Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

Informing Hazard Identification and Risk Characterization of Environmental Chemicals by Combining Transcriptomic and Functional Data from Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocytes.

Environmental chemicals may contribute to the global burden of cardiovascular disease, but experimental data are lacking to determine which substances pose the greatest risk. Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a high-throughput cardiotoxicity model that is widely used to test drugs and chemicals; however, most studies focus on exploring electro-physiological readouts. Gene expression data may provide additional molecular insights to be used for both mechanistic interpretation and dose-response analyses. Therefore, we hypothesized that both transcriptomic and functional data in human iPSC-derived cardiomyocytes may be used as a comprehensive screening tool to identify potential cardiotoxicity hazards and risks of the chemicals. To test this hypothesis, we performed concentration-response analysis of 464 chemicals from 12 classes, including both pharmaceuticals and nonpharmaceutical substances. Functional effects (beat frequency, QT prolongation, and asystole), cytotoxicity, and whole transcriptome response were evaluated. Points of departure were derived from phenotypic and transcriptomic data, and risk characterization was performed. Overall, 244 (53%) substances were active in at least one phenotype; as expected, pharmaceuticals with known cardiac liabilities were the most active. Positive chronotropy was the functional phenotype activated by the largest number of tested chemicals. No chemical class was particularly prone to pose a potential hazard to cardiomyocytes; a varying proportion (10-44%) of substances in each class had effects on cardiomyocytes. Transcriptomic data showed that 69 (15%) substances elicited significant gene expression changes; most perturbed pathways were highly relevant to known key characteristics of human cardiotoxicants. The bioactivity-to-exposure ratios showed that phenotypic- and transcriptomic-based POD led to similar results for risk characterization. Overall, our findings demonstrate how the integrative use of in vitro transcriptomic and phenotypic data from iPSC-derived cardiomyocytes not only offers a complementary approach for hazard and risk prioritization, but also enables mechanistic interpretation of the in vitro test results to increase confidence in decision-making.

Genome and transcriptome sequencing of the green bottle fly, Lucilia sericata, reveals underlying factors of sheep flystrike and maggot debridement therapy.

The common green bottle blow fly Lucilia sericata (family, Calliphoridae) is widely used for maggot debridement therapy, which involves the application of sterile maggots to wounds. The larval excretions and secretions are important for consuming necrotic tissue and inhibiting bacterial growth in wounds of patients. Lucilia sericata is also of importance as a pest of sheep and in forensic studies to estimate a postmortem interval. Here we report the assembly of a 565.3 Mb genome from long read PacBio DNA sequencing of genomic DNA. The genome contains 14,704 predicted protein coding genes and 1709 non-coding genes. Targeted annotation and transcriptional analyses identified genes that are highly expressed in the larval salivary glands (secretions) and Malpighian tubules (excretions) under normal growth conditions and following heat stress. The genomic resources will underpin future genetic studies and in development of engineered strains for genetic control of L. sericata and for biotechnology-enhanced maggot therapy.

High-resolution characterization of the structural features and genetic variation of six feline leukocyte antigen class I loci via single molecule, real-time (SMRT) sequencing.

Of the 12 full-length feline leukocyte antigen class I (FLAI) loci, 3 are presumed to be classical: FLAI-E, FLAI-H, and FLAI-K. As diversity is a class Ia hallmark, multi-allelism is an important surrogate supporting a classical designation, in the absence of direct demonstration of T-cell restriction. Conversely, limited polymorphism at an expressed locus suggests regulation of immune effectors with invariant receptors, and non-classical status. FLAI-A, FLAI-J, FLAI-L, and FLAI-O are putative class Ib genes in cats. For both classes, identifying prevalent variants across outbred populations can illuminate specific genotypes to be prioritized for immune studies, as shared alleles direct shared responses. Since variation is concentrated in exons 2 and 3, which encode the antigen-binding domains, partial-length cloning/sequencing can be used for allele discovery, but is laborious and occasionally ambiguous. Here we develop a targeted approach to FLAI genotyping, using the single-molecule real-time (SMRT) platform, which allows full-length (3.4-kb) reads without assembly. Consensus sequences matched full-length Sanger references. Thirty-one new class Ia genes were found in 17 cats. Alleles segregated strongly by loci, and the origins of formerly difficult-to-assign sequences were resolved. Although not targeted, FLAI-L and FLAI-J, and the pseudogene FLAI-F, were also returned. Eighteen class Ib alleles were identified. Diversity was restricted and outside hypervariable regions. Both class Ib genes were transcriptionally active. Novel alternative splicing of FLAI-L was observed. SMRT sequencing of FLAI amplicons is useful for full-length genotyping at feline class Ia loci. High-throughput sequencing could allow highly accurate allele surveys in large cat cohorts.

Assessment of the non-lactic acid bacteria microbiota in fresh cucumbers and commercially fermented cucumber pickles brined with 6% NaCl.

Limited documentation of the cucumber fermentation microbiome has impeded the understanding of the role of microbes on the quality of finished products. We characterized the microbiome of fresh and fermented cucumber samples using culture dependent and independent techniques, with an emphasis on the non-lactic acid bacteria (non-LAB) population. Insubstantial microbiome variations were observed among fresh cucumber types with Rhizobium (31.04%), Pseudomonas (14.08%), Pantoea (9.25%), Stenotrophomonas (6.83%), and Acinetobacter (6.5%) prevailing. The relative abundance of LAB remained below 0.4% and 4.0% on fresh cucumbers and day 3 of the fermentations brined with 6% sodium chloride, respectively. Fermentation cover brine samples collected on day 1 harbored Pseudomonas, Pantoea, Stenotrophomonas, Acinetobacter, Comamonas, Wautersiella, Microbacterium, Flavobacterium, Ochrobactrum and the Enterobacteriaceae, Citrobacter, Enterobacter and Kluyvera. Plate counts for presumptive Klebsiella and Pseudomonas from fermentation cover brine samples reached 2.80 ±â€¯0.36 and 2.78 ±â€¯0.83 log of CFU/mL, respectively, in 30% and 60% of the nine tanks scrutinized with selective media. Both genera were found in cover brine samples with pH values at 4.04 ±â€¯0.15. We aim at elucidating whether the low relative abundance of non-LAB in commercial cucumber fermentations, in particular Pseudomonas and Enterobacteriaceae, impacts the quality of fermented cucumbers.

Complete genome sequences of Lactobacillus acidophilus strain P42 and Limosilactobacillus reuteri strain P43 isolated from chicken cecum.

The gut microflora contains a diverse microbial population that is influenced by the host and the environment. We report the complete circular genome sequences of Lactobacillus acidophilus strain P42 and Limosilactobacillus reuteri strain P43 isolated from chicken cecal samples. P42 and P43 could potentially serve as poultry probiotic strains.

Risk-based prioritization of PFAS using phenotypic and transcriptomic data from human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes.

Per- and polyfluoroalkyl substances (PFAS) are chemicals with important applications; they are persistent in the environment and may pose human health hazards. Regulatory agencies are con­sidering restrictions and bans of PFAS; however, little data exists for informed decisions. Several prioritization strategies were proposed for evaluation of potential hazards of PFAS. Structure-based grouping could expedite the selection of PFAS for testing; still, the hypothesis that structure-effect relationships exist for PFAS requires confirmation. We tested 26 structurally diverse PFAS from 8 groups using human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes, and tested concentration-response effects on cell function and gene expression. Few phenotypic effects were observed in hepatocytes, but negative chronotropy was observed in cardiomyocytes for 8 PFAS. Substance- and cell type-dependent transcriptomic changes were more prominent but lacked substantial group-specific effects. In hepatocytes, we found upregulation of stress-related and extracellular matrix organization pathways, and down-regulation of fat metabolism. In car­diomyocytes, contractility-related pathways were most affected. We derived phenotypic and transcriptomic points of departure and compared them to predicted PFAS exposures. Conservative estimates for bioactivity and exposure were used to derive a bioactivity-to-exposure ratio (BER) for each PFAS; 23 of 26 PFAS had BER > 1. Overall, these data suggest that structure-based PFAS grouping may not be sufficient to predict their biological effects. Testing of individual PFAS may be needed for scientifically-supported decision-making. Our proposed strategy of using two human cell types and considering phenotypic and transcriptomic effects, combined with dose-response analysis and calculation of BER, may be used for PFAS prioritization.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many products. However, most of these substances have not been tested for safety, and concerns exist that they may be harmful to human health and/or the environment. This study aimed to use human cell-based models to investigate if some of the PFAS may exhibit hazardous properties and if similarities among substance groups are observed. Few effects were observed in liver cells, but a decrease in beating frequency was observed in heart cells for some PFAS. Gene expression changes were substance-and cell type-dependent. We did not find convincing structure-based similarities among PFAS; this suggests that testing of individual PFAS may be necessary in the future to inform health decisions. Overall, this study showed that a test strategy of using two human cell types, from liver and heart, may inform PFAS prioritization without a need for testing in animals.

Yearly variation coupled with social interactions shape the skin microbiome in free-ranging rhesus macaques.

While skin microbes are known to mediate human health and disease, there has been minimal research on the interactions between skin microbiota, social behavior, and year-to-year effects in non-human primates-important animal models for translational biomedical research. To examine these relationships, we analyzed skin microbes from 78 rhesus macaques living on Cayo Santiago Island, Puerto Rico. We considered age, sex, and social group membership, and characterized social behavior by assessing dominance rank and patterns of grooming as compared to nonsocial behaviors. To measure the effects of a shifting environment, we sampled skin microbiota (based on sequence analysis of the 16S rRNA V4 region) and assessed weather across sampling periods between 2013 and 2015. We hypothesized that, first, monkeys with similar social behavior and/or in the same social group would possess similar skin microbial composition due, in part, to physical contact, and, second, microbial diversity would differ across sampling periods. We found significant phylum-level differences between social groups in the core microbiome as well as an association between total grooming rates and alpha diversity in the complete microbiome, but no association between microbial diversity and measures of rank or other nonsocial behaviors. We also identified alpha and beta diversity differences in microbiota and differential taxa abundance across two sampling periods. Our findings indicate that social dynamics interact with yearly environmental changes to shape the skin microbiota in rhesus macaques, with potential implications for understanding the factors affecting the microbiome in humans, which share many biological and social characteristics with these animals. IMPORTANCE Primate studies are valuable for translational and evolutionary insights into the human microbiome. The majority of primate microbiome studies focus on the gut, so less is known about the factors impacting the microbes on skin and how their links affect health and behavior. Here, we probe the impact of social interactions and the yearly environmental changes on food-provisioned, free-ranging monkeys living on a small island. We expected animals that lived together and groomed each other would have more similar microbes on their skin, but surprisingly found that the external environment was a stronger influence on skin microbiome composition. These findings have implications for our understanding of the human skin microbiome, including potential manipulations to improve health and treat disease.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Complete Genome Sequences of Six Lactobacilli Isolated from American Quarter Horses.

We report the complete circular genome sequences of six Lactobacillus strains and their plasmids, if any, from the fecal material of quarter horses at different ages.

Complete Genome Sequences of Lactobacillus Strains C25 and P38, Isolated from Chicken Cecum.

We report the complete circular genome sequences of Lactobacillus crispatus strain C25, its plasmid, and Lactobacillus animalis strain P38; both strains were isolated from the cecum of 4-week-old chickens. These isolates represent potential probiotic strains for poultry.

Using ergosterol to mitigate the deleterious effects of ethanol on bilayer structure.

In wine fermentations, yeast is exposed to concentrated ethanol solutions. Ergosterol, a sterol that is found in lower eukaryotic membranes, helps preserve the structural integrity of yeast membranes in stressful environmental conditions. A premature arrest in ethanol production due to unknown metabolic changes in yeasts results in undesirably large concentrations of residual sugar and may be caused by the formation of an ethanol-induced interdigitated phase. We use atomistic molecular dynamics simulations to examine the induction of the interdigitated phase in model yeast membranes that contain either 0, 10, 20, 25 mol % ergosterol in ethanol concentrations of 0, 10, 15 vol %. The 25 mol % ergosterol system shows a similar level of interdigitation for the 0 and 10 vol % ethanol solutions, indicating that ergosterol molecules in this system are able to effectively counteract the disruptive behavior of ethanol molecules. However, at a 15 vol % ethanol solution, the amount of interdigitation triples and this ethanol concentration is similar to the concentrations found in stuck fermentations. The other three ergosterol concentrations studied (0, 10, 20 mol %) show larger quantities of interdigitation in the 10 vol % ethanol solution than the 0 vol % solution. Thus, the 25 mol % ergosterol bilayer, which is representative of the ergosterol concentrations seen in yeast membranes, is unique in the systems examined in its ability to delay the onset of ethanol-induced interdigitation. The concentration of ergosterol affects the permeability of a fluid-phase bilayer, where the 10 mol % ergosterol bilayer is more permeable to ethanol than either a bilayer containing no ergosterol molecules or a bilayer containing 20/25 mol % ergosterol. This lipid permeability appears to be correlated with the existence of a lipid region whose lipids neither have direct contact with ergosterol molecules nor exhibit bulk lipid/lipid interactions.

Behavioral differences between phosphatidic acid and phosphatidylcholine in the presence of the nicotinic acetylcholine receptor.

It has been found experimentally that negatively charged phosphatidic acid (PA) lipids and cholesterol molecules stabilize the nicotinic acetylcholine receptor (nAChR) in a functional resting state that can participate in an agonist-induced conformational change. In this study, we compare phosphatidylcholine (PC) and PA lipid behavior in the presence of the nAChR to determine why PC lipids do not support a functional nAChR. For lipids that are located within 1.0 nm of the protein, both PC and PA lipids have very similar order-parameter and bilayer-thickness values, which indicate that the annular lipid properties are protein-dependent. The most significant difference between the PC and PA bilayers is the formation of a lipid domain around the protein, which is visible in the PA bilayer but not the PC bilayer. This suggests that the PA domain may help stabilize the nAChR resting state. The PA lipids in the microdomain have a decreased order compared to a homogeneous PA bilayer and the lipids near the protein attempt to increase the free space in their vicinity by residing in multiple lateral planes.

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C16:1ω7c/C16:1ω6c, C16:0, and C18:1ω7c, and were consistent with the genus. Genomic G+C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3T as the type strain (=LMG 30136, =DSM 105008).

Simulations of Biomembranes and Water: Important Technical Aspects.

In this contribution, we discuss several important technical aspects which are relevant for the molecular modeling of biomembranes in aqueous environments. We study the effect of coarse-grained water models on free and supported systems and show that the choice of water model has dramatic repercussions on the phase behavior. We characterize the phase behavior of a widely used water model and discuss the technical implications of modeling solid supports for biomembrane simulations. Finally, we compare the effect of anisotropic pressure coupling with surface tension coupling in atomistic bilayer simulations including alcohols.

Complete Genome Sequence of a Bacterium Representing a Deep Uncultivated Lineage within the Gammaproteobacteria Associated with the Degradation of Polycyclic Aromatic Hydrocarbons.

The bacterial strain TR3.2, representing a novel deeply branching lineage within the Gammaproteobacteria, was isolated and its genome sequenced. This isolate is the first cultivated representative of the previously described "Pyrene Group 2" (PG2) and represents a variety of environmental sequences primarily associated with petrochemical contamination and aromatic hydrocarbon degradation.

Complete Genome Sequence of NC983, a Live Attenuated Strain of Salmonella enterica Serovar Typhimurium.

Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate.

Complete Genome Sequence of a Novel Bacterium within the Family Rhodocyclaceae That Degrades Polycyclic Aromatic Hydrocarbons.

A polycyclic aromatic hydrocarbon-degrading bacterium designated strain Ca6, a member of the family Rhodocyclaceae and a representative of the uncharacterized pyrene group 1 (PG1), was isolated and its genome sequenced. The presence of several genes suspected to be associated with PG1 was confirmed, and additional genes for aromatic compound metabolism were detected.

The effect of pyridine modification of Ni-DOBDC on CO2 capture under humid conditions.

The metal-organic framework Ni-DOBDC was modified with pyridine molecules to make the normally hydrophilic internal surface more hydrophobic. Experiments and molecular simulations show that the pyridine modification reduces H2O adsorption while retaining substantial CO2 capacity under the conditions of interest for carbon capture from flue gas.