Indicator Of Viability Research Articles

BAO-1 - Expanding human blastocyst expansion takes longer to expand compared to expanded blastocysts after warming on biopsied and non-biopsied embryos: a time lapse analysis

Objective: Blastocoel expansion has been shown to be an important indicator of human embryo viability and implantation potential which may be affected by mechanical manipulations like assisted hatching and embryo biopsy. Current clinical practice uses trophectoderm biopsy to screen for genetic abnormalities, but may introduce additional mechanical stressors to the preimplantation embryo. The present study seeks to understand if embryo stage and biopsy modifies the rate of blastocoel expansion after warming.

Non‐invasive physiological markers demonstrate link between habitat quality, adult sex ratio and poor population growth rate in a vulnerable species, the Cape mountain zebra

Abstract Effective conservation and species management require an understanding of the causes of poor population growth. Conservation physiology uses biomarkers to identify factors that contribute to low individual fitness and population declines. Building on this, macrophysiology can use the same markers to assess how individual physiology varies with different ecological or demographic factors over large temporal and spatial scales. Here, we use a macrophysiological approach to identify the ecological and demographic correlates of poor population growth rates in the Cape mountain zebra metapopulation. We use two non‐invasive biomarkers: faecal glucocorticoids as a measure of chronic stress, and faecal androgens as an indicator of male physiological status. We found that faecal glucocorticoid concentrations were highest in the spring prior to summer rainfall, and were elevated in individuals from populations associated with low‐quality habitat (lower grass abundance). In addition, faecal androgen concentrations were higher in populations with a high proportion of non‐breeding stallions (where male:female adult sex ratios exceed 2:1) suggesting sex ratio imbalances may intensify male competition. Finally, population growth rate was negatively associated with faecal glucocorticoid concentrations and female fecundity was negatively associated with faecal androgens, indicating a relationship between hormone profiles and fitness. Together, our results provide cross‐population evidence for how poor population growth rates in Cape mountain zebra can be linked to individual physiological biomarkers. More broadly, we advocate physiological biomarkers as indicators of population viability, and as a way to evaluate the impact of variable ecological and demographic factors. In addition, conservation physiology can be used to assess the efficacy of management interventions for this subspecies, and this approach could inform models of species’ responses to future environmental change. A plain language summary is available for this article.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Impact of large-scale rooftop solar PV integration: An algorithm for hydrothermal-solar scheduling (HTSS)

Hydrothermal scheduling is a standard problem in the operation of power systems. With the integration of renewable resources, like solar and/or wind to the existing hydro-thermal system, there is a need for a dedicated approach for long-term generation scheduling of the integrated system. Long-term generation scheduling of hydro-thermal-solar systems at the grid level has not been reported so far. This paper discusses a Hydrothermal-Solar Scheduling (HTSS) algorithm for the same. The algorithm is a two-stage formulation using dynamic programming and linear programming techniques. This generic methodology can be used for assessing the impact of large-scale rooftop solar-PV integration with a hydrothermal system, in terms of the electricity energy-mix, generation schedules and the annual generation-costs of the concerned system. It has been illustrated by application to the problem of integration of a large-scale rooftop solar photovoltaic scenario with the existing Mumbai sub-system.The results show a significantly higher annual generation-cost saving, achieved by integrating the large-scale rooftop solar photovoltaic scenario via the proposed HTSS algorithm. The benefits, however, reduce with increasing penetrations of solar in the electricity energy-mix. The annual generation-cost savings have also been used for estimating the upper cap on feed-in tariffs for solar generation, and the financial viability indicators for the large-scale rooftop solar photovoltaic scenario; the corresponding internal rate of return is found to decline from 18.4% to 16.6% as the share of solar-PV in the electricity energy-mix of Mumbai increases from 2.8% to 13.9%.

Zinc oxide-Iron-Aluminum nanostructured cover for photoelectrochemical water splitting

ABSTRACTThe purpose of this research is to study the viability of photocatalytic water splitting using ASTM A792 Zn-Al-Fe commercial metallic sheets as substrates for electrodeposited and corroded electrodes. The nanostructures were synthesized in two different procedures: via electrodeposition of migrating species from one electrode to another and from the remaining materials after corrosion of electrodes during electrodeposition, both procedures were done immersing the metallic electrodes in FeCl3 salts dissolved in water as cell electrolyte. The released or remaining Zinc-Aluminum-Iron can be used for the construction of nanostructures or as co-catalyst on the coating over the substrate. Actual EDS-SEM data reveals incorporation of Zinc on dendrite-like structures with traces of Al-Fe due to material release and further electrodeposition on working electrode, meanwhile, dendrite-like structures with an increased amount of Iron were obtained from the corrosion in the auxiliary electrode. Finally, samples were tested with lineal voltammetry to measure the photocurrent activity as indicator of photocatalytic viability for water splitting, obtaining an improvement of 31 mA/cm2 over natural photovoltaic current generation of substrates with higher Zinc concentrations under UV-Visible radiation.

Application of green fluorescent protein to measure antimicrobial efficacy and the kinetics of cell death against Escherichia coli

Industrial antimicrobials have been extensively used to control unwanted microbial growth by incorporation into a variety of products such as plastics and paints, reducing biodeterioration and biofouling and extending the lifespan of the product. Industrial antimicrobials generally have broad sites of action affecting core cellular functions such as central metabolism, enzyme function, cell wall or DNA synthesis and can either be biocidal or biostatic. In addition, susceptibility can be affected by the metabolic state of the microbe, with metabolically inactive cells generally more resistant than metabolically active cells. Previously it was demonstrated that cytosolically expressed green fluorescent protein could be used as a real-time viability indicator in the yeast Aureobasidium pullulans based on the pH dependent fluorescence of GFP and the collapse of the proton gradient across the cell membrane during cell death. In this study we report on the development and validation of an equivalent GFP fluorescence viability assay in Escherichia coli and used this assay to study the effect of five antimicrobials commonly used in plastics; 4,5-dichloro-2-octyl-isothiazol-3-one (DCOIT), sodium pyrithione, 1,2-benzisothiazol-3-one (BIT), 2-octyl-isothiazol-3-one (OIT) and n-butyl-1,2-benzisothiazol-3-one (BBIT). The results demonstrate broad differences amongst the antimicrobials in both relative efficacy, rate of effect and for some antimicrobials, marked differences in sensitivity toward growing and non-growing cells.

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants – ultraviolet (UV) and chlorine – on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm2). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm2) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability.

Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells.

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.

Epithelial ciliated beating cells essential for ex vivo ALI culture growth

BackgroundBronchial epithelium plays a key role in orchestrating innate and adaptive immunity. The fate of ex vivo airway epithelial cultures growing at the air liquid interface (ALI) derived from human endobronchial biopsies or brushings is not easy to predict. Calibrating and differentiating these cells is a long and expensive process requiring rigorous expertise. Pinpointing factors associated with ALI culture success would help researchers gain further insight into epithelial progenitor behavior.MethodsA successful ALI culture was defined as one in which a pseudostratified epithelium has formed after 28 days in the presence of all differentiated epithelial cell types. A 4-year prospective bi-center study was conducted with adult subjects enrolled in different approved research protocols.Results463 consecutive endobronchial biopsies were obtained from normal healthy volunteers, healthy smokers, asthmatic patients and smokers with COPD. All demographic variables, the different fiber optic centers and culture operators, numbers of endo-bronchial biopsies and the presence of ciliated cells were carefully recorded. Univariate and multivariate models were developed. A stepwise procedure was used to select the final logistic regression model. ALI culture success was independently associated with the presence of living ciliated cells within the initial biopsy (OR = 2.18 [1.50–3.16], p < 0.001).ConclusionThis finding highlights the properties of the cells derived from the epithelium dedifferentiation process. The preferential selection of samples with ciliated beating cells would probably save time and money. It is still unknown whether successful ALI culture is related to indicators of general cell viability or a purported stem cell state specifically associated with ciliated beating cells.

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

Biofilm formation represents a successful survival strategy for bacteria. In biofilms, cells are embedded in a matrix of extracellular polymeric substances (EPS). As they are often more stress-tolerant than single cells, biofilm cells might survive the conditions present in space and on Mars. To investigate this topic, the bacterium Deinococcus geothermalis was chosen as a model organism due to its tolerance toward desiccation and radiation. Biofilms cultivated on membranes and, for comparison, planktonically grown cells deposited on membranes were air-dried and exposed to individual stressors that included prolonged desiccation, extreme temperatures, vacuum, simulated martian atmosphere, and UV irradiation, and they were exposed to combinations of stressors that simulate space (desiccation + vacuum + UV) or martian (desiccation + Mars atmosphere + UV) conditions. The effect of sulfatic Mars regolith simulant on cell viability during stress was investigated separately. The EPS produced by the biofilm cells contained mainly polysaccharides and proteins. To detect viable but nonculturable (VBNC) cells, cultivation-independent viability indicators (membrane integrity, ATP, 16S rRNA) were determined in addition to colony counts. Desiccation for 2 months resulted in a decrease of culturability with minor changes of membrane integrity in biofilm cells and major loss of membrane integrity in planktonic bacteria. Temperatures between -25°C and +60°C, vacuum, and Mars atmosphere affected neither culturability nor membrane integrity in both phenotypes. Monochromatic (254 nm; ≥1 kJ m-2) and polychromatic (200-400 nm; >5.5 MJ m-2 for planktonic cells and >270 MJ m-2 for biofilms) UV irradiation significantly reduced the culturability of D. geothermalis but did not affect cultivation-independent viability markers, indicating the induction of a VBNC state in UV-irradiated cells. In conclusion, a substantial proportion of the D. geothermalis population remained viable under all stress conditions tested, and in most cases the biofilm form proved advantageous for surviving space and Mars-like conditions. Key Words: Biofilms-Desiccation-UV radiation-Mars-Lithopanspermia. Astrobiology 17, 431-447.

IMPACT OF ULTRAVIOLET RADIATION ON GERMINATION AND GROWTH PROCESSES OF WHEAT SEEDS

Scientists did not come to a consensus when studying the influence of ultraviolet (UV) radiation on the crop properties and the biometrics of various crops and optimal exposure time and wavelength. The authors studied the influence of UV radiation from a source VL-6.LC with a wavelength of 365 nm for 5 and 30 min for seeds of spring and winter wheat varieties. For seeds of each variety there is the optimum amount of the absorbed radiation energy resulting in the maximum effect. The stimulating influence of UV radiation is not revealed on the seeds of wheat variety Esther having low indicators of viability. The germination of other 4 varieties increased by 1-3 percent. However, biometric indicators of all studied varieties were improved after irradiation. The weight of all varieties wheat seedlings increased after 30 min of irradiation, but especially in case of Moskovskaya 39 and Yubileynaya 100 this indicator reaches up to 20 percent. Five-minute radiation did not caused the similar effect. Height of seedlings depending on the variety is many-valued. After 30 min irradiation this indicator in case of varieties Moskovskaya 39 increased by 30 percent, and of Yubileynaya 100, Dar'ya and Afina it accrues only by 3-5 percen. Root system depend on UV radiation. Root length of all studied varieties except Dar'ya increased up to 60 percent. Therefore the UV radiation does not affect the germination of seeds of the investigated wheat varieties, but significantly changes the seedlings biometrics. It is shown that the action of ultraviolet light is selectively and highly dependent on a variety. The winter wheat Moskovskaya 39 is the most susceptible variety when ultraviolet radiation with a wavelength of 365 nm for 30 min.

Screening of Natural Product Library for the Discovery of Novel Leukemia Therapeutics

KAYLA HANCHER, DEANNA SCHMITT, and JOSEPH HORZEMPA. Department of Natural Sciences and Mathematics, West Liberty University, West Liberty, WV 26074. Screening of a Natural Product Library for the Discovery of Novel Leukemia Therapeutics Leukemia is the leading cause of death in children. The discovery of effective cancer therapies is imperative to combat this deadly disease. The National Center for Natural Products Research (NCNPR) has comprised a library of over 4,000 organic extracts derived from plants, fungi, marine, and algae species with potential anti-cancer properties. In partnership with NCNPR, we have developed a rapid and effective method for screening large quantities of these organic compounds for anti-cancer efficacy against THP-1 cells (a monocytic leukemia cell line). A microtiter-based AlamarBlue Assay was used to identify extracts that reduced the viability of the THP-1 cells. For this assay, THP-1 cells were treated with an extract from the NCNPR library and were incubated at 37˚C, 5% CO2 for 24 hours. Subsequently, resazurin was added and incubated for an additional 24 hours. Change to a red color was an indicator of viability. Cells treated with extracts that produced little to no color change were identified as leads. Lead extracts will be further evaluated for toxicity to non-cancer cells.

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals

Ethnopharmacological relevanceLeaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. Aim of the studyTo investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. Material and methodsPlants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. ResultsSeven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. ConclusionWe have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species.

Analysis of a sequential production of electricity, ice and drying of agricultural products by cascading geothermal energy

In this paper, it is presented an analysis for the sequential production of electricity, ice and drying of agricultural products, by the concept of cascade for medium and low temperature geothermal energy. To carry out the analysis, a set of practical assumptions for the integration of technologies operated by cascade method for geothermal energy were defined. The geothermal cascade is composed of three thermal levels, each one operating under different temperatures in decreasing form. Additionally, the thermal cascade is composed at the first level by an Organic Rankine Cycle (ORC) for electricity production. In the second thermal level, by an absorption refrigeration cycle for ice production, and in the third level includes a dehydrator for drying of agricultural products. The agricultural products considered for dehydration are: avocado, green chile and tomato. Once the main assumptions were set, five integration alternatives and five different modes of operation of the system were proposed. The alternatives have different features for the activation of the first thermal level of the cascade, different nominal capacities of electricity production, cold, and quantity of product to be dehydrated. Subsequently, a technical-economic analysis is carried out to obtain the performance and energy characteristics of the different thermal levels of the cascade, different modes of operation, carbon dioxide emissions, cost estimations and indicators of economic viability. The results indicate that the dehydration process improves drastically the economic profits of all alternatives, especially for dehydration of tomato, achieving simple payback periods of around one year, overall energy efficiency of up to 17.84% and greenhouse emissions reduction of 537.7 tonnes of CO2 per year. Concerning the modes of operation, it was determined that the one based on only-electricity production is not desirable due to the worst energy and economic performance.

Distinct signaling programs control human hematopoietic stem cell survival and proliferation

AbstractSeveral growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38−CD45RA−CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90−CD49f− cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

Correlation or cause? The limitations of population density as an indicator for public transport viability in the context of a rapidly growing developing city

Abstract In response to high urbanization rates in developing countries, many city authorities are embarking upon large scale public transport improvement programmes. Supportive land use environments will be necessary to reduce operating subsidies to within sustainable limits for these developing economies. In past decades, the literature on the relationship between land use and public transport viability has been dominated by city population density, which is posited to have a strong causal link. This has led to the recommendation of minimum density thresholds for viability. However, for some rapidly growing cities in developing countries, public transport systems have not attained sustainable subsidy levels despite city population densities significantly above the purported thresholds. The aim of this paper is to investigate the significance of population density as an indicator of public transport viability in rapidly growing developing city contexts generally, and in South African cities in particular. A public transport corridor operating cost model was developed for this purpose. The cost modelling results suggest that the causal link between city population density and public transport viability is only leveraged through particular land use and density distributions, and that many rapidly growing cities have land use and density distributions that are unsupportive. It is estimated that when densification occurs on the periphery of a low-density city, the resulting changes in public transport trip lengths, departure profile and seat renovation can in fact be detrimental to the overall viability of services. The paper demonstrates that population density distribution, land use mix and corridor length can each have considerably stronger causal links to public transport viability, than city population density in isolation. Integrated land use-transport strategies should therefore give equal attention to these three land use characteristics and city population density.

Assessment of Population and ECM Production Using Multiphoton Microscopy as an Indicator of Cell Viability.

Multiphoton microscopy allows continuous depth-resolved, nondestructive imaging of scaffold-seeded cells during cell or tissue culture. Spectrally separated images in high resolution can be provided while cells are conserved in their native state. Here we describe the seeding of mesenchymal stem cells to bacterial nanocellulose hydropolymer scaffolds followed by 2-channel imaging of cellular autofluorescence (AF) and collagen-I formation using second harmonic generation (SHG) signals. With this approach the simultaneous observation of the progression of cell morphology and production of extracellular matrix as hallmarks of viability and cell fitness is possible.

Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells.

Glycine receptor chloride channels (GlyRs) are attractive drug targets for therapeutic intervention and are also more and more recognized in the context of in vitro neurotoxicity and developmental neurotoxicity testing. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system. Human pluripotent NTERA-2 (NT2) stem cells undergo neuronal differentiation upon stimulation with retinoic acid and express a large variety of neuronal proteins-including GlyR. YFP-I152L, a halide-sensitive variant of yellow fluorescent protein, allows high-throughput fluorescence-based functional analysis of GlyRs in NT2 cells. Here we describe a protocol for phenotyping of cellular viability by functional analysis of GlyR in neuronally differentiated NT2 (NT2-N) cells using YFP-I152L as a reporter of functional integrity of GlyRs. The protocol describes neuronal differentiation of NT2 stem cells, transient transfection of NT2-N cells with YFP-I152L as well as functional imaging and analysis of data from high-content imaging.

Erratum to: Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells.

Glycine receptor chloride channels (GlyRs) are attractive drug targets for therapeutic intervention and are also more and more recognized in the context of in vitro neurotoxicity and developmental neurotoxicity testing. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system. Human pluripotent NTERA-2 (NT2) stem cells undergo neuronal differentiation upon stimulation with retinoic acid and express a large variety of neuronal proteins-including GlyR. YFP-I152L, a halide-sensitive variant of yellow fluorescent protein, allows high-throughput fluorescence-based functional analysis of GlyRs in NT2 cells. Here we describe a protocol for phenotyping of cellular viability by functional analysis of GlyR in neuronally differentiated NT2 (NT2-N) cells using YFP-I152L as a reporter of functional integrity of GlyRs. The protocol describes neuronal differentiation of NT2 stem cells, transient transfection of NT2-N cells with YFP-I152L as well as functional imaging and analysis of data from high-content imaging.

Construction of Two mCherry Plasmids (pXG-mCherry) for Transgenic Leishmania: Valuable Tools for Future Molecular Analysis.

Leishmania is the causative agent of leishmaniasis, a neglected tropical disease that affects more than 12 million people around the world. Current treatments are toxic and poorly effective due to the acquisition of resistance within Leishmania populations. Thus, the pursuit for new antileishmanial drugs is a priority. The available methods for drug screening based on colorimetric assays using vital dyes are time-consuming. Currently, the use of fluorescent reporter proteins is replacing the use of viability indicator dyes. We have constructed two plasmids expressing the red fluorescent protein mCherry with multiple cloning sites (MCS), adequate for N- and C-terminal fusion protein constructs. Our results also show that the improved pXG-mCherry plasmid can be employed for drug screening in vitro. The use of the red fluorescent protein, mCherry, is an easier tool for numerous assays, not only to test pharmacological compounds, but also to determine the subcellular localization of proteins.