A Single Amino Acid Substitution Changes Antigenicity of ORF2-Encoded Proteins of Hepatitis E Virus

Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452–617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype 2), US (genotype 3) and China (genotype 4), indicating the existence of pan-genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation-dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins

Comprehensive molecular and morphological resolution of blubber stratification in a deep-diving, fasting-adapted seal

Blubber is a modified subcutaneous adipose tissue in marine mammals that provides energy storage, thermoregulation, hydrodynamic locomotion, and buoyancy. Blubber displays vertical stratification by lipid content, fatty acid composition, and vascularization, leading to the assumption that deeper blubber layers are metabolically active, while superficial layers are mainly structural and thermoregulatory. However, few studies have examined functional stratification of marine mammal blubber directly, especially in pinnipeds. We characterized morphological and transcriptional differences across blubber layers in the northern elephant seal, a deep-diving and fasting-adapted phocid. We collected blubber from seals early in their fasting period and divided blubber cores into three similarly sized portions. We hypothesized that the innermost blubber portion would have higher 1) heterogeneity in adipocyte size, 2) microvascular density, and 3) expression of genes associated with metabolism and hormone signaling than outer blubber. We found that adipocyte area and variance increased from outermost (skin-adjacent) to innermost (muscle-adjacent) blubber layers, suggesting that inner blubber has a higher capacity for lipid storage and turnover than outer blubber. Inner blubber had a higher proportion of CD144+ endothelial cells, suggesting higher microvascular density. In contrast, outer blubber had a higher proportion of CD4+ immune cells than inner blubber, suggesting higher capacity for response to tissue injury. Transcriptome analysis identified 61 genes that were differentially expressed between inner and outer blubber layers, many of which have not been studied previously in marine mammals. Based on known functions of these genes in other mammals, we suggest that inner blubber has potentially higher 1) adipogenic capacity, 2) cellular diversity, and 3) metabolic and neuroendocrine signaling activity, while outer blubber may have higher 1) extracellular matrix synthesis activity and 2) responsiveness to pathogens and cell stressors. We further characterized expression of nine genes of interest identified by transcriptomics and two adipokines with higher precision across blubber layers using targeted assays. Our study provides functional insights into stratification of blubber in marine mammals and a molecular key, including CD144, CD4, HMGCS2, GABRG2, HCAR2, and COL1A2, for distinguishing blubber layers for physiological and functional studies in seals

Blubber transcriptome response to acute stress axis activation involves transient changes in adipogenesis and lipolysis in a fasting-adapted marine mammal

Stress can compromise an animal\u27s ability to conserve metabolic stores and participate in energy-demanding activities that are critical for fitness. Understanding how wild animals, especially those already experiencing physiological extremes (e.g. fasting), regulate stress responses is critical for evaluating the impacts of anthropogenic disturbance on physiology and fitness, key challenges for conservation. However, studies of stress in wildlife are often limited to baseline endocrine measurements and few have investigated stress effects in fasting-adapted species. We examined downstream molecular consequences of hypothalamic-pituitary-adrenal (HPA) axis activation by exogenous adrenocorticotropic hormone (ACTH) in blubber of northern elephant seals due to the ease of blubber sampling and its key role in metabolic regulation in marine mammals. We report the first phocid blubber transcriptome produced by RNAseq, containing over 140,000 annotated transcripts, including metabolic and adipocytokine genes of interest. The acute response of blubber to stress axis activation, measured 2 hours after ACTH administration, involved highly specific, transient (lastinghours) induction of gene networks that promote lipolysis and adipogenesis in mammalian adipocytes. Differentially expressed genes included key adipogenesis factors which can be used as blubber-specific markers of acute stress in marine mammals of concern for which sampling of other tissues is not possible

An Ionic Liquid Facilitates the Proliferation of Antibiotic Resistance Genes Mediated by Class I Integrons

Ionic liquids (ILs), as "environmentally friendly" replacements for industrial volatile organic solvents, have been widely and recently applied in the chemical industry. However, few data have been collected regarding the toxicity and potential environmental effects of ILs, which are fairly important for preparing for their potential release into the environment. In this study, the IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]) was tested for its ability to promote the proliferation and dissemination of antibiotic resistance genes (ARGs) in environmental bacteria. In freshwater microcosms, [BMIm][PF6] (0.5 g/L) significantly enhanced the abundance of the sulI gene (500-fold greater than in untreated controls). Meanwhile, [BMIm][PF6] significantly increased the abundance of class I integrons, which play a key role in ARG dissemination. A positive correlation (p < 0.01) between the intI and sulI genes suggested that [BMIm][PF6]-facilitated sulI propagation was mediated by class I integrons. This idea was supported by sequencing, which showed the sulI locus in the 3′ region of class I integrons. Class I integron transfer experiments between different indigenous strains of Alcaligenes sp. (SMX R ) and Acinetobacter sp. (Str R ) were conducted to show that the horizontal transfer frequency of class I integrons was up to 88-fold higher in the presence of an IL. An IL increased cell membrane permeability as evidenced by flow cytometry, thereby assisting sulI gene transfer mediated by class I integrons. This is the first report that ILs facilitate the proliferation of ARGs in environmental bacteria and thus increase risks to public health. © 2014 American Chemical Society