-concentration measurement and by trypan blue exclusion assay. Results showed that at cooling rates of 0.5 and 1°C/min, the latent heat of microencapsulated PC12 cells was lower than that of suspended cells. This lower latent heat is caused by the fact that the extra-microcapsule froze and the intra-capsule remained unfrozen due to the formation of ice crystals in the extra-capsule space. The post-thaw viability of microencapsulated PC12 cells was improved when the cooling rate was 0.5 or 1°C/min, compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, maintaining the intra-microcapsules in an unfrozen state during freezing reduces the solution effect and thus improves the post-thaw viability.

Bacterial Cell Mechanics.

PubMed

Auer, George K; Weibel, Douglas B

2017-07-25

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Sonication reduces the attachment of Salmonella Typhimurium ATCC 14028 cells to bacterial cellulose-based plant cell wall models and cut plant material.

PubMed

Tan, Michelle S F; Rahman, Sadequr; Dykes, Gary A

2017-04-01

This study investigated the removal of bacterial surface structures, particularly flagella, using sonication, and examined its effect on the attachment of Salmonella Typhimurium ATCC 14028 cells to plant cell walls. S. Typhimurium ATCC 14028 cells were subjected to sonication at 20 kHz to remove surface structures without affecting cell viability. Effective removal of flagella was determined by staining flagella of sonicated cells with Ryu's stain and enumerating the flagella remaining by direct microscopic counting. The attachment of sonicated S. Typhimurium cells to bacterial cellulose-based plant cell wall models and cut plant material (potato, apple, lettuce) was then evaluated. Varying concentrations of pectin and/or xyloglucan were used to produce a range of bacterial cellulose-based plant cell wall models. As compared to the non-sonicated controls, sonicated S. Typhimurium cells attached in significantly lower numbers (between 0.5 and 1.0 log CFU/cm 2 ) to all surfaces except to the bacterial cellulose-only composite without pectin and xyloglucan. Since attachment of S. Typhimurium to the bacterial cellulose-only composite was not affected by sonication, this suggests that bacterial surface structures, particularly flagella, could have specific interactions with pectin and xyloglucan. This study indicates that sonication may have potential applications for reducing Salmonella attachment during the processing of fresh produce. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell viability monitoring using Fano resonance in gold nanoslit array

NASA Astrophysics Data System (ADS)

Wu, Shu-Han; Hsieh, Shu-Yi; Lee, Kuang-Li; Weng, Ruei-Hung; Chiou, Arthur; Wei, Pei-Kuen

2013-09-01

Cell viability is a crucial issue in biological research. We present label-free monitoring of adhesion cells viability by gold nanoslits-based Fano resonance biosensors. Plastic multiple wells with gold nanoslits substrate were made using a thermal nanoimprint method. Adhesion cells in the wells were treated with doxorubicin for inducing cell death and compared with conventional colorimetric assay. The nanoslits method shows better respones of viability tests under low concentration and short interaction time due to its high surface sensitivies. The vinculin labelling indicates that the measured signals are in good agreement with the adhesion abilities of cells.

The effect of ultrasound-related stimuli on cell viability in microfluidic channels

PubMed Central

2013-01-01

Background In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Results Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. Conclusion The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that

The effect of ultrasound-related stimuli on cell viability in microfluidic channels.

PubMed

Ankrett, Dyan N; Carugo, Dario; Lei, Junjun; Glynne-Jones, Peter; Townsend, Paul A; Zhang, Xunli; Hill, Martyn

2013-06-28

In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Bacterial Viability within Dental Calculus: An Untrodden, Inquisitive Clinico-Patho- Microbiological Research.

PubMed

Gupta, Swati; Jain, P K; Kumra, Madhumani; Rehani, Shweta; Mathias, Yulia; Gupta, Ramakant; Mehendiratta, Monica; Chander, Anil

2016-07-01

Chronic inflammatory periodontal diseases i.e. gingivitis and periodontitis are one of the most common afflictions faced by human beings. Dental plaque, which is a pool of pathogenic microorganisms, remains to be current mainstay in etiopathogenesis. Dental calculus, which is a mineralized product of this plaque remains ignored and is considered merely as an ash heap of minor significance. However, the intriguing array in disease etiopathogenesis bulldozed researchers to suspect the role of calculus in disease chrysalis but still the viability of bacteria inside calculus and thus its pathogenicity remains an intricacy; the answer to which lies in the Pandora's Box. The present study was undertaken to investigate the viability of bacteria within dental calculus along with their identification. Also, to classify dental calculus on the basis of mineralization and to observe the variation of viable microflora found in dental calculus with the extent of mineralization and disease severity. A total of 60 samples were obtained, by harvesting two samples of supragingival calculus from each patient having chronic inflammatory periodontal disease. These samples were divided into two groups (Group A and Group B). Samples of Group A were kept non-irradiated and samples of Group B were exposed to UV radiation. The samples were categorized into less, moderately and highly mineralized according to the force required for crushing them. All the crushed calculus samples were then divided into three parts. These were used for dark-field microscopy, gram staining and bacterial cultures. Bacterial identification of the cultures obtained was also carried out by performing various biochemical assays. The present study revealed the presence of motile spirochaetes within the samples under dark-field microscope. Gram staining revealed presence of numerous gram positive cocci and gram negative bacilli. Bacterial cultures showed growth of variety of aerobic and capnophilic microorganisms. The

Viability of HEK 293 cells on poly-β-hydroxybutyrate (PHB) biosynthesized from a mutant Azotobacter vinelandii strain. Cast film and electrospun scaffolds.

PubMed

Romo-Uribe, Angel; Meneses-Acosta, Angelica; Domínguez-Díaz, Maraolina

2017-12-01

Sterilization, cytotoxicity and cell viability are essential properties defining a material for medical applications and these characteristics were investigated for poly(β-hydroxybutyrate) (PHB) of 230kDa obtained by bacterial synthesis from a mutant strain of Azotobacter vinelandii. Cell viability was investigated for two types of PHB scaffolds, solution cast films and non-woven electrospun fibrous membranes, and the efficiency was compared against a culture dish. The biosynthesized PHB was sterilized by ultraviolet radiation and autoclave, it was found that the thermal properties and intrinsic viscosity remained unchanged indicating that the sterilization methods did not degrade the polymer. Sterilized scaffolds were then seeded with human embryonic kidney 293 (HEK 293) cells to evaluate the cytotoxic response. The cell viability of these cells was evaluated for up to six days, and the results showed that the cell morphology was normal, with no cytotoxic effects. The films and electrospun membranes exhibited over 95% cell viability whereas the viability in culture dishes reached only ca. 90%. The electrospun membrane, however, exhibited significantly higher cell density than the cast film suggesting that the fibrous morphology enables better nutrients transfer. The results indicate that the biosynthesized PHB stands UV and autoclave sterilization methods, it is biocompatible and non-toxic for cell growth of human cell lines. Furthermore, cell culture for up to 18 days showed that 62% and 90% of mass was lost for the film and fibrous electrospun scaffold, respectively. This is a favorable outcome for use in tissue engineering where material degradation, as tissue regenerates, is desirable. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of 99 GHz continuous millimeter wave electro-magnetic radiation on E. coli viability and metabolic activity.

PubMed

Cohen, Irena; Cahan, Rivka; Shani, Gad; Cohen, Eyal; Abramovich, Amir

2010-05-01

To investigate time exposure dependence of continuous millimeter wave (CW) 99 GHz radiation on Escherichia coli bacterial cell viability and metabolic activity. Suspensions of E. coli bacterial cells with an optical density of OD(660 nm) = 0.1 were used for viability tests and OD(660 nm) = 1.0 for metabolic activity tests. These suspensions were exposed to 99 GHz CW electromagnetic radiation, generated by a Backward Wave Oscillator (BWO) tube base instrument with a horn antenna at the BWO exit, to obtain an almost ideal Gaussian beam. Calculations of the Gaussian beam show that a power of 0.2 mW/cm(2) was obtained at the bacterial plane. The experimental results show that 1 hour of exposure to 99 GHz CW electromagnetic radiation had no effect on E. coli viability and colony characterisation. In 19 h of radiation, the number of colonies forming units was half order of magnitude higher than the sham-exposed and the control. However, 19 h of exposure did not affect the E. coli metabolic activity. Exposure of E. coli to millimeter wave (MW) CW 99 GHz radiation for a short period did not affect the viability of E. coli bacterial cells. However, exposure for 19 h caused a slight proliferation but did not influence the metabolic activities of about 90 biochemical reactions that were examined. Hence, we assume that the slight proliferation (half order of magnitude) after 19 h of exposure dose not have a biological meaning.

An approach for cell viability online detection based on the characteristics of lensfree cell diffraction fingerprint.

PubMed

Li, Guoxiao; Zhang, Rongbiao; Yang, Ning; Yin, Changsheng; Wei, Mingji; Zhang, Yecheng; Sun, Jian

2018-06-01

To overcome the drawbacks such as low automation and high cost, an approach for cell viability online detection is proposed, based on the extracted lensfree cell diffraction fingerprint characteristics. The cell fingerprints are acquired by a constructed large field-of-view (FOV) diffraction imaging platform without any lenses. The approach realizes distinguishing live and dead cells online and calculating cell viability index based on the number of live cells. With theoretical analysis and simulation, diffraction fingerprints of cells with different morphology are simulated and two characteristics are discovered to be able to reflect cell viability status effectively. Two parameters, fringe intensity contrast (FIC) and fringe dispersion (FD), are defined to quantify these two characteristics. They are verified to be reliable to identify live cells. In a cytotoxicity assay of different methyl mercury concentration on BRL cells, the proposed approach is used to detect cell viability. MTT method is also employed and the results of correlational analysis and Bland-Altman analysis prove the validity of the proposed approach. By comparison, it can be revealed that the proposed approach has some advantages over other present techniques. Therefore it may be widely used as a cell viability measurement method in drug screening, nutritional investigation and cell toxicology studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

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

PubMed

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

2017-01-01

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

Effects of Fluid Shear Stress on Cancer Stem Cell Viability

NASA Astrophysics Data System (ADS)

Sunday, Brittney; Triantafillu, Ursula; Domier, Ria; Kim, Yonghyun

2014-11-01

Cancer stem cells (CSCs), which are believed to be the source of tumor formation, are exposed to fluid shear stress as a result of blood flow within the blood vessels. It was theorized that CSCs would be less susceptible to cell death than non-CSCs after both types of cell were exposed to a fluid shear stress, and that higher levels of fluid shear stress would result in lower levels of cell viability for both cell types. To test this hypothesis, U87 glioblastoma cells were cultured adherently (containing smaller populations of CSCs) and spherically (containing larger populations of CSCs). They were exposed to fluid shear stress in a simulated blood flow through a 125-micrometer diameter polyetheretherketone (PEEK) tubing using a syringe pump. After exposure, cell viability data was collected using a BioRad TC20 Automated Cell Counter. Each cell type was tested at three physiological shear stress values: 5, 20, and 60 dynes per centimeter squared. In general, it was found that the CSC-enriched U87 sphere cells had higher cell viability than the CSC-depleted U87 adherent cancer cells. Interestingly, it was also observed that the cell viability was not negatively affected by the higher fluid shear stress values in the tested range. In future follow-up studies, higher shear stresses will be tested. Furthermore, CSCs from different tumor origins (e.g. breast tumor, prostate tumor) will be tested to determine cell-specific shear sensitivity. National Science Foundation Grant #1358991 supported the first author as an REU student.

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

PubMed

Pérez, L M; Alvarez, B L; Codony, F; Fittipaldi, M; Adrados, B; Peñuela, G; Morató, J

2010-09-01

It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

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

Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de; Salamon, Achim; Adam, Stefanie

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiationmore » of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.« less

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

PubMed

Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

2005-08-01

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

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.

Concentration and Viability of Airborne Bacteria Over the Kuroshio Extension Region in the Northwestern Pacific Ocean: Data From Three Cruises

NASA Astrophysics Data System (ADS)

Hu, Wei; Murata, Kotaro; Fukuyama, Shinichiro; Kawai, Yoshimi; Oka, Eitarou; Uematsu, Mitsuo; Zhang, Daizhou

2017-12-01

Airborne bacteria have been shown to act as condensation and ice nuclei in mixed-phase clouds and are consequently hypothesized to have significant effects on atmospheric processes and even the global climate. However, few data are available regarding their concentration and variation in the air over the open ocean. Aerosol samples were collected during three cruises in the early summers of 2013, 2014, and 2016 over the Kuroshio Extension region of the northwest Pacific Ocean. The concentrations of viable and nonviable bacterial cells in the marine surface air were quantified using epifluorescence enumeration with the LIVE/DEAD BacLight stain. The concentrations of total bacteria varied between 1.0 × 104 and 2.5 × 105 cells m-3 and averaged 5.2 × 104, 1.0 × 105, and 7.5 × 104 cells m-3 in the three respective cruises. The viabilities, i.e., the ratios of the concentration of viable bacterial cells to that of total bacterial cells, ranged from 80% to 100% (average 93%), and the respective means were 93%, 89%, and 96% in the cruises. The total bacterial concentration had a close correlation with the wind speed near the sea surface, and the bacterial viability correlated negatively with the air temperature, sea surface temperature, and concentration of coarse particles (size > 1 μm). The deposition and sea spray fluxes of bacteria were roughly estimated as hundreds of cells m-2 s-1 on average. The limited data on bacterial concentration and viability from the three cruises indicate the rapid air-sea exchange of bacteria over the Kuroshio Extension region of the northwest Pacific Ocean.

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

PubMed

Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

2015-07-28

Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We

BID is a critical factor controlling cell viability regulated by IFN-α.

PubMed

Tsuno, Takaya; Mejido, Josef; Zhao, Tongmao; Phillips, Terry; Myers, Timothy G; Bekisz, Joseph; Zoon, Kathryn C

2012-01-01

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

Morphology based scoring of chromosomal instability and its correlation with cell viability.

PubMed

Yadav, Shubhlata; Bhatia, Alka

2017-09-01

The aim of this study was to devise the quantitative scoring system for Chromosomal instability (CIN) based on morphological indicators like MPM, NB, NPB, CS, La and MN in cancer cell line and to correlate it with cell viability and death. Human hepatocellular carcinoma (HepG2) cells were treated with drugs like Diethylstilbestrol 0-100μM, Griseofulvin 0-40μg/ml, Vincristine sulphate 0-25μg/ml, Mitomycin C 0-600ng/ml, Bleomycin 0-10μg/ml, Doxorubicin 0-30μg/ml for 24h. Following this, the CIN was assessed by counting the morphological indicators like Micronuclei (MN), Nuclear Buds (NB), Nucleoplasmic bridges, Laggards, Multipolar mitosis and chromatin strings/1000 cells in Giemsa stained smears by light microscopy and by determining the percentage of aneuploid cells by flow cytometry. The cell viability was assessed by MTT assay and percentage of apoptotic cells was determined by flow cytometry. The MN and NB were most frequently seen indicators and main determinants of morphological CIN. However, the morphological CIN score did not show any correlation with cell viability and apoptosis. Aneuploidy however was found to correlate positively with cell viability and NB score in our study (P-value <0.05). The study for the 1st time attempted to develop a scoring system for CIN based on morphological parameters. However, a no correlation was observed between the later and cell viability or apoptosis. More robust techniques to quantify CIN may perhaps be more helpful in exploring the true link between CIN and cell viability in future. Copyright © 2017 Elsevier GmbH. All rights reserved.

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

PubMed Central

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-01-01

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

PubMed

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-07-07

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

Bacterial properties of rainwater associated with cyclones, stationary fronts and typhoons in southwestern Japan

NASA Astrophysics Data System (ADS)

Zhang, D.; Hu, W.; Niu, H.

2016-12-01

The activities and role of bioaerosols in aerosol-cloud-precipitation links are important but unresolved issues in atmospheric and microbiological sciences. Bacteria, a main part of bioaerosols, are ubiquitous in atmospheric water. They are considered to be involved in the processes of cloud condensation and ice nuclei formation. However, to date, little information on rainwater bacteria is available. Rainwater samples were collected at a suburban site in southwestern Japan during October 2014 to September 2015. Results show that the cell concentration of rainwater bacteria was 2.3±1.5×104 cells ml-1, with a viability of 80±10% on average. The bacterial abundance and viability systematically differed with the weather systems causing rain. In cold-front-derived rain, the average bacterial concentration was the highest (3.5±1.6×104 cells ml-1), with the lowest viability as 75%. In the stationary-front-derived rain during Meiyu period and typhoon rain, the average bacterial concentrations were lower, but with higher viability. In stationary-front-derived rain during non-Meiyu period, the average abundance was higher (2.4±1.6×104 cells ml-1), while the viability was lower (78%) than those during Meiyu period. It was suggested that clouds produced by air mass from ocean areas carried fewer bacteria but with higher viability than those originated from continental regions. Bacterial concentrations in rainwater did not show good correlations with the ratios of total and decreased airborne particle concentrations to rainfall. Combining the univariate and factorial analysis of chemical compositions and bacterial abundance, we found that bacteria in rainwater were mainly associated with nss-SO42-, nss-Ca2+, and NO3-, which can act as nuclei or be produced within clouds. The cultured heterotrophic marine bacteria were of much higher abundance in stationary-front-derived rain than those in cold-front-derived rain. Bacterial genera containing ice nucleation active

Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

PubMed

Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

2016-09-16

3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.

PubMed

Chaytor, Jennifer L; Tokarew, Jacqueline M; Wu, Luke K; Leclère, Mathieu; Tam, Roger Y; Capicciotti, Chantelle J; Guolla, Louise; von Moos, Elisabeth; Findlay, C Scott; Allan, David S; Ben, Robert N

2012-01-01

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

PubMed

Fabian, Dusan; Dusan, Fabian; Sabol, Marián; Marián, Sabol; Domaracká, Katarína; Katarína, Domaracká; Bujnáková, Dobroslava; Dobroslava, Bujnáková

2006-12-01

Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

PubMed

Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

2016-09-15

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

PubMed Central

Prakash, Satya; Malgorzata Urbanska, Aleksandra

2008-01-01

There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

Measurement of cell viability in in vitro cultures.

PubMed

Castro-Concha, Lizbeth A; Escobedo, Rosa María; Miranda-Ham, María de Lourdes

2006-01-01

An overview of the methods for assessing cell viability in in vitro cultures is presented. The protocols of four of the most commonly used assays are described in detail, so the readers may be able to determine which assay is suitable for their own projects using plant cell cultures.

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

PubMed Central

Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

2015-01-01

ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

Study of wettability and cell viability of H implanted stainless steel

NASA Astrophysics Data System (ADS)

Shafique, Muhammad Ahsan; Ahmad, Riaz; Rehman, Ihtesham Ur

2018-03-01

In the present work, the effect of hydrogen ion implantation on surface wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are implanted in the near-surface of stainless steel to facilitate hydrogen bonding at different doses with constant energy of 500 KeV, which consequently improve the surface wettability. Treated and untreated sample are characterized for surface wettability, incubation of hydroxyapatite and cell viability. Contact angle (CA) study reveals that surface wettability increases with increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite over the surface increase with increasing dose of H-ions. Cell viability study using MTT assay describes improved cell viability in treated samples as compared to the untreated sample. It is found that low dose of H-ions is more effective for cell proliferation and the cell count decreases with increasing ion dose. Our study demonstrates that H ion implantation improves the surface wettability and biocompatibility of stainless steel.

Fluorescein Diacetate Microplate Assay in Cell Viability Detection.

PubMed

Chen, Xi; Yang, Xiu-Ying; Fang, Lian-Hua; DU, Guan-Hua

2016-12-20

Objective To investigate the application of the fluorescein diacetate (FDA) microplate assay in cell viability detection. Methods Cells were seeded in a 96-well culture plate until detection. After incubated with FDA,the plate was detected by fluorescence microplate analyzer. The effects of FDA incubation duration,concentration,and other factors on the assay's accuracy and stability were assessed. We also compared the results of FDA with methyl thiazolyl(MTT) in terms of cell numbers and H 2 O 2 injury. Results Within 0-30 minutes,the fluorescence-cell number coefficient of FDA assay increased with duration and reached 0.99 in 27-30 minutes. The optimum concentration of final FDA in this study was 10-30 μg/ml. On cell viability detection,the result of FDA method was equivalent to MTT method. As to H 2 O 2 injury assay,the sensitivity of FDA method was superior to MTT on the higher concentration H 2 O 2 treatment due to a relative shorter duration for detection. Conclusion As a stable and reliable method,FDA is feasible for cell variability detection under varied conditions.

The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment.

PubMed

Wang, Juan; Wei, Yun; Zhao, Shasha; Zhou, Ying; He, Wei; Zhang, Yang; Deng, Wensheng

2017-01-01

Mammalian cells are very important experimental materials and widely used in biological and medical research fields. It is often required that mammalian cells are transported from one laboratory to another to meet with various researches. Conventional methods for cell shipment are laborious and costive despite of maintaining high viability. In this study we aimed to develop a simple and low-cost method for cell shipment by investigating the viabilities of different cell lines treated at different temperatures. We show that the viability of mammalian cells incubated at 1°C or 5°C significantly reduced when compared with that at 16°C or 22°C. Colony formation assays revealed that preservation of mammalian cells at 1°C or 5°C led to a poorer recovery than that at 16°C or 22°C. The data from proliferation and apoptotic assays confirmed that M2 cells could continue to proliferate at 16°C or 22°C, but massive death was caused by apoptosis at 1°C or 5°C. The morphology of mammalian cells treated under hypothermia showed little difference from that of the untreated cells. Quantitative RT-PCR and alkaline phosphatase staining confirmed that hypothermic treatment did not change the identity of mouse embryonic stem cells. A case study showed that mammalian cells directly suspended in culture medium were able to be shipped for long distance and maintained a high level of viability and recovery. Our findings not only broaden the understanding to the effect of hypothermia on the viability of mammalian cells, but also provide an alternative approach for cell shipment.

Is cell viability always directly related to corrosion resistance of stainless steels?

PubMed

Salahinejad, E; Ghaffari, M; Vashaee, D; Tayebi, L

2016-05-01

It has been frequently reported that cell viability on stainless steels is improved by increasing their corrosion resistance. The question that arises is whether human cell viability is always directly related to corrosion resistance in these biostable alloys. In this work, the microstructure and in vitro corrosion behavior of a new class of medical-grade stainless steels were correlated with adult human mesenchymal stem cell viability. The samples were produced by a powder metallurgy route, consisting of mechanical alloying and liquid-phase sintering with a sintering aid of a eutectic Mn-Si alloy at 1050 °C for 30 and 60 min, leading to nanostructures. In accordance with transmission electron microscopic studies, the additive particles for the sintering time of 30 min were not completely melted. Electrochemical impedance spectroscopic experiments suggested the higher corrosion resistance for the sample sintered for 60 min; however, a better cell viability on the surface of the less corrosion-resistant sample was unexpectedly found. This behavior is explained by considering the higher ion release rate of the Mn-Si additive material, as preferred sites to corrosion attack based on scanning electron microscopic observations, which is advantageous to the cells in vitro. In conclusion, cell viability is not always directly related to corrosion resistance in stainless steels. Typically, the introduction of biodegradable and biocompatible phases to biostable alloys, which are conventionally anticipated to be corrosion-resistant, can be advantageous to human cell responses similar to biodegradable metals. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

PubMed Central

Varghese, Divya S.; Parween, Shama; Ardah, Mustafa T.; Emerald, Bright Starling

2017-01-01

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results. PMID:29147115

Nanodiamonds on tetrahedral amorphous carbon significantly enhance dopamine detection and cell viability.

PubMed

Peltola, Emilia; Wester, Niklas; Holt, Katherine B; Johansson, Leena-Sisko; Koskinen, Jari; Myllymäki, Vesa; Laurila, Tomi

2017-02-15

We hypothesize that by using integrated carbon nanostructures on tetrahedral amorphous carbon (ta-C), it is possible to take the performance and characteristics of these bioelectrodes to a completely new level. The integrated carbon electrodes were realized by combining nanodiamonds (NDs) with ta-C thin films coated on Ti-coated Si-substrates. NDs were functionalized with mixture of carboxyl and amine groups ND andante or amine ND amine , carboxyl ND vox or hydroxyl groups ND H and drop-casted or spray-coated onto substrate. By utilizing these novel structures we show that (i) the detection limit for dopamine can be improved by two orders of magnitude [from 10µM to 50nM] in comparison to ta-C thin film electrodes and (ii) the coating method significantly affects electrochemical properties of NDs and (iii) the ND coatings selectively promote cell viability. ND andante and ND H showed most promising electrochemical properties. The viability of human mesenchymal stem cells and osteoblastic SaOS-2 cells was increased on all ND surfaces, whereas the viability of mouse neural stem cells and rat neuroblastic cells was improved on ND andante and ND H and reduced on ND amine and ND vox. The viability of C6 cells remained unchanged, indicating that these surfaces will not cause excess gliosis. In summary, we demonstrated here that by using functionalized NDs on ta-C thin films we can significantly improve sensitivity towards dopamine as well as selectively promote cell viability. Thus, these novel carbon nanostructures provide an interesting concept for development of various in vivo targeted sensor solutions. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial Acclimation Inside an Aqueous Battery.

PubMed

Dong, Dexian; Chen, Baoling; Chen, P

2015-01-01

Specific environmental stresses may lead to induced genomic instability in bacteria, generating beneficial mutants and potentially accelerating the breeding of industrial microorganisms. The environmental stresses inside the aqueous battery may be derived from such conditions as ion shuttle, pH gradient, free radical reaction and electric field. In most industrial and medical applications, electric fields and direct currents are used to kill bacteria and yeast. However, the present study focused on increasing bacterial survival inside an operating battery. Using a bacterial acclimation strategy, both Escherichia coli and Bacillus subtilis were acclimated for 10 battery operation cycles and survived in the battery for over 3 days. The acclimated bacteria changed in cell shape, growth rate and colony color. Further analysis indicated that electrolyte concentration could be one of the major factors determining bacterial survival inside an aqueous battery. The acclimation process significantly improved the viability of both bacteria E. coli and B. subtilis. The viability of acclimated strains was not affected under battery cycle conditions of 0.18-0.80 mA cm(-2) and 1.4-2.1 V. Bacterial addition within 1.0×10(10) cells mL(-1) did not significantly affect battery performance. Because the environmental stress inside the aqueous battery is specific, the use of this battery acclimation strategy may be of great potential for the breeding of industrial microorganisms.

Bacterial Acclimation Inside an Aqueous Battery

PubMed Central

Dong, Dexian; Chen, Baoling; Chen, P.

2015-01-01

Specific environmental stresses may lead to induced genomic instability in bacteria, generating beneficial mutants and potentially accelerating the breeding of industrial microorganisms. The environmental stresses inside the aqueous battery may be derived from such conditions as ion shuttle, pH gradient, free radical reaction and electric field. In most industrial and medical applications, electric fields and direct currents are used to kill bacteria and yeast. However, the present study focused on increasing bacterial survival inside an operating battery. Using a bacterial acclimation strategy, both Escherichia coli and Bacillus subtilis were acclimated for 10 battery operation cycles and survived in the battery for over 3 days. The acclimated bacteria changed in cell shape, growth rate and colony color. Further analysis indicated that electrolyte concentration could be one of the major factors determining bacterial survival inside an aqueous battery. The acclimation process significantly improved the viability of both bacteria E. coli and B. subtilis. The viability of acclimated strains was not affected under battery cycle conditions of 0.18-0.80 mA cm-2 and 1.4-2.1 V. Bacterial addition within 1.0×1010 cells mL-1 did not significantly affect battery performance. Because the environmental stress inside the aqueous battery is specific, the use of this battery acclimation strategy may be of great potential for the breeding of industrial microorganisms. PMID:26070088

Effect of IFN-gamma on the killing of S. aureus in human whole blood. Assessment of bacterial viability by CFU determination and by a new method using alamarBlue.

PubMed

DeForge, L E; Billeci, K L; Kramer, S M

2000-11-01

Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.

Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

PubMed Central

2015-01-01

Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

PubMed

Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

2017-07-01

This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction.

PubMed

Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells. The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest). Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells. The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.

The reducibility of heLa cell viability by Sargassum polycystum extracts

NASA Astrophysics Data System (ADS)

Firdaus, M.; Setijawati, D.; Islam, I.; Nursyam, H.; Kartikaningsih, H.; Yufidasari, H. S.; Prihanto, A. A.; Nurdiani, R.; Jaziri, A. A.

2018-04-01

Cervical cancer is the second largest cause of death-related cancer in women. The efficacy of cancer drugs is still low. Bioactive of brown seaweed has been studied by in vitro and in vivo as anticancer. The aim of this study was to evaluate the cytotoxicity of Sargassum polycystum extracts on HeLa cell, to recognize bioactive on extract and estimate the interaction between the bioactive and target protein. S. polycystum was found from Talango Island waters and HeLa cell was obtained from Indonesian Science Institute. Sample was extracted by ethanol, ethyl acetate and hexane, concentrated and finally, extracts were assayed on HeLa cell. The viability of this cell was quantified on ELISA-Reader. The bioactive compounds of the extract were elucidated by GC-MS. The interaction between bioactive and target protein was evaluated by using in silico method. The result showed that the lowest viability of HeLa cell on n-hexane extracts treatment. The n-hexane extract of this seaweed contained benzenepropanoic acid. This compound reduced HeLa cell viability by reducing of thrombin concentration. In conclusion, the benzene propanoic acid of S. polycystum was the cytotoxic agent and it is potential agent for anti-cervical cancer.

Viability of 3h grown bacterial micro-colonies after direct Raman identification.

PubMed

Mathey, R; Dupoy, M; Espagnon, I; Leroux, D; Mallard, F; Novelli-Rousseau, A

2015-02-01

Clinical diagnostics in routine microbiology still mostly relies on bacterial growth, a time-consuming process that prevents test results to be used directly as key decision-making elements for therapeutic decisions. There is some evidence that Raman micro-spectroscopy provides clinically relevant information from a limited amount of bacterial cells, thus holding the promise of reduced growth times and accelerated result delivery. Indeed, bacterial identification at the species level directly from micro-colonies at an early time of growth (6h) directly on their growth medium has been demonstrated. However, such analysis is suspected to be partly destructive and could prevent the further growth of the colony needed for other tests, e.g. antibiotic susceptibility testing (AST). In the present study, we evaluated the effect of the powerful laser excitation used for Raman identification on micro-colonies probed after very short growth times. We show here, using envelope integrity markers (Syto 9 and Propidium Iodide) directly on ultra-small micro-colonies of a few tens of Escherichia coli and Staphylococcus epidermidis cells (3h growth time), that only the cells that are directly impacted by the laser lose their membrane integrity. Growth kinetics experiments show that the non-probed surrounding cells are sometimes also affected but that the micro-colonies keep their ability to grow, resulting in normal aspect and size of colonies after 15h of growth. Thus, Raman spectroscopy could be used for very early (<3h) identification of grown micro-organisms without impairing further antibiotics susceptibility characterization steps. Copyright © 2014 Elsevier B.V. All rights reserved.

Human periodontal ligament cell viability in milk and milk substitutes.

PubMed

Pearson, Robert M; Liewehr, Frederick R; West, Leslie A; Patton, William R; McPherson, James C; Runner, Royce R

2003-03-01

The purpose of this study was to determine the efficacy of several milk substitutes compared to whole milk in maintaining the viability of human periodontal ligament (PDL) cells on avulsed teeth. PDL cells were obtained from freshly extracted, healthy third molars and cultured in Eagle's minimal essential media (EMEM). The cells were plated onto 24-well culture plates and allowed to attach for 24 h. EMEM was replaced with refrigerated whole milk (positive control), reconstituted powdered milk, evaporated milk, or one of two baby formulas (Similac or Enfamil). Tap water served as the negative control. Tissue culture plates were incubated with the experimental media at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined by a cell proliferation assay (CellTiter 96 AQ Assay), with absorbance read at 450 nM. A two-way ANOVA (p < 0.001) indicated that at 1 h there was no difference in the effect on PDL cell viability between any of the materials and whole milk. At 2 h, Enfamil and Similac performed significantly better than whole milk, whereas evaporated milk performed worse. At 4 h, Enfamil performed better than whole milk, whereas all other milk substitutes performed worse. At 8 h, all substitutes performed worse than whole milk. These results suggest that Enfamil, which is supplied in powder form that does not require special storage and has a shelf life of 18 months, is a more effective storage medium for avulsed teeth than pasteurized milk for at least 4 h.

Effect of extracorporeal shock wave lithotripsy on bacterial viability. Relationship to the treatment of struvite stones.

PubMed

Reid, G; Jewett, M A; Nickel, J C; McLean, R J; Bruce, A W

1990-01-01

The aim of this study was to determine whether extracorporeal shock wave lithotripsy (ESWL) affected the viability of the infecting bacteria within a simulated struvite stone matrix. A strain, Proteus mirabilis 28cii, was prepared in three forms: (1) suspended in saline and urine, (2) artificially encapsulated by suspending in agar beads and (3) artificially encapsulated and mineralised by suspending in agar beads with calcium carbonate crystals. The preparations were placed in capped vials partially immersed in degassed water and held in the focal point of the Siemens Lithostar and given 1,000 shocks. Subsequent viability testing showed that bacteria suspended in urine were greatly affected by shock treatments (55% loss in viability), but incorporation into agar beads negated this effect (even if the cells were exposed to 2000 shocks). Mineralisation of the beads with calcium carbonate crystals caused a decrease in viability of 82% that was significantly different from controls. However, this still left 2.3 X 10(8) viable organisms (82% of 2.8 X 10(8], easily enough to form the focus for further infections. A series of control experiments carried out using an ultrasonic cell sonicator probe gave comparable results to those obtained with ESWL. These results demonstrate the ESWL treatment of infected stones must be accompanied by antimicrobial coverage.

Microencapsulation in Alginate and Chitosan Microgels to Enhance Viability of Bifidobacterium longum for Oral Delivery

PubMed Central

Yeung, Timothy W.; Üçok, Elif F.; Tiani, Kendra A.; McClements, David J.; Sela, David A.

2016-01-01

Probiotic microorganisms are incorporated into a wide variety of foods, supplements, and pharmaceuticals to promote human health and wellness. However, maintaining bacterial cell viability during storage and gastrointestinal transit remains a challenge. Encapsulation of bifidobacteria within food-grade hydrogel particles potentially mitigates their sensitivity to environmental stresses. In this study, Bifidobacterium longum subspecies and strains were encapsulated in core-shell microgels consisting of an alginate core and a microgel shell. Encapsulated obligate anaerobes Bifidobacterium longum subsp. infantis and Bifidobacterium longum subsp. longum exhibited differences in viability in a strain-dependent manner, without a discernable relationship to subspecies lineage. This includes viability under aerobic storage conditions and modeled gastrointestinal tract conditions. Coating alginate microgels with chitosan did not improve viability compared to cells encapsulated in alginate microgels alone, suggesting that modifying the surface charge alone does not enhance delivery. Thus hydrogel beads have great potential for improving the stability and efficacy of bifidobacterial probiotics in various nutritional interventions. PMID:27148184

A study of different buffers to maximize viability of an oral Shigella vaccine.

PubMed

Chandrasekaran, Lakshmi; Lal, Manjari; Van De Verg, Lillian L; Venkatesan, Malabi M

2015-11-17

Live, whole cell killed and subunit vaccines are being developed for diarrheal diseases caused by V. cholerae, Shigella species, ETEC, and Campylobacter. Some of these vaccines can be administered orally since this route best mimics natural infection. Live vaccines administered orally have to be protected from the harsh acidic gastric environment. Milk and bicarbonate solutions have been administered to neutralize the stomach acid. For many Shigella vaccine trials, 100-120 ml of a bicarbonate solution is ingested followed by the live vaccine candidate, which is delivered in 30 ml of bicarbonate, water or saline. It is not clear if maximum bacterial viability is achieved under these conditions. Also, volumes of neutralizing buffer that are optimal for adults may be unsuitable for children and infants. To address these questions, we performed studies to determine the viability and stability of a Shigella sonnei vaccine candidate, WRSS1, in a mixture of different volumes of five different buffer solutions added to hydrochloric acid to simulate gastric acidity. Among the buffers tested, bicarbonate solution, rotavirus buffer and CeraVacx were better at neutralizing acid and maintaining the viability of WRSS1. Also, a much smaller volume of the neutralizing buffer was sufficient to counteract stomach acid while maintaining bacterial viability. Published by Elsevier Ltd.

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

PubMed

Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

2014-01-01

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell viability in optical tweezers: high power red laser diode versus Nd:YAG laser

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Hendinger, Anita; Sailer, Reinhard; Gschwend, Michael H.; Strauss, Wolfgang S.; Bauer, Manfred; Schuetze, Karin

2000-01-01

Viability of cultivated Chinese hamster ovary cells in optical tweezers was measured after exposure to various light doses of red high power laser diodes ((lambda) equals 670 - 680 nm) and a Nd:yttrium-aluminum-garnet laser ((lambda) equals 1064 nm). When using a radiant exposure of 2.4 GJ/cm2, a reduction of colony formation up to a factor 2 (670 - 680 nm) or 1.6 (1064 nm) as well as a delay of cell growth were detected in comparison with nonirradiated controls. In contrast, no cell damage was found at an exposure of 340 MJ/cm2 applied at 1064 nm. Cell viabilities were correlated with fluorescence excitation spectra and with literature data of wavelength dependent cloning efficiencies. Fluorescence excitation maxima of the coenzymes NAD(P)H and flavins were detected at 365 and 450 nm, respectively. This is half of the wavelengths of the maxima of cell inactivation, suggesting that two-photon absorption by these coenzymes may contribute to cellular damage. Two-photon excitation of NAD(P)H and flavins may also affect cell viability after exposure to 670 - 680 nm, whereas one-photon excitation of water molecules seems to limit cell viability at 1064 nm.

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.

Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

PubMed

Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

2013-09-01

The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally modified calcium alginate hydrogel.

PubMed

Wright, Bernice; Cave, Richard A; Cook, Joseph P; Khutoryanskiy, Vitaliy V; Mi, Shengli; Chen, Bo; Leyland, Martin; Connon, Che J

2012-05-01

Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for 'on-demand' use. In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells.

PubMed

Falah, Mizied; Rayan, Anwar; Srouji, Samer

2015-09-01

In our recent studies, the transplantation of human adipose tissue-derived stromal cells (ASCs) has shown promise for treatment of diseases related to bone and joint disorders. For the current clinical applications, ASCs were formulated and suspended in PlasmaLyte A supplemented with heparin, glucose and human serum albumin, balanced to pH 7.4 with sodium bicarbonate. This cell solution constitutes 20% of the overall transplanted mixture and is supplemented with hyaluronic acid (60%) and OraGraft particles (20%). We intended to investigate the effect of this transplantation mixture on the viability and biofunctionality of ASCs in bone formation. Freshly harvested cells were resuspended and incubated in the indicated mixture for up to 48 h at 4°C. Cell viability was assessed using trypan blue and AlamarBlue, and cell functionality was determined by quantifying their adhesion rate in vitro and bone formation in an ectopic mouse model. More than 80% of the ASCs stored in the transplantation mixture were viable for up to 24 h. Cell viability beyond 24 h in storage decreased to approximately 50%. In addition, an equal degree of bone formation was observed between the cells transplanted following incubation in transplantation mixture for up to 24 h and zero-time non-incubated cells (control). The viability and functionality of ASCs stored in the presented formulation will make such cell therapy accessible to larger and more remote populations. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

PubMed

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

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

PubMed

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

2012-01-01

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

Effect of smokeless tobacco products on human oral bacteria growth and viability

PubMed Central

Liu, Min; Jin, Jinshan; Pan, Hongmiao; Feng, Jinhui; Cerniglia, Carl E.; Yang, Maocheng; Chen, Huizhong

2017-01-01

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32–2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3–1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3–2.11 log10 fold, 18 strains showed enhanced growth of 0.3–0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3–2.96 log10 fold, 8 strains showed enhanced growth of 0.3–1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1–66.5% decrease for 4 strains at 1 mg/ml, 20.3–85.7% decrease for 10 strains at 10 mg/ml, 20.0–93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 μg/ml; while the growth of P. micros was enhanced 0.31–0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33–0.36 log10 fold; and the

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

PubMed

Jacobsson, S O; Rongård, E; Stridh, M; Tiger, G; Fowler, C J

2000-12-15

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

PubMed

Germann, Anja; Oh, Young-Joo; Schmidt, Tomm; Schön, Uwe; Zimmermann, Heiko; von Briesen, Hagen

2013-10-01

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells.

PubMed

Bankier, Claire; Cheong, Yuen; Mahalingam, Suntharavathanan; Edirisinghe, Mohan; Ren, Guogang; Cloutman-Green, Elaine; Ciric, Lena

2018-01-01

Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens. Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments. Strong antimicrobial effects of the three NPCs (AMNP0-2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification. Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.

Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs.

PubMed

Powell, Heather M; Armour, Alexis D; Boyce, Steven T

2011-01-01

Quantification of cell viability and distribution within engineered tissues currently relies on representative histology, phenotypic assays, and destructive assays of viability. To evaluate uniformity of cell density throughout 3D collagen scaffolds prior to in vivo use, a nondestructive, field assessment of cell viability is advantageous. Here, we describe a field measure of cell viability in lyophilized collagen-glycosaminoglycan (C-GAG) scaffolds in vitro using fluorescein diacetate (FdA). Fibroblast-C-GAG constructs are stained 1 day after cellular inoculation using 0.04 mg/ml FdA followed by exposure to 366 nm UV light. Construct fluorescence quantified using Metamorph image analysis is correlated with inoculation density, MTT values, and histology of corresponding biopsies. Construct fluorescence correlates significantly with inoculation density (p  <  0.001) and MTT values (p  <  0.001) of biopsies collected immediately after FdA staining. No toxicity is detected in the constructs, as measured by MTT assay before and after the FdA assay at different time points; normal in vitro histology is demonstrated for the FdA-exposed constructs. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered tissue.

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

PubMed Central

Perdomo-Celis, Federico; Salgado, Doris M.; Castañeda, Diana M.

2016-01-01

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3+ CD8+ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3+ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = −0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. PMID:26961858

Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

PubMed Central

ZHAO, BING; HU, MENGCAI

2013-01-01

Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer. PMID:24843386

Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

PubMed

Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

2012-01-01

Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

Effect of fluoride on the cell viability, cell organelle potential, and photosynthetic capacity of freshwater and soil algae.

PubMed

Chae, Yooeun; Kim, Dokyung; An, Youn-Joo

2016-12-01

Although fluoride occurs naturally in the environment, excessive amounts of fluoride in freshwater and terrestrial ecosystems can be harmful. We evaluated the toxicity of fluoride compounds on the growth, viability, and photosynthetic capacity of freshwater (Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata) and terrestrial (Chlorococcum infusionum) algae. To measure algal growth inhibition, a flow cytometric method was adopted (i.e., cell size, granularity, and auto-fluorescence measurements), and algal yield was calculated to assess cell viability. Rhodamine123 and fluorescein diacetate were used to evaluate mitochondrial membrane potential (MMA, ΔΨ m ) and cell permeability. Nine parameters related to the photosynthetic capacity of algae were also evaluated. The results indicated that high concentrations of fluoride compounds affected cell viability, cell organelle potential, and photosynthetic functions. The cell viability measurements of the three algal species decreased, but apoptosis was only observed in C. infusionum. The MMA (ΔΨ m ) of cells exposed to fluoride varied among species, and the cell permeability of the three species generally decreased. The decrease in the photosynthetic activity of algae may be attributable to the combination of fluoride ions (F - ) with magnesium ions (Mg 2+ ) in chlorophyll. Our results therefore provide strong evidence for the potential risks of fluoride compounds to microflora and microfauna in freshwater and terrestrial ecosystems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of NFkappaB reduces cellular viability in GH3 pituitary adenoma cells.

PubMed

Vender, John R; Laird, Melissa D; Dhandapani, Krishnan M

2008-05-01

Adenomas of the pituitary gland are among the most common types of tumors of the adult brain. Although adenomas are histologically benign, they may be associated with significant morbidity and mortality, mostly because of their invasive growth pattern and hormone hypersecretion. Current medical therapies are suppressive, acting at a receptor level. Thus, there is a need to identify novel cellular and molecular targets for pituitary tumors. We investigated the possible role of the NFkappaB transcription factor in pituitary tumor cell growth. The effect of NFkappaB pathway inhibition on cellular viability was studied in the GH3 pituitary adenoma cell line, a well-characterized rat cell line that secretes growth hormone and prolactin. Cells were treated with mechanistically diverse pharmacological NFkappaB pathway inhibitors or with molecular inhibitors that were overexpressed in tumor cells before the assessment of cellular viability. NFkappaB activity was also assessed in GH3 cells using deoxyribonucleic acid binding assays. GH3 cells exhibited constitutive NFkappaB activity, which contributed to increased cellular proliferation. Treatment with wedelolactone, an IkappaB kinase inhibitor, or overexpression of an IkappaB super-repressor reduced cell viability, further implicating NFkappaB in pituitary tumor cell growth. Pharmacological or molecular inhibition of Akt similarly reduced GH3 viability and NFkappaB binding, suggesting that constitutive activation of NFkappaB may be, at least in part, mediated by Akt. Directed targeting of the Akt and NFkappaB signaling pathways may be a useful adjunct in the clinical management of pituitary tumors. Further elucidation of this pathway may yield novel information regarding the behavior of pituitary tumors in humans.

Effects of inorganic nanoparticles on viability and catabolic activities of Agrobacterium sp. PH-08 during biodegradation of dibenzofuran.

PubMed

Le, Thao Thanh; Murugesan, Kumarasamy; Kim, Eun-Ju; Chang, Yoon-Seok

2014-09-01

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0-1,000 mg L(-1)) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L(-1) showed significant reduction in cell viability (30-93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7-91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.

Bacterial Species and Biochemical Characteristic Investigations of Nostoc flagelliforme Concentrates during its Storage.

PubMed

Yue, Lifang; Lv, Hexin; Zhen, Jing; Jiang, Shengping; Jia, Shiru; Shen, Shigang; Gao, Lu; Dai, Yujie

2016-04-28

Preservation of fresh algae plays an important role in algae seed subculture and aquaculture. The determination and examination of the changes of cell viability, composition, and bacterial species during storage would help to take suitable preservation methods to prolong the preservation time of fresh algae. Nostoc flagelliforme is a kind of edible cyanobacterium with important herbal and dietary values. This article investigated the changes of bacterial species and biochemical characteristics of fresh N. flagelliforme concentrate during natural storage. It was found that the viability of cells decreased along with the storage time. Fourteen bacteria strains in the algae concentrate were identified by PCR-DGGE and were grouped into four phyla, including Cyanobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. Among them, Enterococcus viikkiensis may be a concern in the preservation. Eleven volatile organic compounds were identified from N. flagelliforme cells, in which geosmin could be treated as an indicator of the freshness of N. flagelliforme. The occurrence of indole compound may be an indicator of the degradation of cells.

Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

PubMed

Abbruzzese, L; Agostini, F; Durante, C; Toffola, R T; Rupolo, M; Rossi, F M; Lleshi, A; Zanolin, S; Michieli, M; Mazzucato, M

2013-07-01

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness. © 2013 International Society of Blood Transfusion.

Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

PubMed

Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

2016-03-01

Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative viability of unirradiated and gamma irradiated bacterial cells

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

Maxcy, R.B.

1977-01-01

Gamma radiation injured Escherichia coli, Salmonella typhimurium, and Moraxella sp. were studied under various environmental stresses to determine their fate relative to the parent population. Irradiated cultures formed smaller colonies on surface plates with fewer cells per colony. Unirradiated cultures had a shorter lag phase than irradiated cultures in broth and duration of lag increased as a result of increasing the radiation dose. Repeated irradiation and subculture progressively retarded growth rate. Multiple radiation of highly resistant Moraxella sp. showed radiation injured cells to be more sensitive than uninjured cells. With the three species studied, irradiation raised the lower limits ofmore » growth temperature, increased the sensitivity to freezing and thawing, and increased the susceptibility to lowered water activity. This work indicated that the production of a bizarre, resistant strain of bacteria through recycling in a food processing operation is highly unlikely.« less

Drying process strongly affects probiotics viability and functionalities.

PubMed

Iaconelli, Cyril; Lemetais, Guillaume; Kechaou, Noura; Chain, Florian; Bermúdez-Humarán, Luis G; Langella, Philippe; Gervais, Patrick; Beney, Laurent

2015-11-20

Probiotic formulations are widely used and are proposed to have a variety of beneficial effects, depending on the probiotic strains present in the product. The impact of drying processes on the viability of probiotics is well documented. However, the impact of these processes on probiotics functionality remains unclear. In this work, we investigated variations in seven different bacterial markers after various desiccation processes. Markers were composed of four different viability evaluation (combining two growth abilities and two cytometric measurements) and in three in vitro functionalities: stimulation of IL-10 and IL-12 production by PBMCs (immunomodulation) and bacterial adhesion to hexadecane. We measured the impact of three drying processes (air-drying, freeze-drying and spray-drying), without the use of protective agents, on three types of probiotic bacteria: Bifidobacterium bifidum, Lactobacillus plantarum and Lactobacillus zeae. Our results show that the bacteria respond differently to the three different drying processes, in terms of viability and functionality. Drying methods produce important variations in bacterial immunomodulation and hydrophobicity, which are correlated. We also show that adherence can be stimulated (air-drying) or inhibited (spray-drying) by drying processes. Results of a multivariate analysis show no direct correlation between bacterial survival and functionality, but do show a correlation between probiotic responses to desiccation-rewetting and the process used to dry the bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanical influences in bacterial morphogenesis and cell division

NASA Astrophysics Data System (ADS)

Sun, Sean

2010-03-01

Bacterial cells utilize a ring-like organelle (the Z-ring) to accomplish cell division. The Z-ring actively generates a contractile force and influences cell wall growth. We will discuss a general model of bacterial morphogenesis where mechanical forces are coupled to the growth dynamics of the cell wall. The model suggests a physical mechanism that determines the shapes of bacteria cells. The roles of several bacterial cytoskeletal proteins and the Z-ring are discussed. We will also explore molecular mechanisms of force generation by the Z-ring and how cells can generate mechanical forces without molecular motors.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing

PubMed Central

Krzyzanek, Vladislav; Mravec, Filip; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Marova, Ivana

2016-01-01

Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen–thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences. PMID:27315285

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory †

PubMed Central

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K.; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V. McNeil; Segarra, Verónica A.

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research. PMID:28861134

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

Effects of drinking desalinated seawater on cell viability and proliferation.

PubMed

Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

2017-06-01

Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

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

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

Tiny cells meet big questions: a closer look at bacterial cell biology.

PubMed

Goley, Erin D

2013-04-01

While studying actin assembly as a graduate student with Matt Welch at the University of California at Berkeley, my interest was piqued by reports of surprising observations in bacteria: the identification of numerous cytoskeletal proteins, actin homologues fulfilling spindle-like functions, and even the presence of membrane-bound organelles. Curiosity about these phenomena drew me to Lucy Shapiro's lab at Stanford University for my postdoctoral research. In the Shapiro lab, and now in my lab at Johns Hopkins, I have focused on investigating the mechanisms of bacterial cytokinesis. Spending time as both a eukaryotic cell biologist and a bacterial cell biologist has convinced me that bacterial cells present the same questions as eukaryotic cells: How are chromosomes organized and accurately segregated? How is force generated for cytokinesis? How is polarity established? How are signals transduced within and between cells? These problems are conceptually similar between eukaryotes and bacteria, although their solutions can differ significantly in specifics. In this Perspective, I provide a broad view of cell biological phenomena in bacteria, the technical challenges facing those of us who peer into bacterial cells, and areas of common ground as research in eukaryotic and bacterial cell biology moves forward.

Coconut milk and probiotic milk as storage media to maintain periodontal ligament cell viability: an in vitro study.

PubMed

Saini, Divya; Gadicherla, Prahlad; Chandra, Prakash; Anandakrishna, Latha

2017-06-01

The viability of periodontal ligament (PDL) cells is a significant determinant of the long-term prognosis of replanted avulsed teeth. A storage medium is often required to maintain the viability of these cells during the extra-alveolar period. Many studies have been carried out to search for the most suitable storage medium for avulsed teeth, but an ideal solution has not yet been found. The purpose of the study was to compare and analyze the ability of coconut milk and probiotic milk to maintain PDL cell viability. In an in vitro setting, 69 caries free human premolars with normal periodontium that had been extracted for orthodontic purposes were randomly divided into two experimental groups on the basis of storage media used (i.e., coconut milk or probiotic milk) and a Hanks' balanced salt solution (HBSS) control group (23 samples per group). Immediately after extraction, the teeth were stored dry for 20 min and then immersed for 30 min in one of the storage media. The teeth were then subjected to collagenase-dispase assay and labeled with 0.5% trypan blue staining solution for determination of cell viability. The number of viable cells was counted under a light microscope and statistically analyzed using anova and post hoc Tukey test (P ≤ 0.05). Statistical analysis demonstrated there was a significant difference (P < 0.001) between coconut milk and probiotic milk as well as HBSS in maintaining cell viability. However, there was no significant difference between probiotic milk and HBSS in ability to maintain PDL cell viability (P > 0.05). Coconut milk may not be suitable as an interim transport media due to poor maintenance of cell viability. However, probiotic milk was able to maintain PDL cell viability as well as HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

PubMed Central

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-01-01

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%–3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process. PMID:28772551

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

PubMed

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-02-16

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Isolation of cell-free bacterial inclusion bodies.

PubMed

Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

2010-09-17

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

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

Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.

We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwovenmore » scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.« less

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

Molecular mechanisms of cell-cell spread of intracellular bacterial pathogens.

PubMed

Ireton, Keith

2013-07-17

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell-cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at 'tricellular junctions'--specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

PubMed

Hastings, Jordan; Kenealey, Jason

2017-01-01

Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G 1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G 1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

Band Gap Engineering of Titania Film through Cobalt Regulation for Oxidative Damage of Bacterial Respiration and Viability.

PubMed

Li, Jinhua; Wang, Jiaxing; Wang, Donghui; Guo, Geyong; Yeung, Kelvin W K; Zhang, Xianlong; Liu, Xuanyong

2017-08-23

Biomaterial-related bacterial infections cause patient suffering, mortality, and extended periods of hospitalization and impose a substantial burden on medical systems. In this context, understanding the interactions between nanomaterials and bacteria is clinically significant. Herein, TiO 2 -based heterojunctions, including Co-TiO 2 , CoO-TiO 2 , and Co 3 O 4 -TiO 2 , were first designed by optimizing magnetron sputtering to establish a platform to explore the interactions between nanomaterials and bacteria. We found that the energy band bending and band gap narrowing were effectively promoted at the contact interface of the heterojunctions, which have the ability to induce abiotic reactive oxygen species formation. Using methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, in vitro studies showed that the heterojunctions of Co-TiO 2 , CoO-TiO 2 , and especially Co 3 O 4 -TiO 2 can effectively downregulate the expression levels of bacterial respiratory genes and cause oxidative damage to bacterial membrane respiration and viability. As a result, the surfaces of the heterojunctions possess a favorable antiadherent bacterial activity. Moreover, using an osteomyelitis model, the preclinical study on rats further confirmed the favorable anti-infection effect of the elaborately designed heterojunctions (especially Co 3 O 4 -TiO 2 ). We hope this study can provide new insights into the surface antibacterial design of biomaterials using energy band engineering for both basic research and clinical needs. Meanwhile, this attempt may also contribute to expanding the biomedical applications of cobalt-based nanoparticles for the treatment of antibiotic-resistant infections.

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

Functional microdomains in bacterial membranes.

PubMed

López, Daniel; Kolter, Roberto

2010-09-01

The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid--a known inhibitor of squalene synthases--impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated.

The in vitro impact of toothpaste extracts on cell viability.

PubMed

Cvikl, Barbara; Lussi, Adrian; Gruber, Reinhard

2015-06-01

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity. © 2015 Eur J Oral Sci.

Real time viability detection of bacterial spores

DOEpatents

Vanderberg, Laura A.; Herdendorf, Timothy J.; Obiso, Richard J.

2003-07-29

This invention relates to a process for detecting the presence of viable bacterial spores in a sample and to a spore detection system, the process including placing a sample in a germination medium for a period of time sufficient for commitment of any present viable bacterial spores to occur, mixing the sample with a solution of a lanthanide capable of forming a fluorescent complex with dipicolinic acid, and, measuring the sample for the presence of dipicolinic acid, and the system including a germination chamber having inlets from a sample chamber, a germinant chamber and a bleach chamber, the germination chamber further including an outlet through a filtering means, the outlet connected to a detection chamber, the detection chamber having an inlet from a fluorescence promoting metal chamber and the detection chamber including a spectral excitation source and a means of measuring emission spectra from a sample, the detection chamber further connected to a waste chamber. A germination reaction mixture useful for promoting commitment of any viable bacterial spores in a sample including a combination of L-alanine, L-asparagine and D-glucose is also described.

Innovative Microcapsules for Pancreatic β-Cells Harvested from Mature Double-Transgenic Mice: Cell Imaging, Viability, Induced Glucose-Stimulated Insulin Measurements and Proinflammatory Cytokines Analysis.

PubMed

Mooranian, Armin; Tackechi, Ryu; Jamieson, Emma; Morahan, Grant; Al-Salami, Hani

2017-06-01

Recently we demonstrated that microencapsulation of a murine pancreatic β-cell line using an alginate-ursodeoxycholic acid (UDCA) matrix produced microcapsules with good stability and cell viability. In this study, we investigated if translation of this formulation to microencapsulation of primary β-cells harvested from mature double-transgenic healthy mice would also generate stable microcapsules with good cell viability. Islets of Langerhans were isolated from Ngn3-GFP/RIP-DsRED mice by intraductal collagenase P digestion and density gradient centrifugation, dissociated into single cells and the β-cell population purified by Fluorescence Activated Cell Sorting. β-cells were microencapsulated using either alginate-poly-l-ornithine (F1; control) or alginate-poly-l-ornithine-UDCA (F2; test) formulations. Microcapsules were microscopically examined and microencapsulated cells were analyzed for viability, insulin and cytokine release, 2 days post-microencapsulation. Microcapsules showed good uniformity and morphological characteristics and even cell distribution within microcapsules with or without UDCA. Two days post microencapsulation cell viability, mitochondrial ATP and insulin production were shown to be optimized in the presence of UDCA whilst production of the proinflammatory cytokine IL-1β was reduced. Contradictory to our previous studies, UDCA did not reduce production of any other pro-inflammatory biomarkers. These results suggest that UDCA incorporation improves microcapsules' physical and morphological characteristics and improves the viability and function of encapsulated mature primary pancreatic β-cells.

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 cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

PubMed

Mayer, Fabiana Quoos; Baldo, Guilherme; de Carvalho, Talita Giacomet; Lagranha, Valeska Lizzi; Giugliani, Roberto; Matte, Ursula

2010-05-01

Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.

Effect of berberine on the viability of adipose tissue-derived mesenchymal stem cells in nutrients deficient condition.

PubMed

Ghorbani, Ahmad; Baradaran Rahimi, Vafa; Sadeghnia, Hamid Reza; Hosseini, Azar

2018-03-01

This study was designed to examine whether berberine protects rat adipose tissue-derived stem cells (ASCs) against glucose and serum deprivation (GSD)-induced cell death. ASCs were cultured for 24 h in GSD condition in the presence of berberine and then cell viability, apoptosis and generation of reactive oxygen species (ROS) were evaluated. The GSD condition significantly decreased ASCs viability and increased ROS generation and apoptosis. Incubation with 0.75-3 μM berberine partially increased cell viability and decreased ROS generation and apoptosis in GSD condition. In conclusion, berberine partially protects ASCs in nutrients deficient condition and may help ASCs to preserve their survival during cell therapy of ischemia.

Effect of low-level laser irradiation on proliferation and viability of human dental pulp stem cells.

PubMed

Zaccara, Ivana Maria; Ginani, Fernanda; Mota-Filho, Haroldo Gurgel; Henriques, Águida Cristina Gomes; Barboza, Carlos Augusto Galvão

2015-12-01

A positive effect of low-level laser irradiation (LLLI) on the proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). The aim of this study was to identify the lowest energy density able to promote the proliferation of DPSCs and to maintain cell viability. Human DPSCs were isolated from two healthy third molars. In the third passage, the cells were irradiated or not (control) with an InGaAlP diode laser at 0 and 48 h using two different energy densities (0.5 and 1.0 J/cm²). Cell proliferation and viability and mitochondrial activity were evaluated at intervals of 24, 48, 72, and 96 h after the first laser application. Apoptosis- and cell cycle-related events were analyzed by flow cytometry. The group irradiated with an energy density of 1.0 J/cm² exhibited an increase of cell proliferation, with a statistically significant difference (p < 0.05) compared to the control group at 72 and 96 h. No significant changes in cell viability were observed throughout the experiment. The distribution of cells in the cell cycle phases was consistent with proliferating cells in all three groups. We concluded that LLLI, particularly a dose of 1.0 J/cm², contributed to the growth of DPSCs and maintenance of its viability. This fact indicates this therapy to be an important future tool for tissue engineering and regenerative medicine involving stem cells.

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

Differential Effects of Bevacizumab, Ranibizumab, and Aflibercept on the Viability and Wound Healing of Corneal Epithelial Cells.

PubMed

Kang, Seungbum; Choi, Hyunsu; Rho, Chang Rae

2016-12-01

This study compared the effects of 3 antivascular endothelial growth factor (VEGF) agents (bevacizumab, ranibizumab, and aflibercept) on corneal epithelial cell viability and wound healing using human corneal epithelial cells (HCECs). To determine the cytotoxic effects of anti-VEGF agents on HCECs, HCEC viability was determined at various concentrations of these agents. An in vitro migration assay was used to investigate the migration of HCECs treated with 3 anti-VEGF agents. The protein level of extracellular signal-regulated kinase was used to evaluate the effect of anti-VEGF treatment on cell proliferation. The protein levels of p38 mitogen-activated protein kinase (MAPK) were analyzed by Western blotting to investigate cell migration. After 24 or 48 h of exposure, aflibercept treatment showed no apparent effect on cell viability; however, bevacizumab and ranibizumab treatment decreased cell viability at high concentrations (1 and 2 mg/mL). A migration assay showed that HCEC migration was different among the 3 anti-VEGF treatment groups. Bevacizumab significantly delayed HCEC migration. Western blotting showed that bevacizumab treatment decreased the expression levels of phosphorylated p38 MAPK. Bevacizumab, the most widely used and investigated anti-VEGF agent, decreased epithelial cell migration and viability. Anti-VEGF agents other than bevacizumab might therefore be better for treating corneal neovascularization complicated with epithelial defects.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Osteochondral Tissue Cell Viability Is Affected by Total Impulse during Impaction Grafting

PubMed Central

Balash, Paul; Kang, Richard W.; Schwenke, Thorsten; Cole, Brian J.; Wimmer, Markus A.

2010-01-01

Objective: Osteochondral graft transplantation has garnered significant attention because of its ability to replace the lesion with true hyaline cartilage. However, surgical impaction of the graft to anchor it into the defect site can be traumatic and lead to cell death and cartilage degeneration. This study aimed to test the hypothesis that increasing impulse magnitude during impaction of osteochondral plugs has a direct effect on loss of cell viability. Design: In this controlled laboratory study, the impaction force was kept constant while the impulse was varied. Ninety-six osteochondral plugs were extracted from the trochlea of bovine stifle joints and were randomly assigned into 3 experimental and 1 (nonimpacted) control group. The transferred impulse of the experimental groups reflected the median and the lower and upper quartiles of preceding clinical measurements. Data were obtained at day 0, day 4, and day 8; at each point, cell viability was assessed using the Live/Dead staining kit and histological assessments were performed to visualize matrix structural changes. Results: After impaction, cartilage samples stayed intact and did not show any histological signs of matrix disruption. As expected, higher impulse magnitudes introduced more cell death; however, this relationship was lost at day 8 after impaction. Conclusion: Impulse magnitude has a direct effect on cell viability of the graft. Because impulse magnitude is mostly governed by the press-fit characteristics of the recipient site, this study aids in the definition of optimal insertion conditions for osteochondral grafts. PMID:26069558

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

PubMed

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

2012-01-01

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

Photo-activated disinfection based on indocyanine green against cell viability and biofilm formation of Porphyromonas gingivalis.

PubMed

Pourhajibagher, Maryam; Chiniforush, Nasim; Ghorbanzadeh, Roghayeh; Bahador, Abbas

2017-03-01

Photo-activated disinfection (PAD) is a novel treatment approach, in which bacteria in the root canal system may be exposed to sub-lethal doses of PAD. Such exposure can affect bacterial survival and virulence features, such as biofilm formation ability. The aim of this study was to evaluate the effects of sub-lethal doses of PAD (sPAD) using indocyanine green (ICG) on load and biofilm formation ability of Porphyromonas gingivalis as an anaerobic bacterium associated with endodontic infection. The anti-bacterial and anti-biofilm potential of sPAD against P. gingivalis at sub-lethal doses of ICG as a photosensitizer and using 810nm wavelength of diode laser light via colony forming unit and crystal violet assays, respectively, was determined. High concentrations of ICG and light irradiation time significantly reduced bacteria. High doses of sPAD markedly reduced the number of bacteria and the formation of biofilm, up to 30.4% and 25.1%, respectively. High doses of sPAD affected cell viability and the biofilm formation ability of P. gingivalis; lower doses did not. Thus, selection of appropriate PAD dosage should be considered for the successful treatment of endodontic in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

PubMed

Duan, Qiaonan; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Benes, Cyril H; Ma'ayan, Avi

2014-11-15

Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Bacterial spread from cell to cell: beyond actin-based motility.

PubMed

Kuehl, Carole J; Dragoi, Ana-Maria; Talman, Arthur; Agaisse, Hervé

2015-09-01

Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteus mirabilis viability after lithotripsy of struvite calculi

NASA Astrophysics Data System (ADS)

Prabakharan, Sabitha; Teichman, Joel M. H.; Spore, Scott S.; Sabanegh, Edmund; Glickman, Randolph D.; McLean, Robert J. C.

2000-05-01

Urinary calculi composed of struvite harbor urease-producing bacteria within the stone. The photothermal mechanism of holmium:YAG lithotripsy is uniquely different than other lithotripsy devices. We postulated that bacterial viability of struvite calculi would be less for calculi fragmented with holmium:YAG irradiation compared to other lithotripsy devices. Human calculi of known struvite composition (greater than 90% magnesium ammonium phosphate hexahydrate) were incubated with Proteus mirabilis. Calculi were fragmented with no lithotripsy (controls), or shock wave, intracorporeal ultrasonic, electrohydraulic, pneumatic, holmium:YAG or pulsed dye laser lithotripsy. After lithotripsy, stone fragments were sonicated and specimens were serially plated for 48 hours at 38 C. Bacterial counts and the rate of bacterial sterilization were compared. Median bacterial counts (colony forming units per ml) were 8 X 106 in controls and 3 X 106 in shock wave, 3 X 107 in ultrasonic, 4 X 105 in electrohydraulic, 8 X 106 in pneumatic, 5 X 104 in holmium:YAG and 1 X 106 in pulsed dye laser lithotripsy, p less than 0.001. The rate of bacterial sterilization was 50% for holmium:YAG lithotripsy treated stones versus 0% for each of the other cohorts, p less than 0.01. P. mirabilis viability is less after holmium:YAG irradiation compared to other lithotripsy devices.

Canine adipose-derived stromal cell viability following exposure to synovial fluid from osteoarthritic joints.

PubMed

Kiefer, Kristina M; O'Brien, Timothy D; Pluhar, Elizabeth G; Conzemius, Michael

2015-01-01

Stem cell therapy used in clinical application of osteoarthritis in veterinary medicine typically involves intra-articular injection of the cells, however the effect of an osteoarthritic environment on the fate of the cells has not been investigated. Assess the viability of adipose derived stromal cells following exposure to osteoarthritic joint fluid. Adipose derived stromal cells (ASCs) were derived from falciform adipose tissue of five adult dogs, and osteoarthritic synovial fluid (SF) was obtained from ten patients undergoing surgical intervention on orthopedic diseases with secondary osteoarthritis. Normal synovial fluid was obtained from seven adult dogs from an unrelated study. ASCs were exposed to the following treatment conditions: culture medium, normal SF, osteoarthritic SF, or serial dilutions of 1:1 to 1:10 of osteoarthritic SF with media. Cells were then harvested and assessed for viability using trypan blue dye exclusion. There was no significant difference in the viability of cells in culture medium or normal SF. Significant differences were found between cells exposed to any concentration of osteoarthritic SF and normal SF and between cells exposed to undiluted osteoarthritic SF and all serial dilutions. Subsequent dilutions reduced cytotoxicity. Osteoarthritic synovial fluid in this ex vivo experiment is cytotoxic to ASCs, when compared with normal synovial fluid. Current practice of direct injection of ASCs into osteoarthritic joints should be re-evaluated to determine if alternative means of administration may be more effective.

Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

PubMed

McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

2012-03-01

We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

PubMed Central

Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

2012-01-01

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions

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

Sepulveda-Medina, Paola; Katsenovich, Yelena; Musaramthota, Vishal

Nuclear production facilities during the Cold War have caused liquid waste to leak and soak into the ground creating multiple radionuclide plumes. The Arthrobacter bacteria are one of the most common groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface after uranium exposure and evaluated the effect of bicarbonate ions on U(VI) toxicity of a less uranium tolerant Arthrobacter strain, G968, by investigating changes in adhesion forces and cells dimensions via atomic force microscopy (AFM). AFM and viability studies showed that samples containing bicarbonate aremore » able to acclimate and withstand uranium toxicity. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which might be an indication that the cells are not alive.« less

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

PubMed

Smeringaiova, Ingrida; Trosan, Peter; Mrstinova, Miluse Berka; Matecha, Jan; Burkert, Jan; Bednar, Jan; Jirsova, Katerina

2017-09-01

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

Formation of bacterial nanocells

NASA Astrophysics Data System (ADS)

Vainshtein, Mikhail; Kudryashova, Ekaterina; Suzina, Natalia; Ariskina, Elena; Voronkov, Vadim

1998-07-01

Existence of nanobacteria received increasing attention both in environmental microbiology/geomicro-biology and in medical microbiology. In order to study a production of nanoforms by typical bacterial cells. Effects of different physical factors were investigated. Treatment of bacterial cultures with microwave radiation, or culturing in field of electric current resulted in formation a few types of nanocells. The number and type of nanoforms were determined with type and dose of the treatment. The produced nanoforms were: i) globules, ii) clusters of the globules--probably produced by liaison, iii) nanocells coated with membrane. The viability of the globules is an object opened for doubts. The nanocells discovered multiplication and growth on solidified nutrient media. The authors suggest that formation of nanocells is a common response of bacteria to stress-actions produced by different agents.

Evaluation of goat milk as storage media to preserve viability of human periodontal ligament cells in vitro.

PubMed

Ulusoy, Ayça Tuba; Kalyoncuoglu, Elif; Kaya, Senay; Cehreli, Zafer Cavit

2016-08-01

The purpose of this study was to evaluate the effectiveness of goat milk as a storage media for maintenance of periodontal ligament (PDL) cell viability of avulsed teeth and compare it with commonly used and/or investigated storage media. PDL cells were obtained from the root surface of healthy premolars and were cultured in Eagle's maintenance medium (EMM). Cell cultures were treated with the following storage media: tap water (negative control); EMM (positive control); Hank's balanced salt solution; ultra high temperature (UHT) long-shelf-life lactose-free cow milk; UHT long-shelf-life whole cow milk; UHT long-shelf-life skimmed cow milk; UHT long-shelf-life soy milk; UHT long-shelf-life goat milk, UHT long-shelf-life follow on milk with probiotic, 20% propolis, and egg white. Culture plates were incubated with experimental media at 20°C for 1, 3, 6, 12, and 24 h. PDL cell viability was assessed by tetrazolium salt-based colorimetric (MTT) assay at each test period. One-way anova was used to evaluate the effects of storage solutions at each time point, followed by post hoc Duncan's multiple comparison test (P = 0.05). A dendrogram was constructed to show the arrangement of hierarchical clustering. Goat milk displayed the highest capacity to maintain cell viability at all test intervals (P < 0.001). Between 3 and 24 h, milk with the probiotic showed the lowest time-dependent PDL cell viability among all test media (P < 0.001). Compared with all milks, HBSS performed significantly less effectively in maintaining PDL cell viability during the entire test period (P < 0.001). Based on PDL viability, goat milk can be recommended as a suitable storage medium for avulsed teeth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dragon's Blood Sap (Croton Lechleri) As Storage Medium For Avulsed Teeth: In Vitro Study Of Cell Viability.

PubMed

Martins, Christine Men; Hamanaka, Elizane Ferreira; Hoshida, Thayse Yumi; Sell, Ana Maria; Hidalgo, Mirian Marubayashi; Silveira, Catarina Soares; Poi, Wilson Roberto

2016-01-01

Tooth replantation success depends on the condition of cementum periodontal ligament after tooth avulsion; which is influenced by storage medium. The dragon's blood (Croton lechleri) sap has been suggested as a promising medium because it supports collagen formation and exhibits healing, anti-inflammatory and antimicrobial properties. Thus, the aim of this study was to evaluate the efficacy of dragon's blood sap as a storage medium for avulsed teeth through evaluation of functional and metabolic cell viability. This in vitro study compared the efficacy of different storage media to maintain the viability of human peripheral blood mononuclear and periodontal ligament cells. A 10% dragon's blood sap was tested while PBS was selected as its control. Ultra pasteurized whole milk was used for comparison as a commonly used storage medium. DMEM and distilled water were the positive and negative controls, respectively. The viability was assessed through trypan blue exclusion test and colorimetric MTT assay after 1, 3, 6, 10 and 24 h of incubation. The dragon's blood sap showed promising results due to its considerable maintenance of cell viability. For trypan blue test, the dragon's blood sap was similar to milk (p<0.05) and both presented the highest viability values. For MTT, the dragon's blood sap showed better results than all storage media, even better than milk (p<0.05). It was concluded that the dragon's blood sap was as effective as milk, the gold standard for storage medium. The experimental sap preserved the membrane of all cells and the functional viability of periodontal ligament cells.

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

PubMed Central

Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

2012-01-01

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry.

PubMed

Servais; Courties; Lebaron; Troussellier

1999-08-01

> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

PubMed

Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

2016-07-01

Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

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.

Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.

PubMed

Nyholm, S V; McFall-Ngai, M J

1998-10-01

The symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvironment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate, were collected from anesthetized host animals that had been exposed to a light cue. Microscopy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacteria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bacterial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phagocytic activity of a subset of the host cell population suggested that some of these cells are macrophage-like molluscan hemocytes.

Preventing bacterial growth on implanted device with an interfacial metallic film and penetrating X-rays.

PubMed

An, Jincui; Sun, An; Qiao, Yong; Zhang, Peipei; Su, Ming

2015-02-01

Device-related infections have been a big problem for a long time. This paper describes a new method to inhibit bacterial growth on implanted device with tissue-penetrating X-ray radiation, where a thin metallic film deposited on the device is used as a radio-sensitizing film for bacterial inhibition. At a given dose of X-ray, the bacterial viability decreases as the thickness of metal film (bismuth) increases. The bacterial viability decreases with X-ray dose increases. At X-ray dose of 2.5 Gy, 98% of bacteria on 10 nm thick bismuth film are killed; while it is only 25% of bacteria are killed on the bare petri dish. The same dose of X-ray kills 8% fibroblast cells that are within a short distance from bismuth film (4 mm). These results suggest that penetrating X-rays can kill bacteria on bismuth thin film deposited on surface of implant device efficiently.

Long Noncoding RNA H19 Inhibits Cell Viability, Migration, and Invasion Via Downregulation of IRS-1 in Thyroid Cancer Cells

PubMed Central

Wang, Peng; Xu, Weimin; Liu, Haixia; Bu, Qingao; Sun, Diwen

2017-01-01

Thyroid cancer is a common endocrine gland malignancy which exhibited rapid increased incidence worldwide in recent decades. This study was aimed to investigate the role of long noncoding RNA H19 in thyroid cancer. Long noncoding RNA H19 was overexpressed or knockdown in thyroid cancer cells SW579 and TPC-1, and the expression of long noncoding RNA H19 was detected by real-time polymerase chain reaction. The cell viability, migration, and invasion were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, Transwell assay, and wound healing assay, respectively. Furthermore, cell apoptosis was analyzed by flow cytometry, and expressions of some factors that were related to phosphatidyl inositide 3-kinases/protein kinase B and nuclear factor κB signal pathway were measured by Western blotting. This study revealed that cell viability and migration/invasion of SW579 and TPC-1 were significantly decreased by long noncoding RNA H19 overexpression compared with the control group (P < .05), whereas cell apoptosis was statistically increased (P < .001). Meanwhile, cell viability and migration/invasion were significantly increased after long noncoding RNA H19 knockdown (P < .05). Furthermore, long noncoding RNA H19 negatively regulated the expression of insulin receptor substrate 1 and thus effect on cell proliferation and apoptosis. Insulin receptor substrate 1 regulated the activation of phosphatidyl inositide 3-kinases/AKT and nuclear factor κB signal pathways. In conclusion, long noncoding RNA H19 could suppress cell viability, migration, and invasion via downregulation of insulin receptor substrate 1 in SW579 and TPC-1 cells. These results suggested the important role of long noncoding RNA H19 in thyroid cancer, and long noncoding RNA H19 might be a potential target of thyroid cancer treatment. PMID:29332545

Effect of the oncolytic ECHO-7 virus Rigvir® on the viability of cell lines of human origin in vitro.

PubMed

Tilgase, Andra; Patetko, Liene; Blāķe, Ilze; Ramata-Stunda, Anna; Borodušķis, Mārtiņš; Alberts, Pēteris

2018-01-01

Background: The role of oncolytic viruses in cancer treatment is increasingly studied. The first oncolytic virus (Rigvir®, ECHO-7) was registered in Latvia over a decade ago. In a recent retrospective study Rigvir® decreased mortality 4.39-6.57-fold in stage IB-IIC melanoma patients. The aims of the present study are to test the effect of Rigvir® on cell line viability in vitro and to visualize the cellular presence of Rigvir® by immunocytochemistry. Methods: The cytolytic effect of Rigvir® on the viability of FM-9, RD, AGS, A549, HDFa, HPAF‑II, MSC, MCF7, HaCaT, and Sk-Mel-28 cell lines was measured using live cell imaging. PBMC viability was measured using flow cytometry. The presence of ECHO-7 virus was visualized using immunocytochemistry. Statistical difference between treatment groups was calculated using two-way ANOVA. Results: Rigvir® (10%, volume/volume) reduced cell viability in FM-9, RD, AGS, A549, HDFa, HPAF‑II and MSC cell lines by 67-100%. HaCaT cell viability was partly affected while Rigvir® had no effect on MCF7, Sk-Mel-28 and PBMC viability. Detection of ECHO-7 by immunocytochemistry in FM-9, RD, AGS, A549, HDFa, HPAF-II and Sk-Mel-28 cell lines suggests that the presence of Rigvir® in the cells preceded or coincided with the time of reduction of cell viability. Rigvir® (10%) had no effect on live PBMC count. Conclusions: The results suggest that Rigvir® in vitro reduces the viability of cells of human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma, pancreas adenocarcinoma but not in PBMC. The presence of Rigvir® in the sensitive cells was confirmed using anti-ECHO-7 antibodies. The present results suggest that a mechanism of action for the clinical benefit of Rigvir® is its cytolytic properties. The present results suggest that the effect of Rigvir® could be tested in other cancers besides melanoma. Further studies of possible Rigvir® entry receptors are needed.

Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

PubMed Central

Liu, Qiong; Chen, Jing; Wang, Xu; Yu, Liang; Hu, Li-hong; Shen, Xu

2010-01-01

Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation. Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis. PMID:20644552

In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

PubMed Central

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J.; Rad, Armin Tahmasbi; Madihally, Sundararajan V.; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments. PMID:23630603

In vitro electrochemical corrosion and cell viability studies on nickel-free stainless steel orthopedic implants.

PubMed

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J; Rad, Armin Tahmasbi; Madihally, Sundararajan V; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments.

Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

PubMed

Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

2017-06-01

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

The effect of Aloe vera gel on viability of dental pulp stem cells.

PubMed

Sholehvar, Fatemeh; Mehrabani, Davood; Yaghmaei, Parichehr; Vahdati, Akbar

2016-10-01

Dental pulp stem cells (DPSCs) can play a prominent role in tissue regeneration. Aloe vera L. (Liliaceae) contains the polysaccharide of acemannan that was shown to be a trigger factor for cell proliferation, differentiation, mineralization, and dentin formation. This study sought to determine the viability of DPSCs in Aloe vera in comparison with Hank's balanced salt solution (HBSS). Twelve rabbits underwent anesthesia, and their incisor teeth were extracted; the pulp tissue was removed, chopped, treated with collagenase and plated in culture flasks. DPSCs from passage 3 were cultured in 24-well plates, and after 3 days, the culture media changed to 10, 25, 50, and 100% concentrations of Aloe vera at intervals of 45 and 90 min and 3 and 6 h. Distilled water was used as negative and HBSS as positive control for comparison. The cell morphology, viability, population doubling time (PDT), and growth kinetics were evaluated. RT-PCR was carried out for characterization and karyotyping for chromosomal stability. Aloe vera showed a significant higher viability than HBSS (74.74%). The 50% Aloe vera showed higher viability (97.73%) than other concentrations. PDT in 50% concentration was 35.1 h and for HBSS was 49.5 h. DPSCs were spindle shaped and were positive for CD73 and negative for CD34 and CD45. Karyotyping was normal. Aloe vera as an inexpensive and available herb can improve survival of avulsed or broken teeth in emergency cases as a transfer media. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Simultaneous Gram and viability staining on activated sludge exposed to erythromycin: 3D CLSM time-lapse imaging of bacterial disintegration.

PubMed

Louvet, Jean-Noël; Attik, Ghania; Dumas, Dominique; Potier, Olivier; Pons, Marie-Noëlle

2011-11-01

The effect of erythromycin on activated sludge bacteria according to their Gram type was investigated with 3-dimensional Confocal Laser Scanning Microscopy (CLSM) time-lapse imaging. The fluorescent stains SYTOX Green and Texas Red-X conjugate of wheat germ agglutinin stained dying bacteria and Gram(+) bacteria respectively. Time-lapse imaging allowed an understanding of the staining mechanism and the measurement of the death rate. In presence of erythromycin (10mg/L), Gram(+) bacteria had a higher mortality rate than the Gram(-) bacteria. This result suggests that antibiotic in wastewater could change the activated sludge bacteria composition, according to their Gram type by selecting the bacteria which are the least sensitive to the antibiotics. However bacterial death was followed by bacterial disintegration leading to a decrease in the fluorescence. Results suggested that the viability indicators based on membrane integrity should be used with a correct sampling method, which can give the initial quantity of living bacteria. Copyright © 2011 Elsevier GmbH. All rights reserved.

Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS).

PubMed

Zhao, Wenqiang; Yang, Shanshan; Huang, Qiaoyun; Cai, Peng

2015-04-01

This study investigated the effect of loosely bound extracellular polymeric substances (LB-EPS) on the comprehensive surface properties of four bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida). The removal of LB-EPS from bacterial surfaces by high-speed centrifugation (12,000×g) was confirmed by SEM images. Viability tests showed that the percentages of viable cells ranged from 95.9% to 98.0%, and no significant difference was found after treatment (P>0.05). FTIR spectra revealed the presence of phosphodiester, carboxylic, phosphate, and amino functional groups on bacteria surfaces, and the removal of LB-EPS did not alter the types of cell surface functional groups. Potentiometric titration results suggested the total site concentrations on the intact bacteria were higher than those on LB-EPS free bacteria. Most of the acidity constants (pKa) were almost identical, except the increased pKa values of phosphodiester groups on LB-EPS free S. suis and E. coli surfaces. The electrophoretic mobilities and hydrodynamic diameters of the intact and LB-EPS free bacteria were statistically unchanged (P>0.05), indicating LB-EPS had no influence on the net surface charges and size distribution of bacteria. However, LB-ESP could enhance cell aggregation processes. The four LB-EPS free bacteria all exhibited fewer hydrophobicity values (26.1-65.0%) as compared to the intact cells (47.4-69.3%), suggesting the removal of uncharged nonpolar compounds (e.g., carbohydrates) in LB-EPS. These findings improve our understanding of the changes in cell surface characterizations induced by LB-EPS, and have important implications for assessing the role of LB-EPS in bacterial adhesion and transport behaviors. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxygen Delivery from Hyperbarically Loaded Microtanks Extends Cell Viability in Anoxic Environments

PubMed Central

Cook, Colin A.; Hahn, Kathryn C.; Morrissette-McAlmon, Justin B.F.; Grayson, Warren L.

2016-01-01

Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo. PMID:25818444

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

NASA Astrophysics Data System (ADS)

Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity in milled versus unmilled surface of the femoral head.

PubMed

Rhyu, Kee Hyung; Cho, Chang Hoon; Yoon, Kyung Sik; Chun, Young Soo

2016-12-01

To evaluate cellular activity in milled versus unmilled surface of the femoral head in 21 patients who underwent robot-assisted total hip arthroplasty(THA). The femoral head of 21 consecutive patients who underwent robot-assisted THA for osteonecrosis was used. 10 cc of trabecular bone from the entire milled surface was obtained using a curette. The same amount of trabecular bone was obtained at least 1 cm away from the milled surface and served as a matched control. Cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity in milled versus unmilled surface were assessed. Cell morphology of the milled or unmilled surface was comparable; cells were smaller in the milled surface. Cell viability was a mean of 40% higher in the milled surface (107.4% vs. 67.2%, p<0.001); cell viability at 5 time points was comparable in each group. Osteocalcin activity of cells was slightly higher in the milled surface (1.43 vs. 1.24 ng/ml, p=0.69). Alkaline phosphatase activity of cells was slightly higher in the unmilled surface (150 105 vs. 141 789 U/L, p=0.078). The milled and unmilled surfaces of the femoral head were comparable in terms of cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity.

Cell viability study of thermo-responsive core-shell superparamagnetic nanoparticles for multimodal cancer therapy

NASA Astrophysics Data System (ADS)

Shah, Saqlain A.; Majeed, A.; Shafique, M. A.; Rashid, K.; Awan, Saif-Ullah

2014-02-01

This is a vital extension of our previously published work. Thermo-responsive copolymer coated superparamagnetic MnFe2O4 nanoparticles are tested for cell viability and affinity on HeLa carcinoma cells under different conditions. Nanoparticles were loaded with anticancer drug doxorubicin. Composite nanoparticles of average diameter 45 nm were of core-shell structure having magnetic core of about 18 nm. Magnetic hyperthermia effects on cell viability and drug delivery were studied by exposing the cell suspension to high frequency magnetic field, and living cells were quantified using MTT method. There was almost absence of drug release at 37 °C. Drug was released at temperatures above lower critical solution temperature (LCST) by magnetic heating. LCST of the thermo-responsive copolymer was observed to be around 39 °C. Below this temperature, copolymer was hydrophilic and swelled. But above LCST, copolymer could become hydrophobic, expel water and drug and shrink in volume. Combination of hyperthermia and drug delivery effectively treated cancer cells.

Effect of Irrigation Time of Antiseptic Solutions on Bone Cell Viability and Growth Factor Release.

PubMed

Sawada, Kosaku; Nakahara, Ken; Haga-Tsujimura, Maiko; Fujioka-Kobayashi, Masako; Iizuka, Tateyuki; Miron, Richard J

2018-03-01

Antiseptic solutions are commonly utilized to treat local infection in the oral and maxillofacial region. However, surrounding vital bone is also exposed to antiseptic agents during irrigation and may have a potential negative impact on bone survival. The aim of the present study was therefore to investigate the effect of rinsing time with various antiseptic solutions on bone cell viability, as well as their subsequent release of growth factors important for bone regeneration. The bone samples collected from porcine mandible were rinsed in the following commonly utilized antiseptic solutions; povidone-iodine (0.5%), chlorhexidine digluconate (CHX, 0.2%), hydrogen peroxide (1%), and sodium hypochlorite (0.25%) for 1, 5, 10, 20, 30, or 60 minutes and assessed for cell viability and release of growth factors including vascular endothelial growth factor, transforming growth factor beta 1, bone morphogenetic protein 2, receptor activator of nuclear factor kappa-B ligand, and interleukin-1 beta by enzyme-linked immunosorbent assay. It was found in all the tested groups that the long exposure of any of the tested antiseptic solutions drastically promoted higher cell death. Sodium hypochlorite demonstrated the significantly highest cell death and at all time points. Interestingly, bone cell viability was highest in the CHX group post short-term rinsing of 1, 5, or 10 minutes when compared with the other 4 tested groups. A similar trend was also observed in subsequent growth factor release. The present study demonstrated that of the 4 tested antiseptic solutions, short-term CHX rinsing (ideally within 1 minute) favored bone cell viability and growth factor release. Clinical protocols should be adapted accordingly.

S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

PubMed

Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

2016-11-01

Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

PubMed

Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

2013-09-01

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

Differential eosinophil and mast cell regulation: Mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65

PubMed Central

Zhu, Xiang; Mose, Eucabeth; Hogan, Simon P.

2014-01-01

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. PMID:24742990

Cell Viability and Functionality of Probiotic Bacteria in Dairy Products

PubMed Central

Vinderola, Gabriel; Binetti, Ana; Burns, Patricia; Reinheimer, Jorge

2011-01-01

Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption. PMID:21833320

Growth mechanics of bacterial cell wall and morphology of bacteria

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2010-03-01

The peptidoglycan cell wall of bacteria is responsible for maintaining the cell shape and integrity. During the bacterial life cycle, the growth of the cell wall is affected by mechanical stress and osmotic pressure internal to the cell. We develop a theory to describe cell shape changes under the influence of mechanical forces. We find that the theory predicts a steady state size and shape for bacterial cells ranging from cocci to spirillum. Moreover, the theory suggest a mechanism by which bacterial cytoskeletal proteins such as MreB and crescentin can maintain the shape of the cell. The theory can also explain the several recent experiments on growing bacteria in micro-environments.

Two-dimensional and three-dimensional viability measurements of adult stem cells with optical coherence phase microscopy

NASA Astrophysics Data System (ADS)

Bagnaninchi, Pierre O.; Holmes, Christina; Drummond, Nicola; Daoud, Jamal; Tabrizian, Maryam

2011-08-01

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

The influence of micronutrients in cell culture: a reflection on viability and genomic stability.

PubMed

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Machado, Miriana; Bordin, Diana Lilian; Bergter, Lothar; Prá, Daniel; Henriques, João Antonio Pêgas

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

Escherichia coli viability determination using dynamic light scattering: a comparison with standard methods.

PubMed

Loske, Achim M; Tello, Elba M; Vargas, Susana; Rodriguez, Rogelio

2014-08-01

To determine the concentration of bacteria in a sample is important in the food industry, medicine and biotechnology. A disadvantage of the plate-counting method is that a microorganism colony could arise from one cell or from many cells. The other standard methodology, known as optical density determination, is based on the turbidity of a suspension and registers all bacteria, dead and alive. In this article, dynamic light scattering is proposed as a fast and reliable method to determine bacterial viability and, consequently, time evolution. Escherichia coli was selected because this microorganism is well known and easy to handle. A correlation between the data from these three techniques was obtained. We were able to calculate the growth rate, usually determined by plate counting or optical density measurement, using dynamic light scattering and to predict bacterial behavior. An analytical relationship between the colony forming units and the light scattered intensity was also deduced.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

PubMed Central

Poff, AM; Ari, C; Arnold, P; Seyfried, TN; D’Agostino, DP

2014-01-01

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of β-hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use. PMID:24615175

Comparative Evaluation of Cell Viability Immediately After Osteotomy for Implants With Drills and Piezosurgery: Immunohistochemistry Analysis.

PubMed

Pereira, Cassiano Costa Silva; Batista, Fábio Roberto de Souza; Jacob, Ricardo Garcia Mureb; Nogueira, Lamis Meorin; Carvalho, Abrahão Cavalcante Gomes de Souza; Gealh, Walter Cristiano; Garcia-Júnior, Idelmo Rangel; Okamoto, Roberta

2018-05-08

To evaluate the effect of reusing drills and piezosurgery tips during implant osteotomy on immediate bone cell viability through immunohistochemical analysis. Six male rabbits were divided into 2 groups and then divided into 5 subgroups-correspond to drills and tips used 10, 20, 30, 40, and 50 times, respectively. All animals received 10 osteotomies in each tibia, by use of the classic drilling procedure in one group (G1) and the piezosurgery device in the other group (G2). For immunohistochemical technique were utilized the osteoprotegerin, RANKL, osteocalcin, and caspase 3. Control procedures were performed by omitting the primary antibodies (negative control). Bone formation and resorption responses presented in more intense way during the piezosurgery. The expression of osteocalcin had become quite intense in piezosurgery groups, but with reduced immunostaining from the 30th osteotomy. The caspase 3 showed the viability of the osteoblast from the 20th osteotomy with piezosurgery and remained constant until the 50th. Piezosurgery provides greater osteoblastic cell viability than the system of conventional drilling. This study will provide data so that the authors can recycle the drills and tips for implant placement, thus enabling a better cell viability for osseointegration.

High Modulus Biodegradable Polyurethanes for Vascular Stents: Evaluation of Accelerated in vitro Degradation and Cell Viability of Degradation Products

PubMed Central

Sgarioto, Melissa; Adhikari, Raju; Gunatillake, Pathiraja A.; Moore, Tim; Patterson, John; Nagel, Marie-Danielle; Malherbe, François

2015-01-01

We have recently reported the mechanical properties and hydrolytic degradation behavior of a series of NovoSorb™ biodegradable polyurethanes (PUs) prepared by varying the hard segment (HS) weight percentage from 60 to 100. In this study, the in vitro degradation behavior of these PUs with and without extracellular matrix (ECM) coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C) conditions. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of human umbilical vein endothelial cells (HUVEC) were examined. The results showed that PUs with HS 80% and below completely disintegrated leaving no visual polymer residue at 18 weeks and the degradation medium turned acidic due to the accumulation of products from the soft segment (SS) degradation. As expected the PU with the lowest HS was the fastest to degrade. The accumulated degradation products, when tested undiluted, showed viability of about 40% for HUVEC cells. However, the viability was over 80% when the solution was diluted to 50% and below. The growth of HUVEC cells is similar to but not identical to that observed with tissue culture polystyrene standard (TCPS). The results from this in vitro study suggested that the PUs in the series degraded primarily due to the SS degradation and the cell viability of the accumulated acidic degradation products showed poor viability to HUVEC cells when tested undiluted, however particles released to the degradation medium showed cell viability over 80%. PMID:26000274

Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

PubMed Central

Kminek, Gerhard; Bada, Jeffrey L.; Pogliano, Kit; Ward, John F.

2014-01-01

Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003). When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars. PMID:12751954

Incidence and Predictors of Bacterial infection in Febrile Children with Sickle Cell Disease.

PubMed

Morrissey, Benita J; Bycroft, Thomas P; Almossawi, Ofran; Wilkey, Olufunke B; Daniels, Justin G

2015-01-01

Children with sickle cell disease are at increased risk of developing bacteremia and other serious bacterial infections. Fever is a common symptom in sickle cell disease and can also occur with sickle cell crises and viral infections. We aimed to evaluate the incidence and predictors of bacteremia and bacterial infection in children with sickle cell disease presenting with fever to a district hospital and sickle cell center in London. A retrospective analysis was performed on all attendances of children (aged under 16 years) with sickle cell disease presenting with a fever of 38.5 °C or higher over a 1-year period. Confirmed bacterial infection was defined as bacteremia, bacterial meningitis, urinary tract infection (UTI), pneumonia, osteomyelitis or other bacterial infection with positive identification of organism. Children were defined as having a suspected bacterial infection if a bacterial infection was suspected clinically, but no organism was identified. Over a 1-year period there were 88 episodes analyzed in 59 children. Bacteremia occurred in 3.4% of episodes and confirmed bacterial infection in 7.0%. Suspected bacterial infection occurred in 33.0%. One death occurred from Salmonella typhirium septicemia. C-reactive protein (CRP) level and white blood cell (WBC) count were both significantly associated with bacterial infection (p = 0.004 and 0.02, respectively.) In conclusion, bacterial infections continue to be a significant problem in children with sickle cell disease. C-reactive protein was significantly associated with bacterial infections, and could be included in clinical risk criteria for febrile children with sickle cell disease.

Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.

PubMed

Steele-Mortimer, O; Knodler, L A; Finlay, B B

2000-02-01

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

An In vitro Comparison of Coconut Water, Milk, and Saline in Maintaining Periodontal Ligament Cell Viability

PubMed Central

D’Costa, Vivian Flourish; Bangera, Madhu Keshava; Kini, Shravan; Kutty, Shakkira Moosa; Ragher, Mallikarjuna

2017-01-01

Background and Objectives: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extraoral dry time and the storage media in which the tooth is placed before treatment is rendered. The present study is undertaken to evaluate the periodontal ligament (PDL) cell viability after storage of teeth in different storage media, namely, coconut water, milk, and saline. Materials and Methods: Forty sound human premolars undergoing extraction for orthodontic purpose were selected. The teeth were allowed to lie dry on sand/mud for 30 min followed by which they were randomly divided and stored in three different media, i.e., coconut water, milk, and saline. After 45-min storage in their respective media, the root surface was then scraped for PDL tissue. Results: The ANOVA and Newman–Keuls post hoc procedure for statistical analysis of viable cell count under a light microscope using hemocytometer demonstrated that coconut water preserved significantly more PDL cells viable (P < 0.05) compared with milk and saline. Conclusion: Storage media help in preserving the viability of PDL cells when immediate replantation is not possible. This study evaluated the posttraumatic PDL cells’ viability following storage in three different storage media. Within the parameters of this study, it was found that coconut water is the most effective media for maintaining the viability of PDL. PMID:29284947

Proteomic analysis of the bacterial cell cycle

PubMed Central

Grünenfelder, Björn; Rummel, Gabriele; Vohradsky, Jiri; Röder, Daniel; Langen, Hanno; Jenal, Urs

2001-01-01

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. PMID:11287652

Biosensors for Whole-Cell Bacterial Detection

PubMed Central

Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

2014-01-01

SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells.

PubMed

Grabinski, Nicole; Ewald, Florian

2014-12-01

Ibrutinib (formerly PCI-32765) is a specific, irreversible, and potent inhibitor of Burton's tyrosine kinase (BTK) developed for the treatment of several forms of blood cancer. It is now an FDA-approved drug marketed under the name Imbruvica(TM) (Pharmacyclics, Inc.) and successfully used as an orally administered second-line drug in the treatment of mantle cell lymphoma. Since BTK is predominantly expressed in hematopoietic cells, the sensitivity of solid tumor cells to Ibrutinib has not been analyzed. In this study, we determined the effect of Ibrutinib on breast cancer cells. We demonstrate that Ibrutinib efficiently reduces the phosphorylation of the receptor tyrosine kinases ErbB1, ErbB2 and ErbB3, thereby suppressing AKT and MAPK signaling in ErbB2-positive (ErbB2+) breast cancer cell lines. Treatment with Ibrutinib significantly reduced the viability of ErbB2+ cell lines with IC50 values at nanomolar concentrations, suggesting therapeutic potential of Ibrutinib in breast cancer. Combined treatment with Ibrutinib and the dual PI3K/mTOR inhibitor BEZ235 synergistically reduces cell viability of ErbB2+ breast cancer cells. Combination indices below 0.25 at 50% inhibition of cell viability were determined by the Chou-Talalay method. Therefore, the combination of Ibrutinib and canonical PI3K pathway inhibitors could be a new and effective approach in the treatment of breast cancer with activated ErbB receptors. Ibrutinib could thus become a valuable component of targeted therapy in aggressive ErbB2+ breast cancer.

Elucidating Duramycin's Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope.

PubMed

Hasim, Sahar; Allison, David P; Mendez, Berlin; Farmer, Abigail T; Pelletier, Dale A; Retterer, Scott T; Campagna, Shawn R; Reynolds, Todd B; Doktycz, Mitchel J

2018-01-01

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus -derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin's mode of action and a better understanding of its selectivity.

Lidocaine cytotoxicity to the bovine articular chondrocytes in vitro: changes in cell viability and proteoglycan metabolism.

PubMed

Miyazaki, Tsuyoshi; Kobayashi, Shigeru; Takeno, Kenichi; Yayama, Takafumi; Meir, Adam; Baba, Hisatoshi

2011-07-01

A lot of studies on the effect of intra-articular injections are clinical, but many questions on the effect of lidocaine to articular chondrocytes remain unanswered. This study was performed to determine the effects of varying concentrations and exposure times of lidocaine on the viability and proteoglycan metabolism of chondrocytes in vitro. Cartilage was obtained from metatarsal joints of adult bovines. Chondrocytes in alginate beads were cultured in medium containing 6% fetal calf serum at 370 mOsmol at cell densities of 4 million cells/ml. They were then cultured for 24 h under 21% oxygen with 0.125, 0.25, 0.5, and 1% lidocaine and without lidocaine as control. The cell viability profile across intact beads was determined by manual counting using fluorescent probes and transmission electron microscopy. Lactate production was measured enzymatically as a marker of energy metabolism. Glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Cell viability decreased in a time- and dose-dependent manner in the concentration range of 0.125-1.0% lidocaine under the confocal microscope. Under the electron microscope, apoptosis increased as the concentration of lidocaine increased. GAG accumulation/tissue volume decreases as the concentration of lidocaine increased. However, GAG produced per million cells and the rate of lactate production per live cell were significantly higher for cells cultured at 0.5 and 1% lidocaine than the control group. Bovine chondrocytes cultured in alginate beads under high oxygen pressure are negatively influenced by increasing concentrations of lidocaine. Cell viability and proteoglycan production (GAG accumulation/tissue volume) decreased as the concentration of lidocaine increased. These data suggest caution in prolonged exposure of cartilage to high concentration lidocaine. Repeated joint injection of lidocaine potentially worsens osteoarthrosis by accelerating cartilage degradation.

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring.

PubMed

Halonen, Niina; Kilpijärvi, Joni; Sobocinski, Maciej; Datta-Chaudhuri, Timir; Hassinen, Antti; Prakash, Someshekar B; Möller, Peter; Abshire, Pamela; Kellokumpu, Sakari; Lloyd Spetz, Anita

2016-01-01

Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated.

The addition of albumin improves Schwann cells viability in nerve cryopreservation.

PubMed

González Porto, Sara Alicia; Domenech, Nieves; González Rodríguez, Alba; Avellaneda Oviedo, Edgar Mauricio; Blanco, Francisco J; Arufe Gonda, María C; Álvarez Jorge, Ángel; Sánchez Ibañez, Jacinto; Rendal Vázquez, Esther

2018-04-26

The purpose of the current study was to establish a valid protocol for nerve cryopreservation, and to evaluate if the addition of albumin supposed any advantage in the procedure. We compared a traditional cryopreservation method that uses dimethyl sulfoxide (DMSO) as cryoprotectant, to an alternative method that uses DMSO and albumin. Six Wistar Lewis rats were used to obtain twelve 20 mm fragments of sciatic nerve. In the first group, six fragments were cryopreserved in 199 media with 10% DMSO, with a temperature decreasing rate of 1 °C per minute. In the second group, six fragments were cryopreserved adding 4% human albumin. The unfreezing process consisted of sequential washings with saline in the first group, and saline and 20% albumin in the second group at 37 °C until the crioprotectant was removed. Structural evaluation was performed through histological analysis and electronic microscopy. The viability was assessed with the calcein-AM (CAM) and 4',6-diamino-2-fenilindol (DAPI) staining. Histological results showed a correct preservation of peripheral nerve architecture and no significant differences were found between the two groups. However, Schwann cells viability showed in the CAM-DAPI staining was significantly superior in the albumin group. The viability of Schwann cells was significantly increased when albumin was added to the nerve cryopreservation protocol. However, no significant structural differences were found between groups. Further studies need to be performed to assess the cryopreserved nerve functionality using this new method.

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

PubMed

Lablanche, Sandrine; Cottet-Rousselle, Cécile; Argaud, Laurent; Laporte, Camille; Lamarche, Frédéric; Richard, Marie-Jeanne; Berney, Thierry; Benhamou, Pierre-Yves; Fontaine, Eric

2015-01-01

Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success. Copyright © 2015 Elsevier B.V. All rights reserved.

Polyphenolic extracts of edible flowers incorporated onto atelocollagen matrices and their effect on cell viability.

PubMed

López-García, Jorge; Kuceková, Zdenka; Humpolíček, Petr; Mlček, Jiři; Sáha, Petr

2013-10-30

The phenolic extract of chives flowers (Allium schoenoprasum, Liliaceae), introduced Sage (Salvia pratensis, Lamiaceae), European elderberry (Sambucus nigra, Caprifoliaceae) and common dandelion (Taraxacum officinale, Asteraceae) were characterised by High Performance Liquid Chromatography and incorporated in different concentrations onto atelocollagen thin films. In order to assess the biological impact of these phenolic compounds on cell viability, human immortalised non-tumorigenic keratinocyte cell line was seeded on the thin films and cell proliferation was determined by using an MTT assay. In addition, their antimicrobial activity was estimated by using an agar diffusion test. Data indicated the concomitance between cell viability and concentration of polyphenols. These findings suggest that these phenolic-endowed atelocollagen films might be suitable for tissue engineering applications, on account of the combined activity of polyphenols and collagen.

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

PubMed

Bukhari, Maurish; Burm, Hayley; Samways, Damien S K

2016-10-01

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

PubMed

Zhang, Di; Ren, Li; Chen, Guan-Qun; Zhang, Jie; Reed, Barbara M; Shen, Xiao-Hui

2015-09-01

Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

NASA Astrophysics Data System (ADS)

Catros, Sylvain; Guillotin, Bertrand; Bačáková, Markéta; Fricain, Jean-Christophe; Guillemot, Fabien

2011-04-01

Biofabrication of three dimensional tissues by Laser-Assisted Bioprinting (LAB) implies to develop specific strategies for assembling the extracellular matrix (ECM) and cells. Possible strategies consist in (i) printing cells onto or in the depth of ECM layer and/or (ii) printing bioinks containing both cells and ECM-like printable biomaterial. The aim of this article was to evaluate combinatorial effects of laser pulse energy, ECM thickness and viscosity of the bioink on cell viability. A LAB workstation was used to print Ea.hy926 endothelial cells onto a quartz substrate covered with a film of ECM mimicking Matrigel™. Hence, effect of laser energy, Matrigel™ film thickness and bioink viscosity was addressed for different experimental conditions (8-24 μJ, 20-100 μm and 40-110 mPa s, respectively). Cell viability was assessed by live/dead assay performed 24 h post-printing. Results show that increasing the laser energy tends to augment the cell mortality while increasing the thickness of the Matrigel™ film and the viscosity of the bioink support cell viability. Hence, critical printing parameters influencing high cell viability have been related to the cell landing conditions and more specifically to the intensity of the cell impacts occurring at the air-ECM interface and at the ECM-glass interface.

Bioactive and biocompatible copper containing glass-ceramics with remarkable antibacterial properties and high cell viability designed for future in vivo trials.

PubMed

Popescu, R A; Magyari, K; Vulpoi, A; Trandafir, D L; Licarete, E; Todea, M; Ştefan, R; Voica, C; Vodnar, D C; Simon, S; Papuc, I; Baia, L

2016-07-19

In the present study our interest is focused on finding the efficiency of 60SiO2·(32 - x)CaO·8P2O5·xCuO (mol%) glass-ceramics, with 0 ≤ x ≤ 4 mol%, in terms of bioactivity, biocompatibility, antibacterial properties and cell viability in order to determine the most appropriate composition for their further use in in vivo trials. The sol-gel synthesized samples show a preponderantly amorphous structure with a few crystallization centers associated with the formation of an apatite and calcium carbonate crystalline phases. The Fourier Transform Infrared (FT-IR) spectra revealed slightly modified absorption bands due to the addition of copper oxide, while the information derived from the measurements performed by transmission electron microscopy, UV-vis and electron paramagnetic resonance spectroscopy showed the presence of ions and metallic copper species. X-Ray photoelectron spectroscopic analysis indicated the presence of copper metallic species, in a reduced amount, only on the sample surface with the highest Cu content. Regarding in vitro assessment of bioactivity, the results obtained by X-ray diffraction, FT-IR spectroscopy and scanning electron microscopy, demonstrated the formation of a calcium phosphate layer on all investigated sample surfaces. The inhibitory effect of the investigated samples was more significant on the Pseudomonas aeruginosa than the Staphylococcus aureus strain, the sample with the lowest concentration of copper oxide (0.5 mol%) being also the most efficient in both bacterial cultures. This sample also exhibits a very good bactericidal activity, for the other samples it was necessary to use a higher quantity to inhibit and kill the bacterial species. The secondary structure of adsorbed albumin presents few minor changes, indicating the biocompatibility of the glass-ceramics. The cell viability assay shows a good proliferation rate on samples with 0.5 and 1.5 mol% CuO, although all glass-ceramic samples exhibited a good in vivo

The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

PubMed

Magcwebeba, Tandeka Unathi; Riedel, Sylvia; Swanevelder, Sonja; Swart, Pieter; De Beer, Dalene; Joubert, Elizabeth; Andreas Gelderblom, Wentzel Christoffel

2016-11-01

The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo. © 2016 Royal Pharmaceutical Society.

The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

PubMed Central

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

PubMed

Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

2017-01-01

The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

How plasma induced oxidation, oxygenation, and de-oxygenation influences viability of skin cells

NASA Astrophysics Data System (ADS)

Oh, Jun-Seok; Strudwick, Xanthe; Short, Robert D.; Ogawa, Kotaro; Hatta, Akimitsu; Furuta, Hiroshi; Gaur, Nishtha; Hong, Sung-Ha; Cowin, Allison J.; Fukuhara, Hideo; Inoue, Keiji; Ito, Masafumi; Charles, Christine; Boswell, Roderick W.; Bradley, James W.; Graves, David B.; Szili, Endre J.

2016-11-01

The effect of oxidation, oxygenation, and de-oxygenation arising from He gas jet and He plasma jet treatments on the viability of skin cells cultured in vitro has been investigated. He gas jet treatment de-oxygenated cell culture medium in a process referred to as "sparging." He plasma jet treatments oxidized, as well as oxygenated or de-oxygenated cell culture medium depending on the dissolved oxygen concentration at the time of treatment. He gas and plasma jets were shown to have beneficial or deleterious effects on skin cells depending on the concentration of dissolved oxygen and other oxidative molecules at the time of treatment. Different combinations of treatments with He gas and plasma jets can be used to modulate the concentrations of dissolved oxygen and other oxidative molecules to influence cell viability. This study highlights the importance of a priori knowledge of the concentration of dissolved oxygen at the time of plasma jet treatment, given the potential for significant impact on the biological or medical outcome. Monitoring and controlling the dynamic changes in dissolved oxygen is essential in order to develop effective strategies for the use of cold atmospheric plasma jets in biology and medicine.

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.

Measuring tendon properties in mdx mice: cell viability and viscoelastic characteristics.

PubMed

Rizzuto, E; Musarò, A; Catizone, A; Del Prete, Z

2009-10-16

Muscular dystrophy is a genetic disorder of skeletal muscle characterized by progressive muscle weakness. Here we assessed whether muscle wasting affects cell viability and mechanical properties of extensor digitorum longus (EDL) and of tibialis anterior (TA) tendons from mdx dystrophic mice compared to wild type (WT) mice. mdx mice represent the classical animal model for human Duchenne muscular dystrophy, and show several signs of the pathology, including a decrease in specific force and an increase of fibrotic index. Cell viability of tendons was evaluated by histological analysis, and viscoelastic properties have been assessed by a rapid measurement protocol that allowed us to compute, at the same time, tissue complex compliance for all the frequencies of interest. Confocal microscopy and mechanical properties measurements revealed that mdx tendons, compared to WT ones, have an increase in the number of dead cells and a significant reduction in tissue elasticity for all the frequencies that were tested. These findings indicate a reduced quality of the tissue. Moreover, mdx tendons have an increase in the viscous response, indicating that during dynamic loading, they dissipate more energy compared to WT. Our results demonstrate that muscular dystrophy involves not only muscle wasting, but also alteration in the viscoelastic properties of tendons, suggesting a paracrine effect of altered skeletal muscle on tendinous tissue.

Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression.

PubMed

Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J; Van der Heide, Emile

2017-06-01

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture.

Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression

PubMed Central

Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J.; Van der Heide, Emile

2017-01-01

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture. PMID:28761837

Mixed response in bacterial and biochemical variables to simulated sand mining in placer-rich beach sediments, Ratnagiri, West coast of India.

PubMed

Fernandes, Christabelle E G; Das, Anindita; Nath, B N; Faria, Daphne G; Loka Bharathi, P A

2012-05-01

We investigated the influence on bacterial community and biochemical variables through mechanical disturbance of sediment-akin to small-scale mining in Kalbadevi beach, Ratnagiri, a placer-rich beach ecosystem which is a potential mining site. Changes were investigated by comparing three periods, namely phase I before disturbance, phase II just after disturbance, and phase III 24 h after disturbance as the bacterial generation time is ≤7 h. Cores from dune, berm, high-, mid-, and low-tide were examined for changes in distribution of total bacterial abundance, total direct viability (counts under aerobic and anaerobic conditions), culturability and biochemical parameters up to 40 cm depth. Results showed that bacterial abundance decreased by an order from 10(6) cells g(-1) sediment, while, viability reduced marginally. Culturability on different-strength nutrient broth increased by 155% during phase II. Changes in sedimentary proteins, carbohydrates, and lipids were marked at berm and dune and masked at other levels by tidal influence. Sedimentary ATP reduced drastically. During phase III, Pearson's correlation between these variables evolved from non-significant to significant level. Thus, simulated disturbance had a mixed effect on bacterial and biochemical variables of the sediments. It had a negative impact on bacterial abundance, viability and ATP but positive impact on culturability. Viability, culturability, and ATP could act as important indicators reflecting the disturbance in the system at short time intervals. Culturability, which improved by an order, could perhaps be a fraction that contributes to restoration of the system at bacterial level. This baseline information about the potential mining site could help in developing rational approach towards sustainable harnessing of resources with minimum damage to the ecosystem.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

PubMed

Wang, Kai; Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

2016-01-01

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

PubMed Central

Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

2016-01-01

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis. PMID:27433029

Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells

PubMed Central

Tabatabaei, Fahimeh Sadat

2016-01-01

ABSTRACT Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells. PMID:27099698

Identification of individual biofilm-forming bacterial cells using Raman tweezers

NASA Astrophysics Data System (ADS)

Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

2015-05-01

A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral "Raman fingerprints" obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

Identification of individual biofilm-forming bacterial cells using Raman tweezers.

PubMed

Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

2015-05-01

A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

PubMed

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

Release kinetics and cell viability of ibuprofen nanocrystals produced by melt-emulsification.

PubMed

Fernandes, A R; Dias-Ferreira, J; Cabral, C; Garcia, M L; Souto, E B

2018-06-01

The clinical use of poorly water-soluble drugs has become a big challenge in pharmaceutical development due to the compromised bioavailability of the drugs in vivo. Nanocrystals have been proposed as a formulation strategy to improve the dissolution properties of these drugs. The benefits of using nanocrystals in drug delivery, when compared to other nanoparticles, are related to their production facilities, simple structure, and suitability for a variety of administration routes. High pressure homogenization (HPH) is the most promising production process, which can be employed at low or high temperatures. Ibuprofen nanocrystals with a mean size below 175 nm, and polydispersity below 0.18, have been produced by melt-emulsification, followed by HPH. Two nanocrystal formulations, differing on the surfactant composition, have been produced, their in vitro ibuprofen release tested in Franz diffusion cells and adjusted to several kinetic models (zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas, Baker-Lonsdale and Weibull model). Cell viability was assessed at 3, 6 and 24 h of incubation on human epithelial colorectal cells (Caco-2) by AlamarBlue ® colorimetric assay. For both formulations, Caco-2 cells viability was dependent on the drug concentration and time of exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

Low osmolality and shear stress during liposuction impair cell viability in autologous fat grafting.

PubMed

Ismail, T; Bürgin, J; Todorov, A; Osinga, R; Menzi, N; Largo, R D; Haug, M; Martin, I; Scherberich, A; Schaefer, D J

2017-05-01

Liposuction and subsequent autologous fat grafting have become essential techniques for fat augmentation in plastic surgery. However, standard harvesting techniques that ensure the survival of adipocytes and stromal vascular fraction (SVF) cells and thus preserve the transplanted fat volume are lacking. In particular, the effect of different parameters of the tumescent solution has not been studied in this context. We hypothesized that the osmolality of the tumescent solution could have a significant effect on the survival of adipocytes and SVF cells. We developed two distinct in vitro models based on freshly harvested excision fat from patients undergoing surgical treatment. First, we investigated the effect of osmolality by incubating excision fat in different tumescent solutions and analyzed the total cell survival and the differentiation potential of SVF cells. Vital whole-mount staining, isolation yield of SVF cells, clonogenicity, and osteogenic and adipogenic differentiation capacities were analyzed. Second, we addressed the additional effect of mechanical stress by simulating a liposuction on pieces of excision fat after incubation with the tumescent solutions. Osmolality of the tumescent solution by itself did not have a significant effect on adipocyte and SVF viability or SVF differentiation. However, when osmolality was combined with liposuction, a significant trend toward lower viability and more lipid droplets with lower osmolality was observed. Especially, SVF viability was significantly lower after liposuction with a hypotonic (150 mOsm/kg) solution. This study demonstrates the considerable effect of osmolality during liposuction and may lead to the development of "cell-protective" tumescent solutions. Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

PubMed

Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

2016-06-01

Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target. © 2014 John Wiley & Sons Ltd.

Effects of tocotrienols on cell viability and apoptosis in normal murine liver cells (BNL CL.2) and liver cancer cells (BNL 1ME A.7R.1), in vitro.

PubMed

Har, Chan Hooi; Keong, Chan Kok

2005-01-01

The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32 microg mL(-1). Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T(3)), alpha tocopherol (alpha-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T(3) significantly (P < or = 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a dose-responsive manner (8-16 microg mL(-1)), whereas T did not show any significant (P>0.05) inhibition in cell viability with increasing treatment doses of 0-16 microg mL(-1). The IC(50) for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 microg mL(-1) at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T(3) treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC(50) of 8.98 microg mL(-1) tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98 microg mL(-1)) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T(3)-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T(3)-treatment (8.98 microg mL(-1)) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T(3)-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T(3

Viability of a human melanoma cell after single and combined treatment with fotemustine, dacarbazine, and proton irradiation.

PubMed

Petrović, Ivan M; Korićanac, Lela B; Todorović, Danijela V; Ristić-Fira, Aleksandra M; Valastro, Lucia M; Privitera, Giuseppe; Cuttone, Giacomo

2007-01-01

Viability of human HTB140 melanoma cells after being exposed to fotemustine (FM) and dacarbazine (DTIC) as well as to proton irradiation was studied. Effects of 100 and 250 microM drugs were assessed after incubation of 6, 24, 48, 72, and 96 h. Irradiations were performed with 62 MeV therapeutic protons, delivering to the cell monolayer single doses of 2, 4, 8, 12, and 16 Gy. Viability was evaluated 7 days after irradiation. Inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB) assays. Combined effects of each drug and protons, were carried out using the same drug concentrations. Proton doses applied were those used in therapy, that is, 12 and 16 Gy. With the increase of drug concentration or irradiation dose, level of cell inactivation reached approximately 60%, 48 h after drug treatment or 7 days after irradiation at 16 Gy. Considering the rate of drug concentrations used, as well as the level of doses applied, it appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents tested. The combined treatment with FM or DTIC and protons did not show significant changes of cell viability as compared to the effects of single agents. Since the time point for measuring cumulative effects of drug and irradiation was 48 h post irradiation, it seems that the obtained level of viability could be attributed primarily to the effects of drugs.

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

NASA Astrophysics Data System (ADS)

Avitabile, Concetta; D'Andrea, Luca Domenico; Romanelli, Alessandra

2014-03-01

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells.

PubMed

Zhao, Xinyuan; Xing, Fengjun; Cong, Yewen; Zhuang, Yin; Han, Muxi; Wu, Zhiqiang; Yu, Shali; Wei, Haiyan; Wang, Xiaoke; Chen, Gang

2017-12-01

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-05-01

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10 5 (group A) or 8×10 5 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular

Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

PubMed

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2016-11-01

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm 2 . This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10 5 to 1.4 × 10 6 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

PubMed

Lushnikova, Iryna; Nikandrova, Yelyzaveta; Skibo, Galyna

2017-10-01

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α + ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α + level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies. © 2017 International Federation for Cell Biology.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells.

PubMed

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio; Párraga, Mario; Villena, Joan

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions . These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells

PubMed Central

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells. PMID:29552079

The type and composition of alginate and hyaluronic-based hydrogels influence the viability of stem cells of the apical papilla.

PubMed

Lambricht, Laure; De Berdt, Pauline; Vanacker, Julie; Leprince, Julian; Diogenes, Anibal; Goldansaz, Hadi; Bouzin, Caroline; Préat, Véronique; Dupont-Gillain, Christine; des Rieux, Anne

2014-12-01

The goal of the present work was to evaluate in vitro and in vivo the influence of various types and compositions of natural hydrogels on the viability and metabolic activity of SCAPs. Two alginate, three hyaluronic-based (Corgel™) hydrogel formulations and Matrigel were characterized for their mechanical, surface and microstructure properties using rheology, X-ray photoelectron spectroscopy and scanning electron microscopy, respectively. A characterized SCAP cell line (RP89 cells) was encapsulated in the different experimental hydrogel formulations. Cells were cultured in vitro, or implanted in cyclosporine treated mice. In vitro cell viability was evaluated using a Live/Dead assay and in vitro cellular metabolic activity was evaluated with a MTS assay. In vivo cell apoptosis was evaluated by a TUNEL test and RP89 cells were identified by human mitochondria immunostaining. Hydrogel composition influenced their mechanical and surface properties, and their microstructure. In vitro cell viability was above 80% after 2 days but decreased significantly after 7 days (60-40%). Viability at day 7 was the highest in Matrigel (70%) and then in Corgel 1.5 (60%). Metabolic activity increased over time in all the hydrogels, excepted in alginate SLM. SCAPs survived after 1 week in vivo with low apoptosis (<1%). The highest number of RP89 cells was found in Corgel 5.5 (140cells/mm(2)). Collectively, these data demonstrate that SCAP viability was directly modulated by hydrogel composition and suggest that a commercially available hyaluronic acid-based formulation might be a suitable delivery vehicle for SCAP-based dental pulp regeneration strategies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Comparative Effect Between Laser and Radiofrequency Heating of RGD-Gold Nanospheres on MCF7 Cell Viability.

PubMed

Sánchez-Hernández, Lidia; Ferro-Flores, Guillermina; Jiménez-Mancilla, Nallely P; Luna-Gutiérrez, Myrna A; Santos-Cuevas, Clara L; Ocampo-García, Blanca E; Azorín-Vega, Erika; Isaac-Olivé, Keila

2015-12-01

Gold nanoparticles conjugated to cyclo-[Arg-Gly-Asp-D-Phe-Lys(Cys)] peptides (AuNP-c[RGDfK(C)]) have been reported as systems with specific cell internalization in breast cancer cells. AuNPs have also been proposed as localized heat sources for cancer treatment using laser irradiation or radiofrequency (RF). The aim of this research was to analyze, based on the Mie theory, the AuNP-c[RGDfK(C)] absorption cross-sections (C(abs)) of low-frequency electromagnetic waves (13.56 MHz, λ = 22 m) and optical frequency waves (laser at λ = 532 nm) and to compare their effect on MCF7 cell viability as thermal conversion sources in AuNPs (20 nm) located inside cells. Cell viability was assessed in MCF7 cells treated with AuNP-c[RGDfK(C)] or water after exposure to the RF field (200 W, 100 V/cm) or laser irradiation (Irradiance 0.65 W/cm2). In both cases (RF and laser) the presence of nanoparticles in cells caused a significant increase in the temperature of the medium (RF: AT = 29.9 ± 1.7 degrees C for AuNP compared to ΔT = 13.0 ± 1.4 degrees C for water; laser: ΔT = 13.5 ± 0.7 degrees C for AuNP compared to 3.3 ± 0.5 degrees C for water). Although RF induced a higher increase in the temperature of the medium with nanoparticles, the largest effect on the cell viability was produced by laser when nanoparticles were located inside the cells (8.7?0.7% for laser compared to 19.4 ± 0.9% for RF). The differences obtained in C(abs) values (laser: 3.7 x 10- (16) m2; RF: 7.9 x 10-(23) m2) and the observed effect on MFC7 cell viability support two mechanisms previously proposed "wave energy absorption by AuNPs" when laser is used as a thermal conversion source, and "attenuation of the wave passing through the AuNP suspension" when RF is applied. The AuNP-c[RGDfK(C)] nanosystem shows suitable properties to improve hyperthermia treatments under laser irradiation due to a larger heat release inside cells.

The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis

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

Delijewski, Marcin; Wrześniok, Dorota; Beberok, Ar

Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine onmore » this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells. - Highlights: • Nicotine and UVA induced concentration-dependent loss in melanocytes viability. • Nicotine and UVA modulated melanization process in melanocytes. • Changes in viability and melanization were more pronounced in dark pigmented cells.« less

A stable live bacterial vaccine.

PubMed

Kunda, Nitesh K; Wafula, Denis; Tram, Meilinn; Wu, Terry H; Muttil, Pavan

2016-06-01

Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most. Copyright © 2016 Elsevier B.V. All rights reserved.

The preservative effect of Thai propolis extract on the viability of human periodontal ligament cells.

PubMed

Prueksakorn, Attaporn; Puasiri, Subin; Ruangsri, Supanigar; Makeudom, Anupong; Sastraruji, Thanapat; Krisanaprakornkit, Suttichai; Chailertvanitkul, Pattama

2016-12-01

Tooth avulsion causes an injury to the periodontal ligament (PDL). The success of tooth replantation depends on the quantity and quality of PDL cells. The aim of this study was to examine the preservative and proliferative effects of Thai propolis extract, previously shown to exert anti-inflammatory and antioxidant activities, on human PDL cells. Ninety-six premolars were left to air dry for 30 min and stored in Hank's balanced salt solution (HBSS), milk, or various concentrations of propolis extract from 0.25 to 10 mg ml -1 for 3 h. PDL cells were isolated by collagenase and trypsin digestion, and their viability was determined by a trypan blue dye exclusion assay. PDL tissues were also scraped off the root surface and cultured to determine cell growth and morphology. The alamarBlue ® and BrdU assays were performed to determine the cytotoxic and proliferative effects of the extract on cultured PDL cells, respectively. A non-toxic dose of 2.5 mg ml -1 of propolis extract yielded the greatest percentage of cell viability (78.84 ± 3.34%), which was significantly higher than those of the other concentrations (P < 0.001). Nevertheless, this percentage was not significantly different from that of HBSS (80.14 ± 2.44%; P = 1.00), but was significantly higher than that of milk (71.27 ± 2.79%; P < 0.001). The cells grown from PDL explants looked like fibroblasts. However, 2.5 mg ml -1 of the extract did not induce PDL cell proliferation. Thai propolis extract at 2.5 mg ml -1 appears to be the most effective dose for preserving the viability of PDL cells, and this was comparable to HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

PubMed

Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C

1994-01-01

We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin.

PubMed

Lin, Yueh-Ming; Chen, Chih-I; Hsiang, Yi-Ping; Hsu, Yung-Chia; Cheng, Kung-Chuan; Chien, Pei-Hsuan; Pan, Hsiao-Lin; Lu, Chien-Chang; Chen, Yun-Ju

2018-06-14

Chemotherapeutic 5-fluorouracil (5-FU) combined with oxaliplatin is often used as the standard treatment for colorectal cancer (CRC). The disturbing side effects and drug resistance commonly observed in chemotherapy motivate us to develop alternative optimal therapeutic options for CRC treatment. Chrysin, a natural and biologically active flavonoid abundant in propolis, is reported to have antitumor effects on a few CRCs. However, whether and how chrysin achieves similar effectiveness to the 5-FU combination is not clear. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), western blotting, fluorescence microscopy, and reactive oxygen species (ROS) production were assayed. We found that chrysin exhibited similar inhibition of cell viability as the 5-FU combination in a panel of human CRC cells. Furthermore, the results showed that chrysin significantly increased the levels of LC3-II, an autophagy-related marker, in CRC cells, which was not observed with the 5-FU combination. More importantly, blockage of autophagy induction restored chrysin-attenuated CRC cell viability. Further mechanistic analysis revealed that chrysin, not the 5-FU combination, induced ROS generation, and in turn, inhibited the phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Collectively, these results imply that chrysin may be a potential replacement for the 5-FU and oxaliplatin combination to achieve antitumor activity through autophagy for CRC treatment in the future.

Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor.

PubMed

Cimetta, E; Flaibani, M; Mella, M; Serena, E; Boldrin, L; De Coppi, P; Elvassore, N

2007-05-01

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.

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.

Impact of lithium alone or in combination with haloperidol on oxidative stress parameters and cell viability in SH-SY5Y cell culture.

PubMed

Gawlik-Kotelnicka, Oliwia; Mielicki, Wojciech; Rabe-Jabłońska, Jolanta; Lazarek, Jerry; Strzelecki, Dominik

2016-02-01

It has been reported that lithium may inhibit lipid peroxidation and protein oxidation. Lithium salts also appear to stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress and neurodegeneration may play an important role in the pathophysiology of bipolar disorder and the disease course thereof. The aim of this research is to estimate the influence of lithium (alone and in combination with haloperidol) on the parameters of oxidative stress and viability of SH-SY5Y cell lines in neutral and pro-oxidative conditions. The evaluated oxidative stress parameter was lipid peroxidation. The viability of the cell lines was measured utilising the MTT test. In neutral conditions, higher levels of thiobarbituric acid reactive substances were observed in those samples which contained both haloperidol and lithium than in other samples. However, these differences were not statistically significant. Cell viability was significantly higher in therapeutic lithium samples than in the controls; samples of haloperidol alone as well as those of haloperidol with lithium did not differ from controls. The results of our study may indicate that lithium possess neuroprotective properties that may be partly due to antioxidative effects. The combination of lithium and haloperidol may generate increased oxidative stress.

Monitoring of microbial cell viability using nanostructured electrodes modified with Graphene/Alumina nanocomposite.

PubMed

Hassan, Rabeay Y A; Mekawy, Moataz M; Ramnani, Pankaj; Mulchandani, Ashok

2017-05-15

Microbial infections are rapidly increasing; however most of the existing microbiological and molecular detection methods are time consuming and/or cannot differentiate between the viable and dead cells which may overestimate the risk of infections. Therefore, a bioelectrochemical sensing platform with a high potential to the microbial-electrode interactions was designed based on decorated graphene oxide (GO) sheet with alumina (Al 2 O 3 ) nanocrystals. GO-Al 2 O 3 nanocomposite was synthesized using self-assembly of GO and Al 2 O 3 and characterized using the scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), Raman-spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Enhancement of electrocatalytic activity of the composite-modified electrode was demonstrated. Thus, using the GO-Al 2 O 3 nanocomposite modified electrode, the cell viability was determined by monitoring the bioelectrochemical response of the living microbial cells (bacteria and yeast) upon stimulation with carbon source. The bioelectrochemical assay was optimized to obtain high sensitivity and the method was applied to monitor cell viability and screen susceptibility of metabolically active cells (E. coli, B. subtilis, Enterococcus, P. aeruginosa and Salmonella typhi) to antibiotics such as ampicillin and kanamycin. Therefore, the developed assay is suitable for cell proliferation and cytotoxicity testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

PubMed

Ferreira, Iêda Mendes; de Souza Lacerda, Camila Maria; de Faria, Lígia Santana; Corrêa, Cristiane Rodrigues; de Andrade, Antero Silva Ribeiro

2014-12-01

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

PubMed Central

Bedrosian, Andrea S.; Nguyen, Andrew H.; Hackman, Michael; Connolly, Michael K.; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E.; Henning, Justin R.; Barilla, Rocky; Rehman, Adeel; Pachter, H. Leon; Medina-Zea, Marco V.; Cohen, Steven M.; Frey, Alan B.; Acehan, Devrim; Miller, George

2011-01-01

Background & Aims Acute pancreatitis increases morbidity and mortality from organ necrosis by mechanisms that are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. Methods Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier analysis. Results Numbers of MHC II+CD11c+DC increased 100-fold in pancreas of mice with acute pancreatitis, to account for nearly 15% of intra-pancreatic leukocytes. Intra-pancreatic DC acquired an immune phenotype in mice with acute pancreatitis; they expressed higher levels of MHC II and CD86 and increased production of interleukin-6, membrane cofactor protein (MCP)-1, and tumor necrosis factor (TNF)-α. However, rather than inducing an organ-destructive inflammatory process, DC were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DC and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DC died from acinar cell death within 4 days. Depletion of DC from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DC did not require infiltrating neutrophils, activation of NF-κB, or signaling by mitogen-activated protein kinase or TNF-α. Conclusions DC are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress. PMID:21801698

Conventional and nonconventional strategies for controlling bacterial contamination in fuel ethanol fermentations.

PubMed

Ceccato-Antonini, Sandra Regina

2018-05-25

Ethanol bio-production in Brazil has some unique characteristics that inevitably lead to bacterial contamination, which results in the production of organic acids and biofilms and flocculation that impair the fermentation yield by affecting yeast viability and diverting sugars to metabolites other than ethanol. The ethanol-producing units commonly give an acid treatment to the cells after each fermentative cycle to decrease the bacterial number, which is not always effective. An alternative strategy must be employed to avoid bacterial multiplication but must be compatible with economic, health and environmental aspects. This review analyzes the issue of bacterial contamination in sugarcane-based fuel ethanol fermentation, and the potential strategies that may be utilized to control bacterial growth besides acid treatment and antibiotics. We have emphasized the efficiency and suitability of chemical products other than acids and those derived from natural sources in industrial conditions. In addition, we have also presented bacteriocins, bacteriophages, and beneficial bacteria as non-conventional antimicrobial agents to mitigate bacterial contamination in the bioethanol industry.

Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

NASA Astrophysics Data System (ADS)

Morato de Souza, Letícia; Guilherme Roque Rinco, Ugo; Aparecida Tavares Aguiar, Daniela; Aparecido de Almeida Junior, Luciano; Cosme-Silva, Leopoldo; Marchini Oliveira, Thais; Teixeira Marques, Nádia Carolina; Thiemy Sakai, Vivien

2018-02-01

This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm-2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P  <  0.05). At 24 h, SHED under nutritional deficit showed lower viability and proliferation after 1.2 J cm-2 irradiation. All of the irradiated groups revealed significantly higher viability and proliferation in SHED maintained under nutritional deficit than in regular nutritional conditions, except in the 3.7 and 6.2 J cm-2 groups by MTT assay. In the crystal violet assay, SHED irradiated with 1.2 J cm-2 showed no difference between the different nutritional conditions. Decrease of FBS concentration in the culture medium seems to enhance the sensitivity of SHED to the effects of photobiomodulation therapy. Nutritional stress conditions improved cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm-2.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

PubMed

Tang, Chuan-Xi; Gu, Yan-Xia; Liu, Xin-Feng; Tong, Shu-Yan; Ayanlaja, Abiola A; Gao, Yue; Ji, Guang-Quan; Xiong, Ye; Huang, Lin-Yan; Gao, Dian-Shuai

2018-07-01

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50 ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

PubMed Central

Nothaft, Harald; Zheng, Jing

2013-01-01

Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

FGF1-gold nanoparticle conjugates targeting FGFR efficiently decrease cell viability upon NIR irradiation

PubMed Central

Szlachcic, Anna; Pala, Katarzyna; Zakrzewska, Malgorzata; Jakimowicz, Piotr; Wiedlocha, Antoni; Otlewski, Jacek

2012-01-01

Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy. PMID:23226697

Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

PubMed

Parker, Jennifer K; Wisotsky, Sarah R; Johnson, Evan G; Hijaz, Faraj M; Killiny, Nabil; Hilf, Mark E; De La Fuente, Leonardo

2014-01-01

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

Measuring bacterial cells size with AFM

PubMed Central

Osiro, Denise; Filho, Rubens Bernardes; Assis, Odilio Benedito Garrido; Jorge, Lúcio André de Castro; Colnago, Luiz Alberto

2012-01-01

Atomic Force Microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria revealing surface details and cell integrity. During scanning however, the interactions between the AFM probe and the membrane results in distortion of the images. Such distortions or artifacts are the result of geometrical effects related to bacterial cell height, specimen curvature and the AFM probe geometry. The most common artifact in imaging is surface broadening, what can lead to errors in bacterial sizing. Several methods of correction have been proposed to compensate for these artifacts and in this study we describe a simple geometric model for the interaction between the tip (a pyramidal shaped AFM probe) and the bacterium (Escherichia coli JM-109 strain) to minimize the enlarging effect. Approaches to bacteria immobilization and examples of AFM images analysis are also described. PMID:24031837

Elucidating Duramycin’s Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope

DOE PAGES

Hasim, Sahar; Allison, David P.; Mendez, Berlin; ...

2018-02-14

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity.more » Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.« less

Elucidating Duramycin’s Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope

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

Hasim, Sahar; Allison, David P.; Mendez, Berlin

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity.more » Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.« less

Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

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

Gonzalez-Montalban, Nuria; Departament de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona; Ciber de Bioingenieria, Biomateriales y Nanomedicina

The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-likemore » structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone {beta}-galactosidase fusion protein are clearly toxic for mammalian cells but the {beta}-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.« less

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

PubMed

Stockert, Juan C; Horobin, Richard W; Colombo, Lucas L; Blázquez-Castro, Alfonso

2018-04-01

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described. Copyright © 2018 Elsevier GmbH. All rights reserved.

Effects of hydrostatic pressure and supercritical carbon dioxide on the viability of Botryococcus braunii algae cells.

PubMed

Yildiz-Ozturk, Ece; Ilhan-Ayisigi, Esra; Togtema, Arnoud; Gouveia, Joao; Yesil-Celiktas, Ozlem

2018-05-01

In bio-based industries, Botryococcus braunii is identified as a potential resource for production of hydrocarbons having a wide range of applications in chemical and biopolymer industries. For a sustainable production platform, the algae cultivation should be integrated with downstream processes. Ideally the algae are not harvested, but the product is isolated while cultivation and growth is continued especially if the doubling time is slow. Consequently, hydrocarbons can be extracted while keeping the algae viable. In this study, the effects of pressure on the viability of B. braunii cells were tested hydrostatically and under supercritical CO 2 conditions. Viability was determined by light microscopy, methylene blue uptake and by re-cultivation of the algae after treatments to follow the growth. It was concluded that supercritical CO 2 was lethal to the algae, whereas hydrostatic pressure treatments up to 150 bar have not affected cell viability and recultivation was successful. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microbial whole‐cell arrays

PubMed Central

Elad, Tal; Lee, Jin Hyung; Belkin, Shimshon; Gu, Man Bock

2008-01-01

Summary The coming of age of whole‐cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines – the whole‐cell array. In the present review, we highlight the state‐of‐the‐art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high‐performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis and – most importantly – enhanced long‐term maintenance of viability and activity on the fabricated biochips. PMID:21261831

Structure of a bacterial cell surface decaheme electron conduit

USDA-ARS?s Scientific Manuscript database

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

Hyaluronic acid increases tendon derived cell viability and proliferation in vitro: comparative study of two different hyaluronic acid preparations by molecular weight.

PubMed

Gallorini, Marialucia; Berardi, Anna C; Berardocco, Martina; Gissi, Clarissa; Maffulli, Nicola; Cataldi, Amelia; Oliva, Francesco

2017-01-01

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate the effects of two different HA on human rotator cuff tendon derived cells in terms of cell viability, proliferation and apoptosis. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of two different HA preparations: Sinovial HL® (High-Low molecular weight) (MW: 80-100 kDa) and KDa Sinovial Forte SF (MW: 800-1200), at various concentrations. Tendon derived cells morphology was evaluated after 0, 7 and 14 d of culture. Viability and proliferation were analyzed after 0, 24, and 48 h of culture and apoptosis occurrence was assessed after 24 h of culture. All the HAPs tested here increased viability and proliferation, in a dose-dependent manner and they reduced apoptosis at early stages (24 h) compared to control cells (without HAPs). HAPs enhanced viability and proliferation and counteracted apoptosis in tendon derived cells.

Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

PubMed

Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

2017-04-01

In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017. © 2017 Wiley Periodicals, Inc.

Bee venom induced cytogenetic damage and decreased cell viability in human white blood cells after treatment in vitro: a multi-biomarker approach.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2011-09-01

The aim of this study was to evaluate cytogenotoxic effects of bee venom to human lymphocytes and take a look into the mechanisms behind them. Bee venom was tested in concentrations ranging from 0.1μg/ml to 20μg/ml over different lengths of time. Cell viability, type of the cell death, and morphological alterations were evaluated using phase-contrast and fluorescent microscopy in addition to DNA diffusion assay, whereas cytogenotoxic effects were assessed with the micronucleus test. DNA damage and its relation to oxidative stress were evaluated combining the standard alkaline and the Fpg-modified comet assay. Our results showed lower cell viability, morphological cell alterations, cytogenotoxicity, and dominantly necrotic type of cell death in human lymphocytes after treatment with bee venom. All the effects were time- and dose-dependent. These results provide an insight into the effects of bee venom on the cell structure that could be relevant for therapeutic purposes. Copyright © 2011 Elsevier B.V. All rights reserved.

Species-specific viability analysis of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus in mixed culture by flow cytometry

PubMed Central

2014-01-01

Background Bacterial species coexist commonly in mixed communities, for instance those occurring in microbial infections of humans. Interspecies effects contribute to alterations in composition of communities with respect to species and thus, to the course and severity of infection. Therefore, knowledge concerning growth and viability of single species in medically-relevant mixed communities is of high interest to resolve complexity of interspecies dynamics and to support development of treatment strategies. In this study, a flow cytometric method was established to assess the species-specific viability in defined three-species mixed cultures. The method enables the characterization of viability of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus, which are relevant to lung infections of Cystic Fibrosis (CF) patients. The method combines fluorescence detection by antibody and lectin labeling with viability fluorescence staining using SYBR®Green I and propidium iodide. In addition, species-specific cell enumeration analysis using quantitative terminal restriction fragment length polymorphisms (qT-RFLP) was used to monitor the growth dynamics. Finally, to investigate the impact of substrate availability on growth and viability, concentrations of main substrates and metabolites released were determined. Results For each species, the time course of growth and viability during mixed culture cultivations was obtained by using qT-RFLP analysis in combination with flow cytometry. Comparison between mixed and pure cultures revealed for every species differences in growth properties, e.g. enhanced growth of P. aeruginosa in mixed culture. Differences were also observed for B. cepacia and S. aureus in the time course of viability, e.g. an early and drastic reduction of viability of S. aureus in mixed culture. Overall, P. aeruginosa clearly dominated the mixed culture with regard to obtained cell concentrations. Conclusions In combination with q

Surface-modified bacterial nanofibrillar PHB scaffolds for bladder tissue repair.

PubMed

Karahaliloğlu, Zeynep; Demirbilek, Murat; Şam, Mesut; Sağlam, Necdet; Mızrak, Alpay Koray; Denkbaş, Emir Baki

2016-01-01

The aim of the study is in vitro investigation of the feasibility of surface-modified bacterial nanofibrous poly [(R)-3-hydroxybutyrate] (PHB) graft for bladder reconstruction. In this study, the surface of electrospun bacterial PHB was modified with PEG- or EDA via radio frequency glow discharge method. After plasma modification, contact angle of EDA-modified PHB scaffolds decreased from 110 ± 1.50 to 23 ± 0.5 degree. Interestingly, less calcium oxalate stone deposition was observed on modified PHB scaffolds compared to that of non-modified group. Results of this study show that surface-modified scaffolds not only inhibited calcium oxalate growth but also enhanced the uroepithelial cell viability and proliferation.

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

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

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

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells.

PubMed

Kujawska, T; Secomski, W; Bilmin, K; Nowicki, A; Grieb, P

2014-07-01

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm(2) to 8 W/cm(2) (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm(2). Copyright © 2014 Elsevier B.V. All rights reserved.

Bacterial cell identification in differential interference contrast microscopy images.

PubMed

Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

2013-04-23

Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

PubMed

Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

2007-06-01

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles

NASA Astrophysics Data System (ADS)

Fede, C.; Albertin, Giovanna; Petrelli, L.; De Caro, R.; Fortunati, I.; Weber, V.; Ferrante, Camilla

2017-09-01

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

PubMed

Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

2014-04-01

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Lead Toxicity to the Performance, Viability, And Community Composition of Activated Sludge Microorganisms

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

Yuan, L; Zhi, W; Liu, YS

Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (<= 24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3-N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake ratemore » responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies.« less

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.

Harnessing cell-to-cell variations to probe bacterial structure and biophysics

NASA Astrophysics Data System (ADS)

Cass, Julie A.

Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

PubMed

Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

2015-12-01

We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

LncRNA-LET inhibits cell viability, migration and EMT while induces apoptosis by up-regulation of TIMP2 in human granulosa-like tumor cell line KGN.

PubMed

Han, Qingfang; Zhang, Wenke; Meng, Jinlai; Ma, Li; Li, Aihua

2018-04-01

Polycystic ovary syndrome (PCOS) is a common endocrine disease characterized by hyperandrogenism, irregular menses, and polycystic ovaries. Several long non-coding RNAs (lncRNAs) are aberrantly expressed in PCOS patients; however, little is known about the effects of the lncRNA-low expression in tumor (lncRNA-LET) on PCOS. We aimed to explore the effects of lncRNA-LET on human granulosa-like tumor cell line, KGN. Expression of lncRNA-LET in normal IOSE80 cells and granulosa cells was determined by qRT-PCR. KGN cell viability, apoptosis and migration were measured by trypan blue exclusion method, flow cytometry assay and wound healing assay, respectively. TGF-β1 was used to induce epithelial-mesenchymal transition (EMT) process. LncRNA-LET expression and mRNA expressions of TIMP2 and EMT-related proteins were measured by qRT-PCR. Western blot analysis was used to measure the protein expression of apoptosis-related proteins, EMT-related proteins, TIMP2, and the proteins in the Wnt/β-catenin and Notch signaling pathways. lncRNA-LET was down-regulated in KGN cells, and its overexpression inhibited cell viability and migration, and promoted apoptosis in KGN cells. Overexpression of lncRNA-LET increased the expression of E-cadherin and decreased the expressions of N-cadherin and vimentin in KGN cells. These effects of lncRNA-LET on KGN cells were reversed by TIMP2 suppression. Overexpression of TIMP2 inhibited cell viability, migration and EMT process, and increased apoptosis by activating the Wnt/β-catenin and Notch pathways. Overexpression of lncRNA-LET inhibits cell viability, migration and EMT process, and increases apoptosis in KGN cells by up-regulating the expression of TIMP2 and activating the Wnt/β-catenin and notch signaling pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Preliminary study on an innovative, simple mast cell-based electrochemical method for detecting foodborne pathogenic bacterial quorum signaling molecules (N-acyl-homoserine-lactones).

PubMed

Jiang, Donglei; Feng, Dongdong; Jiang, Hui; Yuan, Limin; Yongqi, Yin; Xu, Xin; Fang, Weiming

2017-04-15

This paper reports the a novel and simple mast cell-based electrochemical method for detecting of bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs), which can be utilized to preliminarily evaluate the toxicity of food-borne pathogenic bacteria. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide hydrogel were immobilized on a gold electrode, while mast cells as recognition elements were cultured in a 3D cell culture system. Electrochemical impedance spectroscopy (EIS) was utilized to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). The results indicated that cellular activities such as cell viability, apoptosis, intracellular calcium, and degranulation were markedly influenced by the AHLs. Importantly, the exposure of 3OC 12 -HSL to mast cells induced a marked decrease in the electrochemical impedance signal in a dose-dependent manner. The detection limit for 3OC 12 -HSL was 0.034μM with a linear range of 0.1-1μM. These results were confirmed via conventional cell assay and transmission electron microscope (TEM) analysis. Altogether, the proposed method appears to be an innovative and effective approach to the quantitative measurement of Gram-negative bacterial quorum signaling molecules; to this effect, it also may serve as a primary evaluation of the cytotoxicity of food-borne pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

PubMed

Wajid, Nadia; Naseem, Rashida; Anwar, Sanam Saiqa; Awan, Sana Javaid; Ali, Muhammad; Javed, Sara; Ali, Fatima

2015-09-01

Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

PubMed

Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

2018-07-01

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

PubMed Central

Davis, Grace L.; Ray, Nashone A.; Lahiri, Ramanuj; Gillis, Thomas P.; Krahenbuhl, James L.; Williams, Diana L.; Adams, Linda B.

2013-01-01

Background The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Methodology/Principle Findings Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. Conclusions hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

PubMed

Davis, Grace L; Ray, Nashone A; Lahiri, Ramanuj; Gillis, Thomas P; Krahenbuhl, James L; Williams, Diana L; Adams, Linda B

2013-01-01

The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and

The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

PubMed

Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

2016-03-01

It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded

Cell viability viscoelastic measurement in a rheometer used to stress and engineer tissues at low sonic frequencies.

PubMed

Klemuk, Sarah A; Jaiswal, Sanyukta; Titze, Ingo R

2008-10-01

Effects of vibration on human vocal fold extracellular matrix composition and the resultant tissue viscoelastic properties are difficult to study in vivo. Therefore, an in vitro bioreactor, simulating the in vivo physiological environment, was explored. A stress-controlled commercial rheometer was used to administer shear vibrations to living tissues at stresses and frequencies corresponding to male phonation, while simultaneously measuring tissue viscoelastic properties. Tissue environment was evaluated and adjustments made in order to sustain cell life for short term experimentation up to 6 h. Cell nutrient medium evaporation, osmolality, pH, and cell viability of cells cultured in three-dimensional synthetic scaffolds were quantified under comparably challenging environments to the rheometer bioreactor for 4 or 6 h. The functionality of the rheometer bioreactor was demonstrated by applying three vibration regimes to cell-seeded three-dimensional substrates for 2 h. Resulting strain was quantified throughout the test period. Rheologic data and cell viability are reported for each condition, and future improvements are discussed.

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality.

PubMed

Angel, Stephanie; von Briesen, Hagen; Oh, Young-Joo; Baller, Marko K; Zimmermann, Heiko; Germann, Anja

2016-12-01

Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

Different binarization processes validated against manual counts of fluorescent bacterial cells.

PubMed

Tamminga, Gerrit G; Paulitsch-Fuchs, Astrid H; Jansen, Gijsbert J; Euverink, Gert-Jan W

2016-09-01

State of the art software methods (such as fixed value approaches or statistical approaches) to create a binary image of fluorescent bacterial cells are not as accurate and precise as they should be for counting bacteria and measuring their area. To overcome these bottlenecks, we introduce biological significance to obtain a binary image from a greyscale microscopic image. Using our biological significance approach we are able to automatically count about the same number of cells as an individual researcher would do by manual/visual counting. Using the fixed value or statistical approach to obtain a binary image leads to about 20% less cells in automatic counting. In our procedure we included the area measurements of the bacterial cells to determine the right parameters for background subtraction and threshold values. In an iterative process the threshold and background subtraction values were incremented until the number of particles smaller than a typical bacterial cell is less than the number of bacterial cells with a certain area. This research also shows that every image has a specific threshold with respect to the optical system, magnification and staining procedure as well as the exposure time. The biological significance approach shows that automatic counting can be performed with the same accuracy, precision and reproducibility as manual counting. The same approach can be used to count bacterial cells using different optical systems (Leica, Olympus and Navitar), magnification factors (200× and 400×), staining procedures (DNA (Propidium Iodide) and RNA (FISH)) and substrates (polycarbonate filter or glass). Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

PubMed

Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

2015-05-01

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells.

PubMed

Peternel, Spela; Komel, Radovan

2010-09-10

In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process.To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared.During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation.During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity.High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

Potential of coconut water and soy milk for use as storage media to preserve the viability of periodontal ligament cells: an in vitro study.

PubMed

Moura, Camilla Cristhian Gomes; Soares, Priscilla Barbosa Ferreira; de Paula Reis, Manuella Verdinelli; Fernandes Neto, Alfredo Júlio; Zanetta Barbosa, Darceny; Soares, Carlos José

2014-02-01

There is no consensus regarding the ability of coconut water and soy milk to maintain long-term cell viability. This study investigated the ability of pH-adjusted coconut water and soy milk to maintain the viability of periodontal ligament cells over a short and a longer period and compared these abilities with those of other solutions. Dog premolar teeth were extracted, dried for 30 min, and stored in the following media for 50 min or 24 h: long shelf-life whole milk (SWM), long shelf-life skim milk (SSM), Hank's Balanced Salt Solution (HBSS), soy milk (SM), and pH-adjusted coconut water (CW). The positive and two negative control groups corresponded to 0-min, 30-min (short-term), and 24-h (long-term) dry times, respectively. Cell viability was analyzed by trypan blue exclusion. Data were statistically analyzed using the Kruskal-Wallis test with post-analysis using the Dunn method. In the short-term experiment, the SSM resulted in significantly lower cell viability than SM and CW. At 24 h, SM and CW resulted in higher viability than HBSS and SSM and in comparable performance with the positive control group. Cell viability decreased over time, except in SM and CW. Soy milk and pH-adjusted coconut water showed promising results as storage solutions for avulsed teeth, preserving the viability for up to 24 h. © 2013 John Wiley & Sons A/S.

Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells.

PubMed

Sasaki, Natsuki; Nakamura, Masayuki; Kodama, Akiko; Urata, Yuka; Shiokawa, Nari; Hayashi, Takehiro; Sano, Akira

2016-11-01

The autophagy pathway has recently been implicated in several neurodegenerative diseases. Recently, it was reported that chorein-depleted cells showed accumulation of autophagic markers and impaired autophagic flux. Here, we demonstrate that chorein overexpression preserves cell viability from starvation-induced cell death in human embryonic kidney 293 (HEK293) cells. Subsequent coimmunoprecipitation and reverse coimmunoprecipitation assays using extracts from chorein that stably overexpressed HEK293 cells revealed that chorein interacts with α-tubulin and histone deacetylase 6, a known α-tubulin deacetylater and central component of basal autophagy. Indeed, acetylated α-tubulin immunoreactivity was significantly decreased in chorein that stably overexpressed HEK293 cells. These results suggest that chorein/histone deacetylase 6/α-tubulin interactions may play an important role in starvation-induced cell stress, and their disruption may be one of the molecular pathogenic mechanisms of chorea-acanthocytosis.-Sasaki, N., Nakamura, M., Kodama, A., Urata, Y., Shiokawa, N., Hayashi, T., Sano, A. Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells. © FASEB.

Effect of pulsed ultrasound in combination with gentamicin on bacterial viability in biofilms on bone cements in vivo.

PubMed

Ensing, G T; Roeder, B L; Nelson, J L; van Horn, J R; van der Mei, H C; Busscher, H J; Pitt, W G

2005-01-01

The aim of this study is to investigate whether pulsed ultrasound (US) in combination with gentamicin yields a decreased viability of bacteria in biofilms on bone cements in vivo. Bacterial survival on bone cement in the presence and absence of ultrasound was compared in a rabbit model. Two bone cement samples with an Escherichia coli ATCC 10798 biofilm were implanted in a total of nine rabbits. In two groups bone cement discs loaded with gentamicin, freshly prepared and aged were used, and in one group unloaded bone cement discs in combination with systemically administered gentamicin. Pulsed ultrasound with a frequency of 28.48 kHz and a maximum acoustic intensity of 500 mW cm(-2) was applied continuously from 24 h till 72 h postsurgery on one of the two implanted discs. After euthanization and removal of the bacteria from the discs, the number of viable bacteria were quantified and skin samples were analysed for histopathological examination. Application of ultrasound, combined with gentamicin, reduced the viability of the biofilms in all three groups varying between 58 and 69% compared with the negative control. Histopathological examinations showed no skin lesions. Ultrasound resulted in a tendency of improved efficacy of gentamicin, either applied locally or systemically. Usage of ultrasound in this model proved to be safe. This study implies that ultrasound could improve the prevention of infection immediately after surgery, especially because the biomaterials, gentamicin and ultrasound used in this model are all in clinical usage, but not yet combined in clinical practice.

Concentrations of and application protocols for hydrogen peroxide bleaching gels: effects on pulp cell viability and whitening efficacy.

PubMed

Soares, Diana Gabriela; Basso, Fernanda Gonçalves; Hebling, Josimeri; de Souza Costa, Carlos Alberto

2014-02-01

To assess the whitening effectiveness and the trans-enamel/trans-dentinal toxicity of experimental tooth-bleaching protocols on pulp cells. Enamel/dentine discs individually adapted to trans-well devices were placed on cultured odontoblast-like cells (MDPC-23) or human dental pulp cells (HDPCs). The following groups were formed: G1 - no treatment (control); G2 to G4 - 35% H2O2, 3 × 15, 1 × 15, and 1 × 5 min, respectively; and G5 to G7 - 17.5% H2O2, 3 × 15, 1 × 15, and 1 × 5 min, respectively. Cell viability and morphology were evaluated immediately after bleaching (T1) and 72 h thereafter (T2). Oxidative stress and cell membrane damage were also assessed (T1). The amount of H2O2 in culture medium was quantified (Mann-Whitney; α=5%) and colour change (ΔE) of enamel was analysed after 3 sessions (Tukey's test; α=5%). Cell viability reduction, H2O2 diffusion, cell morphology alteration, oxidative stress, and cell membrane damage occurred in a concentration-/time-dependent fashion. The cell viability reduction was significant in all groups for HDPCs and only for G2, G3, and G5 in MDPC-23 cells compared with G1. Significant cell viability and morphology recovery were observed in all groups at T2, except for G2 in HDPCs. The highest ΔE value was found in G2. However, all groups presented significant ΔE increases compared with G1. Shortening the contact time of a 35%-H2O2 gel for 5 min, or reducing its concentration to 17.5% and applying it for 45, 15, or 5 min produce gradual tooth colour change associated with reduced trans-enamel and trans-dentinal cytotoxicity to pulp cells. The experimental protocols tested in the present study provided significant tooth-bleaching improvement associated with decreased toxicity to pulp cells, which may be an interesting alternative to be tested in clinical situations intended to reduce tooth sensitivity and pulp damage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of all-trans retinoic acid (ATRA) on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells.

PubMed

Bidad, Katayoon; Salehi, Eisa; Oraei, Mona; Saboor-Yaraghi, Ali-Akbar; Nicknam, Mohammad Hossein

2011-12-01

All-trans retinoic acid (ATRA), as an active metabolite of vitamin A, has been shown to affect immune cells. This study was performed to evaluate the effect of ATRA on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells. CD4+ T cells were separated from heparinized blood of healthy donors and were cultured in conditions, some with, some without ATRA. Viability was assessed by PI flowcytometry and proliferation was measured by MTT assay. CD69 expression was determined by flowcytometry as a measure of cell activation. Lineage-specific transcription factors (FOXP3, RORγt and T-bet) were examined by intracellular staining and flowcytometry. High doses of ATRA (0.1-1 mM) caused extensive cell death in both PBMCs and CD4+ T cells. Doses of ATRA equal to or lower than 10 µM did not adversely affect cell viability and proliferation in comparison to culture medium without ATRA. Doses of ATRA between 10 µM and 1nM significantly increased cell activation when compared to culture medium without ATRA. ATRA could increase FOXP3+ and also FOXP3+RORγt+ T cells while it decreased RORγt+ and T-bet+ T cells. This study showed that doses of ATRA up to 10 µM are safe when using with CD4+ T cells in terms of cell viability, proliferation and activation. We could also show that ATRA diverts the human immune response in neutral conditions (without adding polarizing cytokines) by increasing FOXP3+ cells and decreasing RORγt+ cells. ATRA could be regarded as a potential therapy in inflammatory conditions and autoimmunities.

Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

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

Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi

Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of differentmore » concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.« less

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy.

PubMed

Chan, C W; Hussain, I; Waugh, D G; Lawrence, J; Man, H C

2014-09-01

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by hemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

PubMed

Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

2015-03-03

Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

PubMed

Jiang, Donglei; Liu, Yan; Jiang, Hui; Rao, Shengqi; Fang, Wu; Wu, Mangang; Yuan, Limin; Fang, Weiming

2018-04-15

A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). Experimental results show that 3OC 12 -HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC 12 -HSL in the range of 0.1-1μM, and the detection limit for 3OC 12 -HSL was calculated to be 0.094μM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production. Copyright © 2017 Elsevier B.V. All rights reserved.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

PubMed

Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

2015-12-01

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Myeloid-Derived Suppressor Cells in Bacterial Infections

PubMed Central

Ost, Michael; Singh, Anurag; Peschel, Andreas; Mehling, Roman; Rieber, Nikolaus; Hartl, Dominik

2016-01-01

Myeloid-derived suppressor cells (MDSCs) comprise monocytic and granulocytic innate immune cells with the capability of suppressing T- and NK-cell responses. While the role of MDSCs has been studied in depth in malignant diseases, the understanding of their regulation and function in infectious disease conditions has just begun to evolve. Here we summarize and discuss the current view how MDSCs participate in bacterial infections and how this knowledge could be exploited for potential future therapeutics. PMID:27066459

Comparison of liposomal and 2-hydroxypropyl-β-cyclodextrin-lidocaine on cell viability and inflammatory response in human keratinocytes and gingival fibroblasts.

PubMed

Ferreira, Luiz Eduardo Nunes; Muniz, Bruno Vilela; Dos Santos, Cleiton Pita; Volpato, Maria Cristina; de Paula, Eneida; Groppo, Francisco Carlos

2016-06-01

The aim of this study was to observe the effect multilamellar liposomes (MLV) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in the in-vitro effects of lidocaine in cell viability, pro-inflammatory cytokines and prostaglandin E2 release of both human keratinocytes (HaCaT) and gingival fibroblasts (HGF) cells. HaCaT and HGF cells were exposed to lidocaine 100-1 μm in plain, MLV and HP-β-CD formulations for 6 h or 24 h. The formulation effects in cell viability were measured by XTT assay and by fluorescent labelling. Cytokines (IL-8, IL-6 and TNF-α) and PGE2 release were quantified by ELISA. MLV and HP-β-CD formulations did not affect the HaCaT viability, which was significantly decreased by plain lidocaine after 24 h of exposure. Both drug carriers increased all cytokines released by HGF after 24-h exposure, and none of the carriers was able to reduce the PGE2 release induced by lidocaine. The effect of drug carrier in the lidocaine effects was dependent on the cell type, concentration and time of exposure. MLV and HP-β-CD showed benefits in improving cell viability; however, both of them showed a tendency to increase cytokine release when compared to the plain solution. © 2016 Royal Pharmaceutical Society.

Effects of size and surface of zinc oxide and aluminum-doped zinc oxide nanoparticles on cell viability inferred by proteomic analyses.

PubMed

Pan, Chih-Hong; Liu, Wen-Te; Bien, Mauo-Ying; Lin, I-Chan; Hsiao, Ta-Chih; Ma, Chih-Ming; Lai, Ching-Huang; Chen, Mei-Chieh; Chuang, Kai-Jen; Chuang, Hsiao-Chi

2014-01-01

Although the health effects of zinc oxide nanoparticles (ZnONPs) on the respiratory system have been reported, the fate, potential toxicity, and mechanisms in biological cells of these particles, as related to particle size and surface characteristics, have not been well elucidated. To determine the physicochemical properties of ZnONPs that govern cytotoxicity, we investigated the effects of size, electronic properties, zinc concentration, and pH on cell viability using human alveolar-basal epithelial A549 cells as a model. We observed that a 2-hour or longer exposure to ZnONPs induced changes in cell viability. The alteration in cell viability was associated with the zeta potentials and pH values of the ZnONPs. Proteomic profiling of A549 exposed to ZnONPs for 2 and 4 hours was used to determine the biological mechanisms of ZnONP toxicity. p53-pathway activation was the core mechanism regulating cell viability in response to particle size. Activation of the Wnt and TGFβ signaling pathways was also important in the cellular response to ZnONPs of different sizes. The cadherin and Wnt signaling pathways were important cellular mechanisms triggered by surface differences. These results suggested that the size and surface characteristics of ZnONPs might play an important role in their observed cytotoxicity. This approach facilitates the design of more comprehensive systems for the evaluation of nanoparticles.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

PubMed Central

2010-01-01

Background In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells. PMID:20831775

In vitro effects on biofilm viability and antibacterial and antiadherent activities of silymarin.

PubMed

Evren, Ebru; Yurtcu, Erkan

2015-07-01

Limited treatment options in infectious diseases caused by resistant microorganisms created the need to search new approaches. Several herbal extracts are studied for their enormous therapeutic potential. Silymarin extract, from Silybum marianum (milk thistle), is an old and a new remedy for this goal. The purpose of this study is to evaluate the antibacterial and antiadherent effects of silymarin besides biofilm viability activity on standard bacterial strains. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), antiadherent/antibiofilm activity, and effects on biofilm viability of silymarin were evaluated against standard bacterial strains. MIC values were observed between 60 and >241 μg/mL (0.25->1 mmol/L). Gram-positive bacteria were inhibited at concentrations between 60 and 120 μg/mL. Gram-negative bacteria were not inhibited by the silymarin concentrations included in this study. MBC values for Gram-positive bacteria were greater than 241 μg/mL. Adherence/biofilm formations were decreased to 15 μg/mL silymarin concentration when compared with silymarin-untreated group. Silymarin reduced the biofilm viabilities to 13 and 46 % at 1 and 0.5 mmol/L concentrations, respectively. We demonstrated that silymarin shows antibacterial and antiadherent/antibiofilm activity against certain standard bacterial strains which may be beneficial when used as a dietary supplement or a drug.

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.

Blue light (470 nm) effectively inhibits bacterial and fungal growth

USDA-ARS?s Scientific Manuscript database

The activity of blue light (470nm) alone on (1) bacterial viability, and (2) with a food grade photosensitizer on filamentous fungal viability, was studied. Suspensions of the bacteria Leuconostoc mesenteroides (LM), Bacillus atrophaeus (BA), and Pseudomonas aeruginosa (PA) were prepared and aliquo...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

DOE PAGES

Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

2015-01-19

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

PubMed

Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

2016-12-01

We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

PubMed

Helm, Katharina; Beyreis, Marlena; Mayr, Christian; Ritter, Markus; Jakab, Martin; Kiesslich, Tobias; Plaetzer, Kristjan

2017-01-01

For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments. © 2017 The Author(s)Published by S. Karger AG, Basel.

Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis

PubMed Central

Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

2014-01-01

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. DOI: http://dx.doi.org/10.7554/eLife.01990.001 PMID:24957336

Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer.

PubMed

Elhami, Esmat; Goertzen, Andrew L; Xiang, Bo; Deng, Jixian; Stillwell, Chris; Mzengeza, Shadreck; Arora, Rakesh C; Freed, Darren; Tian, Ganghong

2011-07-01

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells.

PubMed

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

2012-06-22

Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-β in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-α, G-CSF, and TGF-β. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function. Copyright © 2012 Elsevier Inc. All rights reserved.

Lactate calcium salt affects the viability of colorectal cancer cells via betaine homeostasis.

PubMed

Jang, Yeong-Su; Jo, Young-Kwon; Sim, Jae Jun; Ji, Eunhee; Jeong, Keun-Yeong; Kim, Hwan Mook

2016-02-15

Betaine plays an important role in cellular homeostasis. However, the physiological roles of betaine-γ-aminobutyric acid (GABA) transporter (BGT-1) are still being disputed in cancer. In this study, we tried to find the possibility of the antitumor effect on colorectal cancer (CRC) cell via lactate calcium salt (CaLa)-induced BGT-1 downregulation. The CRC cell viability and clonogenic assay was performed using different doses of BGT-1 inhibitor. The expression level of BGT-1 was measured following the treatment of 2.5mM CaLa. Betaine was treated to confirm the resistance of the antitumor activity by CaLa. Tumor growth was also measured using a xenograft animal model. Long-term exposure of 2.5mM CaLa clearly decreased the expression of BGT-1 in the CRC cells. As a result of the downregulation of BGT-1 expression, the clonogenic ability of CRC cells was also decreased in the 2.5mM CaLa-treated group. Reversely, the number of colonies and cell viability was increased by combination treatment with betaine and 2.5mM CaLa, as compared with a single treatment of 2.5mM CaLa. Tumor growth was significantly inhibited in the xenograft model depending on BGT-1 downregulation by 2.5mM CaLa treatment. These results support the idea that long-lasting calcium supplementation via CaLa contributes to disruption of betaine homeostasis in the CRC cells and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC. Copyright © 2016 Elsevier Inc. All rights reserved.

Photodithazine photodynamic effect on viability of 9L/lacZ gliosarcoma cell line.

PubMed

Fontana, Leticia C; Pinto, Juliana G; Pereira, André H C; Soares, Cristina P; Raniero, Leandro J; Ferreira-Strixino, Juliana

2017-08-01

Even with the advances of conventional treatment techniques, the nervous system cancer prognosis is still not favorable to the patient which makes alternative therapies needed to be studied. Photodynamic therapy (PDT) is presented as a promising therapy, which employs a photosensitive (PS) agent, light wavelength suitable for the PS agent, and molecular oxygen, producing reactive oxygen species in order to induce cell death. The aim of this study is to observe the PDT action in gliosarcoma cell using a chlorin (Photodithazine, PDZ). The experiments were done with 9L/lacZ lineage cells, grown in a DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution and put in a culture chamber at 37 °C with an atmosphere of 5% CO 2 . The PS agent used was the PDZ to an LED light source device (Biopdi/IRRAD-LED 660) in the 660-nm region. The location of the PS agent was analyzed by fluorescence microscopy, and cell viability was analyzed by MTT assay (mitochondrial activity), exclusion by trypan blue (cell viability), and morphological examination through an optical microscope (Leica MD 2500). In the analysis of the experiments with PDZ, there was 100% cell death at different concentrations and clear morphological differences in groups with and without treatment. Furthermore, it was observed that the photodithazine has been focused on all nuclear and cytoplasmic extension; however, it cannot be said for sure whether the location is in the inside core region or on the plasma membrane. In general, the PDZ showed a promising photosensitive agent in PDT for the use of gliosarcoma.

Enrichment and Viability Inhibition of Circulating Tumor Cells on a Dual Acid-Responsive Composite Nanofiber Film.

PubMed

Wang, Wenqian; Cheng, Yaya; Li, Yansheng; Zhou, Hao; Xu, Li-Ping; Wen, Yongqiang; Zhao, Liang; Zhang, Xueji

2017-04-06

The formation and metastatic colonization of circulating tumor cells (CTCs) are responsible for the vast majority of cancer-related deaths. Over the last decade, drug-delivery systems (DDSs) have rapidly developed with the emergence of nanotechnology; however, most reported tumor-targeting DDSs are able to deliver drugs only to solid tumor cells and not CTCs. Herein, a novel DDS comprising a composite nanofiber film was constructed to inhibit the viability of CTCs. In this system, gold nanoparticles (Au NPs) were functionalized with doxorubicin (DOX) through an acid-responsive cleavable linker to obtain Au-DOX NPs. Then, the Au-DOX NPs were mixed in a solution of an acid-responsive polymer {i.e., poly[2-(dimethylamino)ethyl methacrylate]} to synthesize the nanofiber film through electrospinning technology. After that, the nanofiber film was modified with a specific antibody (i.e., anti-EpCAM) to enrich the concentration of CTCs on the film. Finally, the Au-DOX NPs were released from the nanofiber film, and they consequently inhibited the viability of CTCs by delivering DOX to the enriched CTCs. This composite nanofiber film was able to decrease the viability of CTCs significantly in the suspended and fluid states, and it is expected to limit the migration and proliferation of tumor cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

PubMed

Cao, Ting-Ting; Zhang, Yu-Qing

2015-09-01

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implants surfaces.

PubMed

Schwarz, Frank; Ferrari, Daniel; Popovski, Kristian; Hartig, Brigitte; Becker, Jürgen

2009-01-01

Studies have indicated that oral biofilm formation at structured titanium surfaces interferes with cell adhesion and proliferation, and its removal by means of conventional treatment procedures may not be sufficient to render these surfaces biologically acceptable. Therefore, the aim of the study was to evaluate the influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implant surfaces. Intraoral splints were used to collect an in vivo biofilm on sandblasted and acid-etched titanium discs for 48 h. A single (1x) and repeated (2x) use of four different powders (amino acid glycine or sodium bicarbonate particles; range of mean particle size (d(v50)):20-75 microm) was applied at two distances (1 and 2 mm) and angles (30 degrees and 90 degrees) to the surfaces. Specimens (2x) were incubated with SaOs-2 cells for 7 days. Residual biofilm (RB) areas (%), and surface alterations (SEM) (1x and 2x), as well as SaOs-2 cell viability, expressed as mitochondrial cell activity (MA) (counts/second) (2x specimens), were assessed. Comparable mean RB areas were observed within and between groups after both 1x (RB: 0.0% +/- 0.0% to 5.7% +/- 5.7%) and 2x (RB: 0.0% +/- 0.0%) treatments. All surface treatments did not lead to MA (2x) values comparable to the sterile control group. However, sodium bicarbonate particles resulted in significantly higher MA (2x) values than amino acid glycine powders of different sizes. This was associated with pronounced alterations of the surface morphology (2x). Within the limits of the present study, it was concluded that SaOs-2 cell viability at biologically contaminated titanium surfaces was mainly influenced by the particle type of the powder. (c) 2008 Wiley Periodicals, Inc.

Bacterial effectors target the plant cell nucleus to subvert host transcription.

PubMed

Canonne, Joanne; Rivas, Susana

2012-02-01

In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells.

Freezing behavior of adherent neuron-like cells and morphological change and viability of post-thaw cells.

PubMed

Uemura, Makoto; Ishiguro, Hiroshi

2015-04-01

Freezing of nerve cells forming a neuronal network has largely been neglected, despite the fact that the cryopreservation of nerve cells benefits the study of cells in the areas of medicine and poison screening. Freezing of nerve cells is also attractive for studying cell morphology because of the characteristic long, thread-like neurites extending from the cell body. In the present study, freezing of neuron-like cells adhering to the substrate (differentiated PC12 cells), in physiological saline, was investigated in order to understand the fundamental freezing and thawing characteristics of nerve cells with neurites. The microscopic freezing behavior of cells under different cooling rates was observed. Next, the post-thaw morphological changes in the cells, including the cytoskeleton, were investigated and post-thaw cell viability was evaluated by dye exclusion using propidium iodide. Two categories of morphological changes, beading and shortening of the neurites, were found and quantified. Also, the morphological changes of neurites due to osmotic stress from sodium chloride were studied to gain a better understanding of causation. The results showed that morphological changes and cell death were promoted with a decrease in end temperature during freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

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

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers includingmore » HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.« less

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.

PubMed

Bircher, Lea; Geirnaert, Annelies; Hammes, Frederik; Lacroix, Christophe; Schwab, Clarissa

2018-04-17

Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (-80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03-2% in protectant-free cultures to 11-37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

[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.

Stoichiometry of mercury-thiol complexes on bacterial cell envelopes

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

Mishra, Bhoopesh; Shoenfelt, Elizabeth; Yu, Qiang

We have examined the speciation of Hg(II) complexed with intact cell suspensions (1013 cells L- 1) of Bacillus subtilis, a common gram-positive soil bacterium, Shewanella oneidensis MR-1, a facultative gram-negative aquatic organism, and Geobacter sulfurreducens, a gram-negative anaerobic bacterium capable of Hg-methylation at Hg(II) loadings spanning four orders of magnitude (120 nM to 350 μM) at pH 5.5 (± 0.2). The coordination environments of Hg on bacterial cells were analyzed using synchrotron based X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy at the Hg LIII edge. The abundance of thiols on intact cells wasmore » determined by a fluorescence-spectroscopy based method using a soluble bromobimane, monobromo(trimethylammonio)bimane (qBBr) to block thiol sites, and potentiometric titrations of biomass with and without qBBr treatment. The chemical forms of S on intact bacterial cells were determined using S k-edge XANES spectroscopy.« less

Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level

PubMed Central

Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A.; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

2015-01-01

Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake. PMID:26656111

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

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

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah

Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cellsmore » by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.« less

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

PubMed Central

Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

2012-01-01

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

Evaluation of cell viability and functionality in vessel-like bioprintable cell-laden tubular channels.

PubMed

Yu, Yin; Zhang, Yahui; Martin, James A; Ozbolat, Ibrahim T

2013-09-01

Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Karwa, Amol; Lusiak, Przemyslaw M.; Srivastava, Kripa; Poreddy, Amruta R.; Pandurangi, Raghootama S.; Galen, Karen P.; Neumann, William L.; Cantrell, Gary E.; Dorshow, Richard B.

2009-06-01

Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

Parallel effect of 4-octylphenol and cyclic adenosine monophosphate (cAMP) alters steroidogenesis, cell viability and ROS production in mice Leydig cells.

PubMed

Jambor, Tomas; Greifova, Hana; Kovacik, Anton; Kovacikova, Eva; Tvrda, Eva; Forgacs, Zsolt; Massanyi, Peter; Lukac, Norbert

2018-05-01

Over the last decade, there is growing incidence of male reproductive malfunctions. It has been documented that numerous environmental contaminants, such as endocrine disruptors (EDs) may adversely affect the reproductive functions of humans as well as wildlife species. The aim of this in vitro study was to examine the effects of 4-octylphenol (4-OP) on the steroidogenesis in mice Leydig cells. We evaluated the impact of this endocrine disruptor on the cholesterol levels and hormone secretion in a primary culture. Subsequently, we determined the cell viability and generation of reactive oxygen species (ROS) following 4-OP treatment. Isolated mice Leydig cells were cultured in the presence of different 4-OP concentrations (0.04-5.0 μg/mL) and 1 mM cyclic adenosine-monophosphate during 44 h. Cholesterol levels were determined from the culture medium using photometry. Quantification of steroid secretion was performed by enzyme-linked immunosorbent assay. The cell viability was assessed using the metabolic activity assay, while ROS production was assessed by the chemiluminescence technique. Slightly increased cholesterol levels were recorded following exposure to the whole applied range of 4-OP, without significant changes (P>0.05). In contrast, the secretion of steroid hormones, specifically dehydroepiandrosterone, androstenedione, and testosterone was decreased following exposure to 4-OP. Experimental doses of 4-OP did not affect cell viability significantly; however a moderate decrease was recorded following the higher doses (2.5 and 5.0 μg/mL) of 4-OP. Furthermore, relative treatment of 4-OP (5.0 μg/mL) caused a significant (P < 0.001) ROS overproduction in the exposed cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evaluation of graft cell viability-efficacy of piezoelectric versus manual bone scraper technique.

PubMed

Pekovits, Karin; Wildburger, Angelika; Payer, Michael; Hutter, Heinz; Jakse, Norbert; Dohr, Gottfried

2012-01-01

The aim of the present study was to compare the influence of 2 different bone scrapers with respect to graft quality. The study was conducted as a prospective, controlled experimental study of patients selected from the outpatient unit of the Department of Oral Surgery and Radiology (Dental Clinic, Medical University, Graz, Austria). Bone samples were obtained during routine lower third molar removal. Both a manual bone scraper (MS) and a piezoelectric device (PD) were used in directly adjacent regions in each case. As variables, the chip morphology, cell viability, and osteogenic differentiation were investigated. For statistical analysis, the Student t test and Fisher's exact test (P < .05) were applied. A total of 20 patients (12 women and 8 men, mean age 28.15 ± 5.8 years) were included in the study. A series of 40 bone samples was obtained during lower third molar removal. MS and PD enabled similar intraoral harvest of bone chips. In vitro outgrowth of adherent cells was found in 90% of the MS and 80% of the PD samples after 7 to 18 days, without statistical significance (P = .67). Similar cell viability of outgrowing cells in both groups was observed (94.7% ± 2.2% in the MS group and 94.1% ± 1.6% in the PD group). Reverse transcriptase-polymerase chain reaction analysis and the staining pattern verified osteopotent cells in both groups. Both manual and piezoelectric techniques are adequate harvesting technologies for limited intraoral augmentations. Our results did not show an advantage for the piezoelectric device. Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation.

PubMed

Boix-Amorós, Alba; Collado, Maria C; Mira, Alex

2016-01-01

Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system.

Effect of microemulsions on cell viability of human dermal fibroblasts

NASA Astrophysics Data System (ADS)

Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

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

Diesel Exhaust Particulate Extracts Inhibit Transcription of Nuclear Respiratory Factor-1 and Cell Viability in Human Umbilical Vein Endothelial Cells

PubMed Central

Mattingly, Kathleen A.; Klinge, Carolyn M.

2011-01-01

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1 regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription and this suppression was not ablated by concomitant treatment with E2, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E2 increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. PMID:22105178

Effects of superheated steam on Geobacillus stearothermophilus spore viability.

PubMed

Head, D S; Cenkowski, S; Holley, R; Blank, G

2008-04-01

To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105-175 degrees C. The decimal reduction time (D-value) and the thermal resistance constant (z-value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z-value of 25.4 degrees C was calculated for both inoculum levels for SS processing temperatures between 130 degrees C and 175 degrees C. Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.

MiR-34a Inhibits Viability and Invasion of Human Papillomavirus-Positive Cervical Cancer Cells by Targeting E2F3 and Regulating Survivin.

PubMed

Geng, Dianzhong; Song, Xiaohua; Ning, Fangling; Song, Qianhua; Yin, Honghua

2015-05-01

Previous studies confirmed that high-risk human papillomavirus (HR-HPV) infection is a risk factor of cervical cancer, and the infection was associated with significantly reduced miR-34a expression during carcinogenesis. However, the downstream targets of miR-34a and their roles are still not well understood. This study explored the regulative role of miR-34a on E2F3 and survivin expression and the viability and invasion of HPV-positive cervical cancer cells. MiR-34a and survivin expression in 56 cases of HR-HPV-positive patients, 28 cases of HR-HPV-negative patients, and 28 normal cases without HR-HPV infections were measured. Human papillomavirus-18-positive HeLa cervical cancer cells and HPV-16-positive SiHa cells were used to explore the effect of miR-34a on cell viability and invasion. The molecular target of miR-34a was also explored in cervical cancer cells. The results showed that miR-34a overexpression could inhibit HPV-positive cancer cell viability, whereas its downregulation promoted cell viability. E2F3 is a direct target of miR-34a in HPV-positive cervical cancer cells. By targeting E2F3, miR-34a could regulate the expression of survivin. Thus, through regulating E2F3 and survivin, miR-34a could reduce the viability and invasion of HPV-positive cervical cancer cells. This study confirmed a novel miR-34a-E2F3-survivin axis in the tumor suppressor role of miR-34a in cervical cancer.

Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

PubMed

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

2016-05-01

Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Controlled laboratory study. After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

Bioactive gel-glasses with distinctly different compositions: Bioactivity, viability of stem cells and antibiofilm effect against Streptococcus mutans.

PubMed

Siqueira, Renato L; Maurmann, Natasha; Burguêz, Daniela; Pereira, Daniela P; Rastelli, Alessandra N S; Peitl, Oscar; Pranke, Patricia; Zanotto, Edgar D

2017-07-01

In this study, an evaluation was performed to determine the in vitro bioactivity, viability of stem cells, and antibiofilm effect against Streptococcus mutans of two bioactive gel-glass 60SiO 2 -36CaO-4P 2 O 5 (BG-A) and 80SiO 2 -15CaO-5P 2 O 5 (BG-B) compositions. Both materials were bioactive and undergo the formation of hydroxycarbonate apatite (HCA) on their surfaces when immersed in simulated body fluid (SBF) after 12h, but the BG-A composition showed a more significant formation rate. The pH variation of the samples during the test in SBF indicated that an abrupt change had occurred for the BG-A composition within the first few hours, and the pH was subsequently maintained over time, supporting its stronger antibacterial effects against S. mutans. For the in vitro viability test using mesenchymal stem cells (MSCs), the BG-B showed significantly higher cell viability compared to the BG-A composition at concentrations of 0.125, 1.25 and 12.50mg/mL for 2days. These results indicated that the higher solubility of the BG-A glass favors bioactivity and antibacterial effects. However, as a result of rapid degradation, the increase in the concentration of ions in the cell culture medium was not favorable for cell proliferation. Thus, by varying the composition of glasses, and consequently their dissolution rate, it is possible to favor bioactivity, antimicrobial activity or stem cell proliferation for a particular application of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow.

PubMed

van der Mei, Henny C; Atema-Smit, Jelly; Jager, Debbie; Langworthy, Don E; Collias, Dimitris I; Mitchell, Michael D; Busscher, Henk J

2008-07-01

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination. (c) 2008 Wiley Periodicals, Inc.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique.

PubMed

Pianetti, Anna; Manti, Anita; Boi, Paola; Citterio, Barbara; Sabatini, Luigia; Papa, Stefano; Rocchi, Marco Bruno Luigi; Bruscolini, Francesca

2008-10-31

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

Treesearch

Rakesh Minocha; Carolyn McQuattie; Wayne Fagerberg; Stephanie Long; Eun Woon Noh

2001-01-01

The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereo-logical and microscopic methods. Exposure to Al for 24-48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al....

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

PubMed

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

2008-03-01

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

PubMed Central

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

2010-01-01

Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

PubMed

Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

2016-12-01

Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media. Copyright © 2016 Elsevier B.V. All rights reserved.

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

The effect of automobile exhaust particulates on cell viability, plating efficiency and cell division of mammalian tissue culture cells.

PubMed

Seemayer, N H; Hadnagy, W; Tomingas, R

1987-03-01

Extract of particulate matter (EPM) of gasoline engine exhaust induced only a slight loss of cell viability of mouse macrophages (line IC-21) in vitro, while a strong dose-dependent reduction of plating efficiency of human cell line A-549 and of Syrian hamster line 14-1b occurred. Cytological investigations of exposed macrophages of line IC-21 revealed an increase in the mitotic index from 1.5% of control values up to 14.6% at the highest tested concentration of EPM. Mitotic arrest is based almost exclusively on C-type mitoses occurring dose-dependently in the presence of EPM. Results indicate disturbances of the spindle apparatus in the presence of EPM.

Disinfection byproduct formation from chlorination of pure bacterial cells and pipeline biofilms.

PubMed

Wang, Jun-Jian; Liu, Xin; Ng, Tsz Wai; Xiao, Jie-Wen; Chow, Alex T; Wong, Po Keung

2013-05-15

Disinfection byproduct (DBP) formation is commonly attributed to the reaction between natural organic matters and disinfectants, yet few have considered the contribution from disinfecting bacterial materials - the essential process of water disinfection. Here, we explored the DBP formation from chlorination and chloramination of Escherichia coli and found that most selected DBPs were detectable, including trihalomethanes, haloacetonitriles, chloral hydrate, chloropicrin, and 1,1,1-trichloro-2-propanone. A positive correlation (P = 0.08-0.09) between DBP formation and the log reduction of E. coli implied that breaking down of bacterial cells released precursors for DBP formation. As Pseudomonas aeruginosa is a dominant bacterial species in pipeline biofilms, the DBP formation potentials (DBPFPs) from its planktonic cells and biofilms were characterized. Planktonic cells formed 7-11 times greater trihalomethanes per carbon of those from biofilms but significantly lower (P < 0.05) chloral hydrate, highlighting the bacterial phenotype's impact on the bacteria-derived DBPFP. Pipe material appeared to affect the DBPFP of bacteria, with 4-28% lower bromine incorporation factor for biofilms on polyvinyl chloride compared to that on galvanized zinc. This study revealed both the in situ disinfection of bacterial planktonic cells in source water and ex situ reaction between biofilms and residual chlorine in pipeline networks as hitherto unknown DBP sources in drinking water. Copyright © 2013 Elsevier Ltd. All rights reserved.

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

PubMed

Miyano-Kurosaki, Naoko; Ikegami, Kou; Kurosaki, Kunihiko; Endo, Takahiko; Aoyagi, Hoshimi; Hanami, Mari; Yasumoto, Jun; Tomoda, Akio

2009-05-01

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo