Linking void and interphase evolution to electrochemistry in solid-state batteries using operando X-ray tomography.

Despite progress in solid-state battery engineering, our understanding of the chemo-mechanical phenomena that govern electrochemical behaviour and stability at solid-solid interfaces remains limited compared to at solid-liquid interfaces. Here, we use operando synchrotron X-ray computed microtomography to investigate the evolution of lithium/solid-state electrolyte interfaces during battery cycling, revealing how the complex interplay among void formation, interphase growth and volumetric changes determines cell behaviour. Void formation during lithium stripping is directly visualized in symmetric cells, and the loss of contact that drives current constriction at the interface between lithium and the solid-state electrolyte (Li10SnP2S12) is quantified and found to be the primary cause of cell failure. The interphase is found to be redox-active upon charge, and global volume changes occur owing to partial molar volume mismatches at either electrode. These results provide insight into how chemo-mechanical phenomena can affect cell performance, thus facilitating the development of solid-state batteries.

The Role of the Human Microbiome in Chemical Toxicity.

There is overwhelming evidence that the microbiome must be considered when evaluating the toxicity of chemicals. Disruption of the normal microbial flora is a known effect of toxic exposure, and these disruptions may lead to human health effects. In addition, the biotransformation of numerous compounds has been shown to be dependent on microbial enzymes, with the potential for different host health outcomes resulting from variations in the microbiome. Evidence suggests that such metabolism of environmental chemicals by enzymes from the host's microbiota can affect the toxicity of that chemical to the host. Chemical-microbial interactions can be categorized into two classes: Microbiome Modulation of Toxicity (MMT) and Toxicant Modulation of the Microbiome (TMM). MMT refers to transformation of a chemical by microbial enzymes or metabolites to modify the chemical in a way that makes it more or less toxic. TMM is a change in the microbiota that results from a chemical exposure. These changes span a large magnitude of effects and may vary from microbial gene regulation, to inhibition of a specific enzyme, to the death of the microbes. Certain microbiomes or microbiota may become associated with different health outcomes, such as resistance or susceptibility to exposure to certain toxic chemicals, the ability to recover following a chemical-induced injury, the presence of disease-associated phenotypes, and the effectiveness of immune responses. Future work in toxicology will require an understanding of how the microbiome interacts with toxicants to fully elucidate how a compound will affect a diverse, real-world population.

Utility of Serum miR-122, Liver Enzymes, and Hepatic Histopathology in Response to Hepatotoxicants in Sprague-Dawley Rats.

More than 80,000 chemicals are in commercial use worldwide. Hepatic metabolism to toxic intermediates is often a key mechanism leading to tissue damage and organ dysfunction. Effective treatment requires prompt detection of hepatotoxicity, ideally with rapid, minimally invasive diagnostic assays. In this study, archetypal histologic features of chemically induced hepatic injury were compared with clinical chemistries (including liver enzymes) and serum concentrations of microRNA-122 (miR-122, the processed form miR-122-5p), a biomarker of liver injury. The hepatotoxicants 4,4'-methylenedianiline (4,4'-MDA), allyl alcohol (AA), or carbon tetrachloride (CCl4) were orally administered to male Sprague-Dawley rats for 1, 5, 14, or 28 days to induce liver damage. Formalin-fixed, paraffin-embedded liver sections were evaluated histologically for inflammation, fibrosis, necrosis, and lipid accumulation. Liver enzymes were measured in serum, and serum miR-122 concentrations were assessed by quantitative polymerase chain reaction (qPCR). Histologic features of hepatic injury dose-dependently increased in both severity and frequency. Increases in liver enzymes and bilirubin were more pronounced in response to AA or 4,4'-MDA than to CCl4 at early time points. Elevated serum miR-122 levels in animals administered CCl4, AA, or 4,4'-MDA were more strongly associated with degree of hepatic histopathology than with dosage. Given this sensitive expression pattern postexposure, liver-specific miR-122 may improve the diagnostic accuracy of early hepatic injury.

Comparative Proteomic Analysis of Liver Steatosis and Fibrosis after Oral Hepatotoxicant Administration in Sprague-Dawley Rats.

The past decade has seen an increase in the development and clinical use of biomarkers associated with histological features of liver disease. Here, we conduct a comparative histological and global proteomics analysis to identify coregulated modules of proteins in the progression of hepatic steatosis or fibrosis. We orally administered the reference chemicals bromobenzene (BB) or 4,4'-methylenedianiline (4,4'-MDA) to male Sprague-Dawley rats for either 1 single administration or 5 consecutive daily doses. Livers were preserved for histopathology and global proteomics assessment. Analysis of liver sections confirmed a dose- and time-dependent increase in frequency and severity of histopathological features indicative of lipid accumulation after BB or fibrosis after 4,4'-MDA. BB administration resulted in a dose-dependent increase in the frequency and severity of inflammation and vacuolation. 4,4'-MDA administration resulted in a dose-dependent increase in the frequency and severity of periportal collagen accumulation and inflammation. Pathway analysis identified a time-dependent enrichment of biological processes associated with steatogenic or fibrogenic initiating events, cellular functions, and toxicological states. Differentially expressed protein modules were consistent with the observed histology, placing physiologically linked protein networks into context of the disease process. This study demonstrates the potential for protein modules to provide mechanistic links between initiating events and histopathological outcomes.

Dichlorvos exposure results in large scale disruption of energy metabolism in the liver of the zebrafish, Danio rerio.

BACKGROUND: Exposure to dichlorvos (DDVP), an organophosphorus pesticide, is known to result in neurotoxicity as well as other metabolic perturbations. However, the molecular causes of DDVP toxicity are poorly understood, especially in cells other than neurons and muscle cells. To obtain a better understanding of the process of non-neuronal DDVP toxicity, we exposed zebrafish to different concentrations of DDVP, and investigated the resulting changes in liver histology and gene transcription. RESULTS: Functional enrichment analysis of genes affected by DDVP exposure identified a number of processes involved in energy utilization and stress response in the liver. The abundance of transcripts for proteins involved in glucose metabolism was profoundly affected, suggesting that carbon flux might be diverted toward the pentose phosphate pathway to compensate for an elevated demand for energy and reducing equivalents for detoxification. Strikingly, many transcripts for molecules involved in ß-oxidation and fatty acid synthesis were down-regulated. We found increases in message levels for molecules involved in reactive oxygen species responses as well as ubiquitination, proteasomal degradation, and autophagy. To ensure that the effects of DDVP on energy metabolism were not simply a consequence of poor feeding because of neuromuscular impairment, we fasted fish for 29 or 50 h and analyzed liver gene expression in them. The patterns of gene expression for energy metabolism in fasted and DDVP-exposed fish were markedly different. CONCLUSION: We observed coordinated changes in the expression of a large number of genes involved in energy metabolism and responses to oxidative stress. These results argue that an appreciable part of the effect of DDVP is on energy metabolism and is regulated at the message level. Although we observed some evidence of neuromuscular impairment in exposed fish that may have resulted in reduced feeding, the alterations in gene expression in exposed fish cannot readily be explained by nutrient deprivation.

Detection of dichlorvos adducts in a hepatocyte cell line.

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.

Patterns of gene expression associated with recovery and injury in heat-stressed rats.

BACKGROUND: The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model. RESULTS: We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response. CONCLUSIONS: Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

Alteration in circulating metabolites during and after heat stress in the conscious rat: potential biomarkers of exposure and organ-specific injury.

BACKGROUND: Heat illness is a debilitating and potentially life-threatening condition. Limited data are available to identify individuals with heat illness at greatest risk for organ damage. We recently described the transcriptomic and proteomic responses to heat injury and recovery in multiple organs in an in vivo model of conscious rats heated to a maximum core temperature of 41.8°C (Tc,Max). In this study, we examined changes in plasma metabolic networks at Tc,Max, 24, or 48 hours after the heat stress stimulus. RESULTS: Circulating metabolites were identified by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. Bioinformatics analysis of the metabolomic data corroborated proteomics and transcriptomics data in the tissue at the pathway level, supporting modulations in metabolic networks including cell death or catabolism (pyrimidine and purine degradation, acetylation, sulfation, redox alterations and glutathione metabolism, and the urea cycle/creatinine metabolism), energetics (stasis in glycolysis and tricarboxylic acid cycle, ß-oxidation), cholesterol and nitric oxide metabolism, and bile acids. Hierarchical clustering identified 15 biochemicals that differentiated animals with histopathological evidence of cardiac injury at 48 hours from uninjured animals. The metabolic networks perturbed in the plasma corroborated the tissue proteomics and transcriptomics pathway data, supporting a model of irreversible cell death and decrements in energetics as key indicators of cardiac damage in response to heat stress. CONCLUSIONS: Integrating plasma metabolomics with tissue proteomics and transcriptomics supports a diagnostic approach to assessing individual susceptibility to organ injury and predicting recovery after heat stress.

Alterations in gene expression in Caenorhabditis elegans associated with organophosphate pesticide intoxication and recovery.

BACKGROUND: The principal toxicity of acute organophosphate (OP) pesticide poisoning is the disruption of neurotransmission through inhibition of acetylcholinesterase (AChE). However, other mechanisms leading to persistent effects and neurodegeneration remain controversial and difficult to detect. Because Caenorhabditis elegans is relatively resistant to OP lethality--particularly through the inhibition of AChE--studies in this nematode provide an opportunity to observe alterations in global gene expression following OP exposure that cannot be readily observed in less resistant organisms. RESULTS: We exposed cultures of worms in axenic, defined medium to dichlorvos under three exposure protocols. In the first, worms were exposed continuously throughout the experiment. In the second and third, the worms were exposed for either 2 or 8 h, the dichlorvos was washed out of the culture, and the worms were allowed to recover. We then analyzed gene expression using whole genome microarrays from RNA obtained from worms sampled at multiple time points throughout the exposure. The worms showed a time-dependent increase in the expression of genes involved in stress responses. Early in the exposure, the predominant effect was on metabolic processes, while at later times, an immune-like response and cellular repair mechanisms dominated the expression pattern. Following removal of dichlorvos, the gene expression in the worms appeared to relatively rapidly return to steady-state levels. CONCLUSION: The changes in gene expression observed in the worms following exposure to dichlorvos point towards two potential mechanisms of toxicity: inhibition of AChE and mitochondrial disruption.

Aluminum foil negative electrodes with multiphase microstructure for all-solid-state Li-ion batteries.

Metal negative electrodes that alloy with lithium have high theoretical charge storage capacity and are ideal candidates for developing high-energy rechargeable batteries. However, such electrode materials show limited reversibility in Li-ion batteries with standard non-aqueous liquid electrolyte solutions. To circumvent this issue, here we report the use of non-pre-lithiated aluminum-foil-based negative electrodes with engineered microstructures in an all-solid-state Li-ion cell configuration. When a 30-µm-thick Al94.5In5.5 negative electrode is combined with a Li6PS5Cl solid-state electrolyte and a LiNi0.6Mn0.2Co0.2O2-based positive electrode, lab-scale cells deliver hundreds of stable cycles with practically relevant areal capacities at high current densities (6.5 mA cm-2). We also demonstrate that the multiphase Al-In microstructure enables improved rate behavior and enhanced reversibility due to the distributed LiIn network within the aluminum matrix. These results demonstrate the possibility of improved all-solid-state batteries via metallurgical design of negative electrodes while simplifying manufacturing processes.

Biological responses in rats exposed to cigarette smoke and Middle East sand (dust).

Respiratory symptoms are frequently reported in personnel deployed to the Middle East. This project characterized the respiratory toxicity of inhaled Iraqi sand (IS). Adult rats underwent a 6-wk inhalation to air or mainstream cigarette smoke (MSCS) (3 h/d, 5 d/wk) that included exposure to IS or crystalline silica (1 mg/m(3), 19 h/d, 7 d/wk) or air during the last 2 weeks. Assessments included motor activity, whole-body plethysmography, cytological and biochemical analysis of bronchoalveolar lavage fluid, lung metal burden, nasal and lung pathology, and changes in lung protein and gene expression. A number of metals including nickel, manganese, vanadium, and chromium were detected in IS. Elevated lung parenchyma aluminum, silica, barium, manganese, and vanadium concentrations were seen in IS-exposed rats, suggesting that several metals present in IS are bioavailable. Rats exposed to IS only developed mild inflammation in the anterior nose and lung. Silica inhalation was associated with some pulmonary responses that were not seen in IS-exposed rats, such as mild laryngeal and tracheal inflammation, mild tracheal epithelial hyperplasia, and elevated lung silica concentrations. MSCS inhalation with or without co-exposure to either IS or silica resulted in changes consistent with pulmonary inflammation and stress response. Rats exposed to MSCS and silica had more widespread airway lesions when compared with rats exposed to MSCS only. Silica-exposed rats had more robust pulmonary gene expression and proteomic responses than that seen in IS-exposed rat. Our studies show that the respiratory toxicity of IS is qualitatively similar to or less than that seen following short-term silica exposure.

Role of Areal Capacity in Determining Short Circuiting of Sulfide-Based Solid-State Batteries.

Solid-state batteries (SSBs) with lithium metal anodes offer higher specific energy than conventional lithium-ion batteries, but they must utilize areal capacities >3 mAh cm-2 and cycle at current densities >3 mA cm-2 to achieve commercial viability. Substantial research effort has focused on increasing the rate capabilities of SSBs by mitigating detrimental processes such as lithium filament penetration and short circuiting. Less attention has been paid to understanding how areal capacity impacts lithium plating/stripping behavior in SSBs, despite the importance of areal capacity for achieving high specific energy. Here, we investigate and quantify the relationships among areal capacity, current density, and plating/stripping stability using both symmetric and full-cell configurations with a sulfide solid-state electrolyte (Li6PS5Cl). We show that unstable deposition and short circuiting readily occur at rates much lower than the measured critical current density when a sufficient areal capacity is passed. A systematic study of continuous plating under different electrochemical conditions reveals average "threshold capacity" values at different current densities, beyond which short circuiting occurs. Cycling cells below this threshold capacity significantly enhances cell lifetime, enabling stable symmetric cell cycling at 2.2 mA cm-2 without short circuiting. Finally, we show that full cells with LiNi0.8Mn0.1Co0.1O2 also exhibit threshold capacity behavior, but they tend to short circuit at lower current densities and areal capacities. Our results quantify the effects of transferred capacity and demonstrate the importance of using realistic areal capacities in experiments to develop viable solid-state batteries.

Analysis of secreted proteins as an in vitro model for discovery of liver toxicity markers.

Despite the wealth of sequence data and new technologies that can scan large portions of the transcriptome or proteome in a single experiment, attempts to identify human biomarkers of toxicity have been met with limited success. We have adapted an in vitro model system to identify proteins secreted by a human hepatoma-derived cell line (HepG2/C3A) in response to toxicant exposure. Using quantitative proteomics, we can find alterations in the abundance of proteins at the source of damage-liver cells-that are likely to be present in blood samples of exposed animals. In a proof of concept experiment, conditioned medium from cells exposed to ethanol was subjected to quantitative mass spectral analysis after abundant proteins were immunodepleted. Eighty-seven proteins were identified with almost half changing in abundance. Some of these were only identified in the highest treatment condition and presumably result from the release of intracellular proteins into the medium when the cell membrane is disrupted upon cell death. However, the majority of the identified proteins reflect known consequences of ethanol exposure or alcoholism. The analysis of proteins found in conditioned medium after exposure to toxicants appears to be a useful system for the expedited discovery of potential human biomarkers.

Distinct patterns of gene and protein expression elicited by organophosphorus pesticides in Caenorhabditis elegans.

BACKGROUND: The wide use of organophosphorus (OP) pesticides makes them an important public health concern. Persistent effects of exposure and the mechanism of neuronal degeneration are continuing issues in OP toxicology. To elucidate early steps in the mechanisms of OP toxicity, we studied alterations in global gene and protein expression in Caenorhabditis elegans exposed to OPs using microarrays and mass spectrometry. We tested two structurally distinct OPs (dichlorvos and fenamiphos) and employed a mechanistically different third neurotoxicant, mefloquine, as an out-group for analysis. Treatment levels used concentrations of chemical sufficient to prevent the development of 10%, 50% or 90% of mid-vulval L4 larvae into early gravid adults (EGA) at 24 h after exposure in a defined, bacteria-free medium. RESULTS: After 8 h of exposure, the expression of 87 genes responded specifically to OP treatment. The abundance of 34 proteins also changed in OP-exposed worms. Many of the genes and proteins affected by the OPs are expressed in neuronal and muscle tissues and are involved in lipid metabolism, cell adhesion, apoptosis/cell death, and detoxification. Twenty-two genes were differentially affected by the two OPs; a large proportion of these genes encode cytochrome P450s, UDP-glucuronosyl/UDP-glucosyltransferases, or P-glycoproteins. The abundance of transcripts and the proteins they encode were well correlated. CONCLUSION: Exposure to OPs elicits a pattern of changes in gene expression in exposed worms distinct from that of the unrelated neurotoxicant, mefloquine. The functional roles and the tissue location of the genes and proteins whose expression is modulated in response to exposure is consistent with the known effects of OPs, including damage to muscle due to persistent hypercontraction, neuronal cell death, and phase I and phase II detoxification. Further, the two different OPs evoked distinguishable changes in gene expression; about half the differences are in genes involved in detoxification, likely reflecting differences in the chemical structure of the two OPs. Changes in the expression of a number of sequences of unknown function were also discovered, and these molecules could provide insight into novel mechanisms of OP toxicity or adaptation in future studies.

Quantifying the titer and quality of adenovirus stocks.

The broad application of recombinant adenoviruses to the development of vaccines and gene therapy vectors has encouraged the development of molecular assays for the facile quantitation of adenoviral particles and the assignment of their infectious potency. The Genome Quantitation Assay (GQA) and the QPCR-Based Potency Assay (QPA) developed for adenoviruses offer the attributes of precision, rapidity, and high throughput either performed manually or facilitated by simple automated liquid handling systems. These assay attributes allow for accelerated process development support and product characterization and release. The assays for adenovirus could offer the additional advantage in that their quantitation is based on viral replication independent of cytopathology permitting quantitation of serotypes that cause minimal cytopathic effect (CPE) in 293 cells and specificity that allows the components of multivalent vaccines to be discriminated and quantitated for release.

Exposure to toxic metals triggers unique responses from the rat gut microbiota.

Our understanding of the interaction between the gut microbiota and host health has recently improved dramatically. However, the effects of toxic metal exposure on the gut microbiota remain poorly characterized. As this microbiota creates a critical interface between the external environment and the host's cells, it may play an important role in host outcomes during exposure. We therefore used 16S ribosomal RNA (rRNA) gene sequencing to track changes in the gut microbiota composition of rats exposed to heavy metals. Rats were exposed daily for five days to arsenic, cadmium, cobalt, chromium, nickel, or a vehicle control. Significant changes to microbiota composition were observed in response to high doses of chromium and cobalt, and significant dose-dependent changes were observed in response to arsenic, cadmium and nickel. Many of these perturbations were not uniform across metals. However, bacteria with higher numbers of iron-importing gene orthologs were overly represented after exposure to arsenic and nickel, suggesting some possibility of a shared response. These findings support the utility of the microbiota as a pre-clinical tool for identifying exposures to specific heavy metals. It is also clear that characterizing changes to the functional capabilities of microbiota is critical to understanding responses to metal exposure.

Proteomic analysis of bronchoalveolar lavage fluid: effect of acute exposure to diesel exhaust particles in rats.

BACKGROUND: Inhalation of diesel exhaust particles (DEPs) is characterized by lung injury and inflammation, with significant increases in the numbers of polymorphonuclear leukocytes and alveolar macrophages. This influx of cellular infiltrates is associated with the activation of multiple genes, including cytokines and chemokines, and the production of reactive oxygen species. OBJECTIVE: The pathogenesis of the lung injury is not fully understood, but alterations in the presence or abundance of a number of proteins in the lung have been observed. Our objective in this study was to further characterize these changes and to ask whether additional changes could be discerned using modern proteomic techniques. METHODS: The present study investigates global alterations in the proteome of bronchoalveolar lavage fluid taken from rats 1, 7, or 30 days after exposure to 5, 35, or 50 mg/kg of animal weight of DEPs. RESULTS: Analysis by surface-enhanced laser desorption/ionization-time of flight mass spectrometry identified two distinct peaks that appeared as an acute response postexposure at all doses in all animals. We identified these two peaks, with mass to charge ratios (m/z) of 9,100 and 10,100, as anaphylatoxin C3a and calgranulin A by additional mass spectral investigation using liquid chromatography coupled to mass spectrometry. CONCLUSIONS: With this approach, we found a number of inflammatory response proteins that may be associated with the early phases of inflammation in response to DEP exposure. Further studies are warranted to determine whether serum levels of these proteins could be markers of diesel exhaust exposure in workers.

Personalizing Environmental Health: At the Intersection of Precision Medicine and Occupational Health in the Military.

: Recent efforts in precision medicine present unique opportunities for military environmental and occupational health. Risk assessments can be refined by individualized risk factors such as genomics, and health status can be monitored and informed using mobile health (mHealth) devices. The military currently monitors exposures with service-wide databases and has one of the world's largest biobanks of serum samples available for health surveillance. New approaches are being developed for risk assessment, novel exposure-based biomarkers, and mobile applications to combine the facile collection of exposure data with tracking and planning utility. Planning by military leaders and coordination with national efforts puts the Department of Defense (DoD) in a unique position to benefit both Service Members and the nation, as reviewed in a symposium cosponsored by the DoD and the Johns Hopkins University-Applied Physics Laboratory (October 27 to 28, 2015).

Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq).

A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.

Repeated exposure to neurotoxic levels of chlorpyrifos alters hippocampal expression of neurotrophins and neuropeptides.

Chlorpyrifos (CPF), an organophosphorus pesticide (OP), is one of the most widely used pesticides in the world. Subchronic exposures to CPF that do not cause cholinergic crisis are associated with problems in cognitive function (i.e., learning and memory deficits), but the biological mechanism(s) underlying this association remain speculative. To identify potential mechanisms of subchronic CPF neurotoxicity, adult male Long Evans (LE) rats were administered CPF at 3 or 10mg/kg/d (s.c.) for 21 days. We quantified mRNA and non-coding RNA (ncRNA) expression profiles by RNA-seq, microarray analysis and small ncRNA sequencing technology in the CA1 region of the hippocampus. Hippocampal slice immunohistochemistry was used to determine CPF-induced changes in protein expression and localization patterns. Neither dose of CPF caused overt clinical signs of cholinergic toxicity, although after 21 days of exposure, cholinesterase activity was decreased to 58% or 13% of control levels in the hippocampus of rats in the 3 or 10mg/kg/d groups, respectively. Differential gene expression in the CA1 region of the hippocampus was observed only in the 10mg/kg/d dose group relative to controls. Of the 1382 differentially expressed genes identified by RNA-seq and microarray analysis, 67 were common to both approaches. Differential expression of six of these genes (Bdnf, Cort, Crhbp, Nptx2, Npy and Pnoc) was verified in an independent CPF exposure study; immunohistochemistry demonstrated that CRHBP and NPY were elevated in the CA1 region of the hippocampus at 10mg/kg/d CPF. Gene ontology enrichment analysis suggested association of these genes with receptor-mediated cell survival signaling pathways. miR132/212 was also elevated in the CA1 hippocampal region, which may play a role in the disruption of neurotrophin-mediated cognitive processes after CPF administration. These findings identify potential mediators of CPF-induced neurobehavioral deficits following subchronic exposure to CPF at a level that inhibits hippocampal cholinesterase to less than 20% of control. An equally significant finding is that subchronic exposure to CPF at a level that produces more moderate inhibition of hippocampal cholinesterase (approximately 50% of control) does not produce a discernable change in gene expression.