Transcriptomic Analysis of the Differential Nephrotoxicity of Diverse Brominated Flame Retardants in Rat and Human Renal Cells.

Brominated flame retardants (BFRs) are environmentally persistent, are detected in humans, and some have been banned due to their potential toxicity. BFRs are developmental neurotoxicants and endocrine disruptors; however, few studies have explored their potential nephrotoxicity. We addressed this gap in the literature by determining the toxicity of three different BFRs (tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), and tetrabromodiphenyl ether (BDE-47)) in rat (NRK 52E) and human (HK-2 and RPTEC) tubular epithelial cells. All compounds induced time- and concentration-dependent toxicity based on decreases in MTT staining and changes in cell and nuclear morphology. The toxicity of BFRs was chemical- and cell-dependent, and human cells were more susceptible to all three BFRs based on IC50s after 48 h exposure. BFRs also had chemical- and cell-dependent effects on apoptosis as measured by increases in annexin V and PI staining. The molecular mechanisms mediating this toxicity were investigated using RNA sequencing. Principal components analysis supported the hypothesis that BFRs induce different transcriptional changes in rat and human cells. Furthermore, BFRs only shared nine differentially expressed genes in rat cells and five in human cells. Gene set enrichment analysis demonstrated chemical- and cell-dependent effects; however, some commonalities were also observed. Namely, gene sets associated with extracellular matrix turnover, the coagulation cascade, and the SNS-related adrenal cortex response were enriched across all cell lines and BFR treatments. Taken together, these data support the hypothesis that BFRs induce differential toxicity in rat and human renal cell lines that is mediated by differential changes in gene expression.

Insights into brominated flame retardant neurotoxicity: mechanisms of hippocampal neural cell death and brain region-specific transcriptomic shifts in mice.

Brominated flame retardants (BFRs) reduce flammability in a wide range of products including electronics, carpets, and paint, but leach into the environment to result in continuous, population-level exposure. Epidemiology studies have correlated BFR exposure with neurological problems, including alterations in learning and memory. This study investigated the molecular mechanisms mediating BFR-induced cell death in hippocampal cells and clarified the impact of hexabromocyclododecane (HBCD) exposure on gene transcription in the hippocampus, dorsal striatum, and frontal cortex of male mice. Exposure of hippocampus-derived HT-22 cells to various flame retardants, including tetrabromobisphenol-A (current use), HBCD (phasing out), or 2,2',4,4'-tetrabromodiphenyl ether (BDE-47, phased out) resulted in time, concentration, and chemical-dependent cellular and nuclear morphology alterations, alterations in cell cycle and increases in annexin V staining. All 3 BFRs increased p53 and p21 expression; however, inhibition of p53 nuclear translocation using pifthrin-α did not decrease cell death. Transcriptomic analysis upon low (10 nM) and cytotoxic (10 µM) BFR exposure indicated that HBCD and BDE-47 altered genes mediating autophagy-related pathways. Further evaluation showed that BFR exposure increased LC3-II conversion and autophagosome/autolysosome formation, and co-exposure with the autophagy inhibitor 3-methyladenine (3-MA) attenuated cytotoxicity. Transcriptomic assessment of select brain regions from subchronically HBCD-exposed male mice demonstrated alteration of genes mediating vesicular transport, with greater impact on the frontal cortex and dorsal striatum compared with the dorsal and ventral hippocampus. Immunoblot analysis demonstrated no increases in cell death or autophagy markers, but did demonstrate increases in the SNARE binding complex protein SNAP29, specifically in the dorsal hippocampus. These data demonstrate that BFRs can induce chemical-dependent autophagy in neural cells in vitro and provide evidence that BFRs induce region-specific transcriptomic and protein expression in the brain suggestive of changes in vesicular trafficking.

The Brominated Flame Retardant Hexabromocyclododecane Causes Systemic Changes in Polyunsaturated Fatty Acid Incorporation in Mouse Lipids.

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is one brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date the two remain unlinked. As lipids constitute ∼50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male C57BL/6 mice were exposed to 1 mg/kg HBCD every 3 days for 28 days. Major lipid classes were found to change across brain regions, including the membrane glycerolipids phosphatidylcholine and phosphatidylethanolamine, and sphingolipids such as hexosylceramide. In addition, saturated, monounsaturated, and polyunsaturated fatty acids were enriched within brain lipid species. To understand the source of the brain lipidomic alterations, the blood and liver lipidomes and the cecal microbiome were evaluated. The liver and blood demonstrated changes amongst multiple lipid classes, including triacylglycerol suppression, as well as altered esterified fatty acid content. Significant alterations were also detected in the cecal microbiome, with decreases in the Firmicutes to Bacteriodetes ratio, changes in beta diversity, and pathway alterations associated with metabolic pathways and amino acid biosynthesis. These data demonstrate that HBCD can induce lipidomic alterations across brain regions and organs and supports a potential role of the microbiome in these alterations.

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses.

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Abundant proliferating cells within early chicken taste buds indicate a potentially "built-in" progenitor system for taste bud growth during maturation in hatchlings.

Like other epithelial cells, taste bud cells have a short life span and undergo continuous turnover. An active stem or progenitor cell niche is essential for taste bud formation and maintenance. Early taste bud cells have a life span of ~4 days on average in chicken hatchlings when taste buds grow rapidly and undergo maturation. The average life span is shorter than that of mature taste bud cells of rodents (~10-12 days on average). To better understand the mechanism underlying taste bud growth and homeostasis in chickens, we analyzed the distribution of proliferating cells in different tissue compartments, including taste buds, the surrounding epithelium and the underlying connective tissue in P1-3 hatchlings and P45 chickens. Unlike rodents, which lack proliferating cells within both early and mature taste buds, chickens possessed abundant proliferating cells within early taste buds. Further, at post-hatch day 45, when taste buds are mature and undergo continuous cell renewal, taste buds also contained proliferating cells, though to a lesser extent. These proliferating cells in early taste buds, indicated by PCNA⁺ and BrdU⁺ cells, primarily localized to the basal region of taste buds and were largely unlabeled by the two known molecular markers for chicken taste bud cells (Vimentin and α-Gustducin), suggesting their undifferentiated status. Our data indicate that early chicken taste buds have "built-in" progenitors in order to grow to and maintain their large size and rapid cell turnover in hatchlings.

Reduced lysis upon growth of Lactococcus lactis on galactose is a consequence of decreased binding of the autolysin AcmA.

When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.

An Update on the Sense of Taste in Chickens: A Better Developed System than Previously Appreciated.

Taste is important in guiding nutritive choices and motivating food intake. The sensory organs for taste are the taste buds, that transduce gustatory stimuli into neural signals. It has been reported that chickens have a low taste bud number and thus low taste acuity. However, more recent studies indicate that chickens have a well-developed taste system and the reported number and distribution of taste buds may have been significantly underestimated. Chickens, as a well-established animal model for research, are also the major species of animals in the poultry industry. Thus, a clear understanding of taste organ formation and the effects of taste sensation on nutrition and feeding practices is important for improving livestock production strategies. In this review, we provide an update on recent findings in chicken taste buds and taste sensation indicating that the chicken taste organ is better developed than previously thought and can serve as an ideal system for multidisciplinary studies including organogenesis, regenerative medicine, feeding and nutritional choices.

A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data.

BACKGROUND: In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed. RESULTS: A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance. CONCLUSION: Even in cases where 90% of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25%. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

Resistance of Gram-positive bacteria to nisin is not determined by lipid II levels.

Lipid II is essential for nisin-mediated pore formation at nano-molar concentrations. We tested whether nisin resistance could result from different Lipid II levels, by comparing the maximal Lipid II pool in Micrococcus flavus (sensitive) and Listeria monocytogenes (relatively insensitive) and their nisin-resistant variants, with a newly developed method. No correlation was observed between the maximal Lipid II pool and nisin sensitivity, as was further corroborated by using spheroplasts of nisin-resistant and wild-type strains of M. flavus, which were equally sensitive to nisin.

Increased D-alanylation of lipoteichoic acid and a thickened septum are main determinants in the nisin resistance mechanism of Lactococcus lactis.

Nisin is a post-translationally modified antimicrobial peptide produced by Lactococcus lactis which binds to lipid II in the membrane to form pores and inhibit cell-wall synthesis. A nisin-resistant (Nis(R)) strain of L. lactis, which is able to grow at a 75-fold higher nisin concentration than its parent strain, was investigated with respect to changes in the cell wall. Direct binding studies demonstrated that less nisin was able to bind to lipid II in the membranes of L. lactis Nis(R) than in the parent strain. In contrast to vancomycin binding, which showed ring-like binding, nisin was observed to bind in patches close to cell-division sites in both the wild-type and the Nis(R) strains. Comparison of modifications in lipoteichoic acid of the L. lactis strains revealed an increase in d-alanyl esters and galactose as substituents in L. lactis Nis(R), resulting in a less negatively charged cell wall. Moreover, the cell wall displays significantly increased thickness at the septum. These results indicate that shielding the membrane and thus the lipid II molecule, thereby decreasing abduction of lipid II and subsequent pore-formation, is a major defence mechanism of L. lactis against nisin.

Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance.

Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.

An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II.

Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Transcriptome analysis and related databases of Lactococcus lactis.

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL 1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper describes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCye, a metabolic pathway/genome database of Lactococcus lactis.