Determining weathering-induced heterogeneous oxidation profiles of polyethylene, polypropylene and polystyrene using laser-induced breakdown spectroscopy.

Weathering-induced polymer degradation is typically heterogeneous which plays an integral part in fragmentation. Despite that, the current selection of techniques to investigate such heterogeneities, especially beneath the sample surface, is sparse. We introduce Laser-induced Breakdown Spectroscopy (LIBS) as an analytical tool and evaluate its performance for depth profiling. Three types of polymers were selected (polyethylene, polypropylene, and polystyrene) that were aged under controlled conditions. We demonstrate that LIBS can detect heterogeneous oxidation on the surface and inside the samples. The results reveal that different oxidation behaviors are linked to the sample's lattice structure and the subsequent formation of microcracks. This implies that LIBS is beneficial to give additional insights into the weathering and degradation behavior of environmentally relevant plastics.

ATGL-dependent white adipose tissue lipolysis controls hepatocyte PPARα activity.

In hepatocytes, peroxisome proliferator-activated receptor α (PPARα) orchestrates a genomic and metabolic response required for homeostasis during fasting. This includes the biosynthesis of ketone bodies and of fibroblast growth factor 21 (FGF21). Here we show that in the absence of adipose triglyceride lipase (ATGL) in adipocytes, ketone body and FGF21 production is impaired upon fasting. Liver gene expression analysis highlights a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. Adipose tissue lipolysis induced by activation of the ß3-adrenergic receptor also triggers such PPARα-dependent responses not only in the liver but also in brown adipose tissue (BAT). Intact PPARα activity in hepatocytes is required for the cross-talk between adipose tissues and the liver during fat mobilization.

The hepatocyte insulin receptor is required to program the liver clock and rhythmic gene expression.

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression.

Integrative study of diet-induced mouse models of NAFLD identifies PPARα as a sexually dimorphic drug target.

OBJECTIVE: We evaluated the influence of sex on the pathophysiology of non-alcoholic fatty liver disease (NAFLD). We investigated diet-induced phenotypic responses to define sex-specific regulation between healthy liver and NAFLD to identify influential pathways in different preclinical murine models and their relevance in humans. DESIGN: Different models of diet-induced NAFLD (high-fat diet, choline-deficient high-fat diet, Western diet or Western diet supplemented with fructose and glucose in drinking water) were compared with a control diet in male and female mice. We performed metabolic phenotyping, including plasma biochemistry and liver histology, untargeted large-scale approaches (liver metabolome, lipidome and transcriptome), gene expression profiling and network analysis to identify sex-specific pathways in the mouse liver. RESULTS: The different diets induced sex-specific responses that illustrated an increased susceptibility to NAFLD in male mice. The most severe lipid accumulation and inflammation/fibrosis occurred in males receiving the high-fat diet and Western diet, respectively. Sex-biased hepatic gene signatures were identified for these different dietary challenges. The peroxisome proliferator-activated receptor α (PPARα) co-expression network was identified as sexually dimorphic, and in vivo experiments in mice demonstrated that hepatocyte PPARα determines a sex-specific response to fasting and treatment with pemafibrate, a selective PPARα agonist. Liver molecular signatures in humans also provided evidence of sexually dimorphic gene expression profiles and the sex-specific co-expression network for PPARα. CONCLUSIONS: These findings underscore the sex specificity of NAFLD pathophysiology in preclinical studies and identify PPARα as a pivotal, sexually dimorphic, pharmacological target. TRIAL REGISTRATION NUMBER: NCT02390232.

Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis.

Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.

Early life stress induces type 2 diabetes-like features in ageing mice.

Early life stress is known to impair intestinal barrier through induction of intestinal hyperpermeability, low-grade inflammation and microbiota dysbiosis in young adult rodents. Interestingly, those features are also observed in metabolic disorders (obesity and type 2 diabetes) that appear with ageing. Based on the concept of Developmental Origins of Health and Diseases, our study aimed to investigate whether early life stress can trigger metabolic disorders in ageing mice. Maternal separation (MS) is a well-established model of early life stress in rodent. In this study, MS increased fasted blood glycemia, induced glucose intolerance and decreased insulin sensitivity in post-natal day 350 wild type C3H/HeN male mice fed a standard diet without affecting body weight. MS also triggered fecal dysbiosis favoring pathobionts and significantly decreased IL-17 and IL-22 secretion in response to anti-CD3/CD28 stimulation in small intestine lamina propria. Finally, IL-17 secretion in response to anti-CD3/CD28 stimulation was also diminished at systemic level (spleen). For the first time, we demonstrate that early life stress is a risk factor for metabolic disorders development in ageing wild type mice under normal diet.

Consequences of bisphenol a perinatal exposure on immune responses and gut barrier function in mice.

The potent immunomodulatory effect of the endocrine disruptor bisphenol A during development and consequences during life span are of increasing concern. Particular interests have been raised from animal studies regarding the risk of developing food intolerance and infection. We aimed to identify immune disorders in mice triggered by perinatal exposure to bisphenol A. Gravid mice were orally exposed to bisphenol (50 µg/kg body weight/day) from day 15 of pregnancy until weaning. Gut barrier function, local and systemic immunity were assessed in adult female offspring. Mice perinatally exposed to bisphenol showed a decrease in ileal lysozyme expression and a fall of fecal antimicrobial activity. In offspring mice exposed to bisphenol, an increase in colonic permeability was observed associated with an increase in interferon-γ level and a drop of colonic IgA+ cells and fecal IgA production. Interestingly, altered frequency of innate lymphoid cells type 3 occurred in the small intestine, with an increase in IgG response against commensal bacteria in sera. These effects were related to a defect in dendritic cell maturation in the lamina propria and spleen. Activated and regulatory T cells were decreased in the lamina propria. Furthermore, perinatal exposure to bisphenol promoted a sharp increase in interferon-γ and interleukin-17 production in the intestine and elicited a T helper 17 profile in the spleen. To conclude, perinatal exposure to bisphenol weakens protective and regulatory immune functions in the intestine and at systemic level in adult offspring. The increased susceptibility to inflammatory response is an interesting lead supporting bisphenol-mediated adverse consequences on food reactions and infections.

Paneth Cell Defects Induce Microbiota Dysbiosis in Mice and Promote Visceral Hypersensitivity.

BACKGROUND & AIMS: Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages. METHODS: We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction. RESULTS: Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared with mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E coli during maternal separation and visceral hypersensitivity. CONCLUSIONS: Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.

Functional genomics approach for the identification of human host factors supporting dengue viral propagation.

Dengue virus (DENV) is endemic throughout tropical regions of the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high-throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle.

Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication.

BACKGROUND: Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. RESULTS: We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. CONCLUSIONS: Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication.

G protein-coupled receptor kinase 2 promotes flaviviridae entry and replication.

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2 × 2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK(-/-) mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.

Factors affecting reproducibility between genome-scale siRNA-based screens.

RNA interference-based screening is a powerful new genomic technology that addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, the authors performed 2 identically designed small interfering RNA (siRNA)-based, whole-genome screens for host factors supporting yellow fever virus infection. These screens represent 2 separate experiments completed 5 months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole-genome screens. Application of these analysis methodologies within a single screening data set using a fixed threshold equivalent to a p-value < or = 0.001 resulted in hit lists ranging from 82 to 1140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and interscreen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least 2 independent siRNAs for each gene product in primary screens. To facilitate validation, the authors conclude by suggesting methods to reduce false discovery at the primary screening stage. In this study, they present the first comprehensive comparison of multiple analysis strategies and demonstrate the impact of the analysis methodology on the composition of the "hit list." Therefore, they propose that the entire data set derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies.

Polypyrimidine tract binding protein prevents activity of an intronic regulatory element that promotes usage of a composite 3'-terminal exon.

Alternative splicing of 3'-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3'-untranslated regions that regulate their fate and their expression. The Xenopus alpha-tropomyosin pre-mRNA possesses a composite internal/3'-terminal exon (exon 9A9') that is differentially processed depending on the embryonic tissue. Exon 9A9' is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3'-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9' as a 3'-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9' in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9' as a 3'-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3'-end processing of the alpha-tropomyosin pre-mRNA.

Xenopus as a model to study alternative splicing in vivo.

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue-specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole-animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large-scale identification of mRNAs containing alternatively spliced exons, the tissue-specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.

PTB regulates the processing of a 3'-terminal exon by repressing both splicing and polyadenylation.

The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.

Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA.

The Xenopus alpha(fast)-tropomyosin gene contains, at its 3' -end, a composite internal/3' -terminal exon (exon 9A9'), which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles, whereas it is skipped in nonmuscle cells. We have developed an in vivo model based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. We first show that the different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and constitute valuable tools to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in nonmuscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. We show using UV cross-linking and immunoprecipitation experiments that Xenopus PTB (XPTB) interacts with these PTB binding sites. Finally, we show that depletion of endogenous XPTB in Xenopus embryos using a morpholinobased translational inhibition strategy resulted in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' -terminal exon.