Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

The effect of polyamines and silver thiosulphate on micropropagation of date palm followed by genetic stability assessment.

There are some limitations in date palm micropropagation. These include low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. The objective of the study was to determine the effect of the polyamines and Silver Thiosulphate (STS) on the enhancement of shoot multiplication and genetic stability of in vitro cultures of date palm cultivar Quntar. Media supplemented with 75 mg L-1 SPD in combination with 10 mg L-1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective on root formation and the number of roots per shoot, where the best result, 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mg L-1 PUT and 10 mg L-1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 80% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar was beneficial to improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

Rapid and real-time monitoring of bacterial growth against antibiotics in solid growth medium using a contactless planar microwave resonator sensor.

Infection diagnosis and antibiotic susceptibility testing (AST) are pertinent clinical microbiology practices that are in dire need of improvement, due to the inadequacy of current standards in early detection of bacterial response to antibiotics and affordability of contemporarily used methods. This paper presents a novel way to conduct AST which hybridizes disk diffusion AST with microwave resonators for rapid, contactless, and non-invasive sensing and monitoring. In this research, the effect of antibiotic (erythromycin) concentrations on test bacterium, Escherichia coli (E. coli) cultured on solid agar medium (MH agar) are monitored through employing a microwave split-ring resonator. A one-port microwave resonator operating at a 1.76 GHz resonant frequency, featuring a 5 mm2 sensitive sensing region, was designed and optimized to perform this. Upon introducing uninhibited growth of the bacteria, the sensor measured 0.005 dB/hr, with a maximum change of 0.07 dB over the course of 15 hours. The amplitude change decreased to negligible values to signify inhibited growth of the bacteria at higher concentrations of antibiotics, such as a change of 0.005 dB in resonant amplitude variation while using 45 µg of antibiotic. Moreover, this sensor demonstrated decisive results of antibiotic susceptibility in under 6 hours and shows great promise to expand automation to the intricate AST workflow in clinical settings, while providing rapid, sensitive, and non-invasive detection capabilities.

Solution NMR in human embryo culture media as an option for assessment of embryo implantation potential.

NMR offers the potential to holistically screen hundreds of metabolites and has already proved to be a powerful technique able to provide a global picture of metabolic changes in a wide range of biological systems underlying complex and multifactorial matrixes. This review covers the literature until May 2020 centered on the early prediction of the viability of in vitro developed embryos using several analytical techniques, including NMR. Nowadays, the predominant non-invasive technique for selecting viable embryos is based on morphology, where variables associated with the rate of cleavage and blastocyst formation are evaluated by the embryologist following standardized criteria that are somewhat subjective. This morphological approach is therefore inadequate for the prediction of embryo quality, and several studies have focused on developing new non-invasive methods using molecular approaches based particularly on metabolomics. This review outlines the potential of NMR as one of these non-invasive in vitro methods based on the analysis of spent embryo culture media.

An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells.

The protocol provided here describes methodologies for making a highly cost-effective, chemically defined medium for culturing hiPSCs we call B8 medium. The typical cost of B8 medium is US$10 per liter, which with modifications included here is more affordable than standard media. We provide simple protocols for making B8 supplement aliquots, making the basal media DMEM/F12, Matrigel-coated plates, thawing, passaging, culturing, and cryopreserving hiPSCs. We show typical differentiation results and provide a comprehensive troubleshooting guide. For complete details on the use and execution of this protocol, please refer to Kuo et al. (2020).

Assessment of cultured media for desert moss crust by physiological responses.

The physiological responses of desert moss crusts under four artificial media (Beneck, Part, BG11, and Hogland) were investigated to evaluate the function of culture media during different culture periods. The results showed that the value of malondialdehyde (MDA) was at a maximum at 11d, on the contrary, chlorophyll-a, soluble protein, and soluble sugar were at a minimum. As the time increased, the value of MDA and soluble protein decreased faster in the Hogland, while the value of chlorophyll-a and soluble sugar increased. At the end of the culture period, the value of chlorophyll-a and soluble sugar was at a maximum in the Hogland, while the value of MDA and soluble protein was at a minimum. The results suggested that the Hogland medium had a promoting effect on the growth of desert moss crusts. The selected artificial cultivation medium towards wider and larger scale field applications of cultural desert biocrust was widely anticipated.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease. METHODS: Immunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes. RESULTS: Non-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming. CONCLUSIONS: Compared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events.

Enhanced ultrafine nanofibril biosynthesis of bacterial nanocellulose using a low-cost material by the adapted strain of Komagataeibacter xylinus MSKU 12.

Bacterial nanocellulose (BNC) is a renewable and biodegradable biopolymer which has currently received considerable attention due to the rapid increase in environmental issues. In this study, a cost-effective strategy for BNC production was successfully improved in the adapted strain, C30, which was obtained from Komagataeibacter xylinus MSKU 12 by a repetitive cultivation in a low-cost coconut water containing acetic acid and ethanol (CW-AE medium) at 37 °C. The adaptive procedure allowed the strain C30 to be adapted to grow and produce BNC with a higher yield in a limiting nutrient CW-AE medium, than that in a standard HS-AE medium. This strain could produce a high yield of BNC (9.69 g/L dry weight) in a low-cost medium, a modified CW-AE medium supplemented with sucrose and ammonium sulfate. Moreover, SEM images showed that BNC pellicle produced by the strain C30 in the modified CW-AE medium exhibited finer nanofibrils with a narrower range of width compared with those of MSKU 12 while no significant differences in their physicochemical characteristics were detected among these BNCs produced. Therefore, this finding demonstrates, not only the potential strain for the cost-effective BNC production at high temperature, but also the superior ultrafine nanofibrils production useful for further applications.

Provitamin D3 modulation through prebiotics supplementation: simulation based assessment.

Vitamin D is important in multiple health conditions. Vitamin D deficiency is prevalent globally even with exposure to adequate sunlight. Reduction in provitamin D3 (7-dehydrocholesterol, 7-DHC) is an important cause of vitamin D3 deficiency. Vitamin supplementation, food fortification, and use of probiotics are some approaches to reduce vitamin D3 deficiency. This study investigates plausibility of 7-DHC biosynthesis through dietary prebiotics supplementation. Furthermore, it reports mechanistic details and constraints for the biosynthesis using flux balance analysis (FBA) simulations. The FBA simulations using co-metabolism models comprising human host and a resident bacterium (Faecalibacterium prausnitzii or Bacteroides thetaiotamicron) indicated increased flux of 7-DHC with short-chain fructooligosaccharide (scFOS) or inulin supplementation. We observed around 2-fold increase in flux compared to the baseline. Biosynthesis of 7-DHC was primarily modulated through acetate, pyruvate and lactate secreted by the bacterium. We observed diverse mechanisms and dose dependent responses. We extended this assessment to 119 resident bacteria and investigated the metabolites profiles with prebiotics supplementation. In summary, the current study suggests the potential use of applying prebiotics in enhancing 7-DHC biosynthesis. Furthermore, performance of the different gut bacteria with prebiotic supplementation for secreted metabolites profile is reported. These results may be useful to design future clinical studies.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Modulation of culture medium confers high-specificity production of isopentenol in Bacillus subtilis.

Enthusiasm for mining isoprenoid-based flavors, pharmaceuticals, and nutraceuticals from GRAS (Generally Regarded as Safe) status microbial hosts has increased in the past few years due to the limitations associated with their plant-based extraction and chemical synthesis. Bacillus subtilis, a well-known GRAS microbe, is a promising alternative due to its fast growth rate and the ability to metabolize complex carbon sources. The study focused on the high-specificity production of isopentenol in B. subtilis by modulating the culture medium. Media modulation led to a 2.5 folds improvement in isopentenol titer in the wild-type strain. In the recombinant strain, optimization of physico-chemical factors, coupled with overexpression of the nudF enzyme resulted in a maximum isopentenol titer of ∼6 mg/L in a shake flask. The recombinant strain produced ∼5 mg/L isoprenol (∼80% of the total isopentenol production) and ∼1.8 mg/L prenol (∼65% of the total isopentenol production) by utilizing sorbitol and pyruvate as the carbon sources, respectively. Replacement of glucose with sorbitol and pyruvate reduced the production of the undesired metabolites and enhanced high-specificity production of isopentenol. Upon replacement of the carbon source with a low-cost substrate, a non-detoxified rice-straw hydrolysate, the engineered strain produced 2.19 mg/L isopentenol. This proof-of-concept study paves the path for the high-specificity production and cost-effective recovery of isopentenol from industrially competent microbial strains with engineered isoprenoid pathways.

Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model.

BACKGROUND: Bone morphogenetic proteins (BMPs) have played a central role in the regenerative therapies for bone reconstruction, including alveolar cleft and craniofacial surgery. However, the high cost and significant adverse effect of BMPs limit their broad application. Hydroxycholesterols, naturally occurring products of cholesterol oxidation, are a promising alternative to BMPs. The authors studied the osteogenic capability of hydroxycholesterols on human mesenchymal stem cells and the impact of hydroxycholesterols on a rodent alveolar cleft model. METHODS: Human mesenchymal stem cells were treated with control medium or osteogenic medium with or without hydroxycholesterols. Evaluation of cellular osteogenic activity was performed. A critical-size alveolar cleft was created and one of the following treatment options was assigned randomly to each defect: collagen sponge incorporated with hydroxycholesterols, BMP-2, or no treatment. Bone regeneration was assessed by means of radiologic and histologic analyses and local inflammation in the cleft evaluated. Moreover, the role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was examined. RESULTS: All cellular osteogenic activities were significantly increased on human mesenchymal stem cells treated with hydroxycholesterols relative to others. The alveolar cleft treated with collagen sponge with hydroxycholesterols and BMP-2 demonstrated robust bone regeneration. The hydroxycholesterol group revealed histologically complete bridging of the alveolar defect with architecturally mature new bone. The inflammatory responses were less in the hydroxycholesterol group compared with the BMP-2 group. Induction of hydroxycholesterol-mediated in vitro osteogenesis and in vivo bone regeneration were attenuated by hedgehog signaling inhibitor, implicating involvement of the hedgehog signaling pathway. CONCLUSION: Hydroxycholesterols may represent a viable alternative to BMP-2 in bone tissue engineering for alveolar cleft.

Reduction of the fitness cost of antibiotic resistance caused by chromosomal mutations under poor nutrient conditions.

The prevalence of antibiotic resistance in drinking water system is pressing public health risk. Antibiotic resistance conferred by chromosomal mutations often produces fitness cost, which may affect its spread and persistence. In this study, the rifampin-resistant strains were competed with their wild-type counterparts at different nutrient levels. It was observed that the ratio of the absolute number between resistant and wild-type cells quickly decreased under rich nutrient conditions, but it slowly reduced or remained stable in the poor nutrient medium. This finding suggested that poor nutrient conditions resulted in the reduction of fitness cost of antibiotic resistance, i.e. the resistant bacteria became more competitive. Implying mechanisms analysis found that the differences of metabolic activity between wild-type and rifampin-resistant strains was significant smaller (P < 0.05) at low nutrient levels. Additionally, distinguishable large colony size rifampin-resistant strains were observed during competition assay. DNA sequencing of RNA polymerase subunit genes further revealed that these colonies could be adaptive mutants from wild-type strain in rpoB gene. To our knowledge, this is the first study to reveal that the oligotrophic conditions facilitate the persistence of antibiotic resistance in drinking water by reducing the fitness cost of the resistant strains.

Potential use of methane fermentation digested slurry as a low-cost, environmentally-friendly nutrient for bioethanol production from crude glycerol by Klebsiella variicola TB-83D.

A methane fermentation digested slurry (MFDS) was evaluated as a substitute for the commercial nutrient, yeast extract (YE), in ethanol production from glycerol by Klebsiella variicola strain TB-83D. In pH-controlled fed-batch cultures, partial replacement of YE by MFDS did not reduce ethanol productivity significantly. However, non-sterilized MFDS had negative effects on glycerol fermentation by this strain. Although ethanol production decreased when YE was completely replaced by sterilized MFDS, the use of crude glycerol and sterilized MFDS achieved a yield of 14.6 g/L ethanol. This is the first study to report the use of MFDS as the sole nutrient for ethanol production from glycerol, which contributes to the development of a low-cost glycerol biorefinery derived from the biodiesel fuel industry.

A colorimetric broth microdilution method for assessment of Helicobacter pylori sensitivity to antimicrobial agents.

Helicobacter pylori is a major infective etiological agent of the upper gastrointestinal tract diseases. The bacterium exhibits resistance to various conventional antibiotics, being usually challenging for eradication. Since there is an urge to consider alternative therapeutic strategies, the aim of the study was to examine selected essential oils of plants belonging to families Cupressaceae (Juniperus communis) and Lamiaceae (Hyssopus officinalis, Salvia officinalis, Melissa officinalis, Lavandula angustifolia, Ocimum basilicum and Thymus serpyllum) against H. pylori, using an improved microdilution broth method. The oils were examined in concentration range from 0.03 to 4 µL/mL. The method comprises Brain-heart infusion broth supplemented with yeast extract, horse serum and IsoVitaleX. After 3 day incubation, an equal volume of double strengthen Christensen's urea was added into each well and incubated for additional 4 h. In wells with present H. pylori, the medium changed color from yellow to purple, allowing MIC determination even without a microtitre plate reader. The microtitre format method is convenient as it is less expensive, easier to perform and requires less amount of an anti-H. pylori agent. The improved method enhances specificity to H. pylori, as fast urease activity is almost an exclusive property of this bacterium. The application of the second step incubation with Christensen's urea decreases the possibility of false positive/negative results due to contaminant growth or commonly poor H. pylori growth. Among the examined oils, J. communis, H. officinalis and O. basilicum were not active with the highest applied concentrations, while the most active was T. serpyllum, with MIC 2.0-4.0 µL/mL. This is the first report on essential oils activity of T. serpyllum and H. officinalis against H. pylori.

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

Comparative analysis of media effects on human induced pluripotent stem cell-derived cardiomyocytes in proarrhythmia risk assessment.

INTRODUCTION: Cardiotoxicity assessment using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) forms a key component of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). A potentially impactful factor on iPSC-CM testing is the presence of serum in the experimental media. Generally, serum-free media is used to most accurately reproduce "free" drug concentration. However, caution is needed; drug solubility and cardiomyocyte electrophysiology could be affected by media formulation, potentially impacting interpretation of drug-induced effects. METHODS: Effects of 25 drugs on properties of spontaneous field potentials in iPSC-CMs were assayed using a high-throughput microelectrode array (MEA) in two media formulations: serum-containing and serum-free. Comparative analysis was conducted on rate-corrected field potential duration (FPDc) and prevalence of arrhythmic events. Further MEA experiments were conducted, varying percentages of serum as well as carbon substrate components. Comparative LC-MS/MS analysis was done on two compounds to evaluate drug concentrations. RESULTS: In serum-free media, 9 drugs prolonged FPDc. In serum-containing, 11 drugs prolonged FPDc. Eighteen drugs induced arrhythmias, 8 of these induced arrhythmias at lower concentrations in serum-containing media. At the highest non-arrhythmic concentrations, 13 of 25 drugs exhibited significant differences in FPDc prolongation/shortening between the media. Increasing fractions of serum in media yielded higher FPDc measurements. LC-MS/MS analysis of moxifloxacin and quinidine showed higher concentrations in serum-containing media. DISCUSSION: The present study highlights media formulation as an important consideration for cardiac safety testing with iPSC-CMs. Results described here suggest that media formulation influences both compound availability and baseline electrophysiological properties. Special attention should be paid to media for future iPSC-CM assays.

Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate selection.

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates.

Simple low cost differentiation of Candida auris from Candida haemulonii complex using CHROMagar Candida medium supplemented with Pal's medium.

BACKGROUND: Candida auris is unique due to its multidrug resistance and misidentification as Candida haemulonii by commercial systems. Its correct identification is important to avoid inappropriate treatments. AIMS: To develop a cheap method for differentiating C. auris from isolates identified as C. haemulonii by VITEK2. METHODS: Fifteen C. auris isolates, six isolates each of C. haemulonii and Candida duobushaemulonii, and one isolate of Candida haemulonii var. vulnera were tested using CHROMagar Candida medium supplemented with Pal's agar for better differentiation. RESULTS: On CHROMagar Candida medium supplemented with Pal's agar all C. auris strains showed confluent growth of white to cream colored smooth colonies at 37°C and 42°C after 24 and 48h incubation and did not produce pseudohyphae. The isolates of the C. haemulonii complex, on the contrary, showed poor growth of smooth, light-pink colonies at 24h while at 48h the growth was semiconfluent with the production of pseudohyphae. C. haemulonii complex failed to grow at 42°C. CONCLUSIONS: We report a rapid and cheap method using CHROMagar Candida medium supplemented with Pal's agar for differentiating C. auris from isolates identified as C. haemulonii by VITEK2.