Prebiotic Oligosaccharides Potentiate Host Protective Responses against L. Monocytogenes Infection.

Prebiotic oligosaccharides are used to modulate enteric pathogens and reduce pathogen shedding. The interactions with prebiotics that alter Listeria monocytogenes infection are not yet clearly delineated. L. monocytogenes cellular invasion requires a concerted manipulation of host epithelial cell membrane receptors to initiate internalization and infection often via receptor glycosylation. Bacterial interactions with host glycans are intimately involved in modulating cellular responses through signaling cascades at the membrane and in intracellular compartments. Characterizing the mechanisms underpinning these modulations is essential for predictive use of dietary prebiotics to diminish pathogen association. We demonstrated that human milk oligosaccharide (HMO) pretreatment of colonic epithelial cells (Caco-2) led to a 50% decrease in Listeria association, while Biomos pretreatment increased host association by 150%. L. monocytogenes-induced gene expression changes due to oligosaccharide pretreatment revealed global alterations in host signaling pathways that resulted in differential subcellular localization of L. monocytogenes during early infection. Ultimately, HMO pretreatment led to bacterial clearance in Caco-2 cells via induction of the unfolded protein response and eIF2 signaling, while Biomos pretreatment resulted in the induction of host autophagy and L. monocytogenes vacuolar escape earlier in the infection progression. This study demonstrates the capacity of prebiotic oligosaccharides to minimize infection through induction of host-intrinsic protective responses

Inhibition of eIF2α dephosphorylation inhibits ErbB2-induced deregulation of mammary acinar morphogenesis

<p>Abstract</p> <p>Background</p> <p>The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in ~30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2α inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2α can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2α phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.</p> <p>Results</p> <p>We tested whether ErbB2 signaling influenced eIF2α signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt) Neu/ErbB2 or a constitutively active (CA) variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2α signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2α or induction of its downstream target ATF4. However, inhibition of eIF2α dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells.</p> <p>Conclusion</p> <p>Depending on the strength of ErbB2 signaling there is a differential regulation of CHOP and eIF2α phosphorylation. ErbB2 uncouples in basal conditions eIF2α phosphorylation from CHOP induction. However, this signal was restored by salubrinal treatment in Wt-ErbB2 expressing MCF10A cells as these DCIS-like structures underwent luminal clearing. In CA-ErbB2 structures apoptosis is not induced by salubrinal and instead a state of quiescence with reduced proliferation was achieved. Treatments that stabilize P-eIF2α levels may be effective in treating ErbB2 positive cancers without severely disrupting normal tissue function and structure.</p

Quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to Salmonella Typhimurium

The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been established for the first time in pigs. Taken together, our results provide a better understanding of the porcine response to Salmonella infection and the molecular mechanisms underlying Salmonella-host interactions.This research was supported by the National R&D Program of the Spanish Ministry of Science and Innovation (AGL2011-28904 and AGL2014-54089-R).Peer reviewedPeer Reviewe

HIV Exploits Antiviral Host Innate GCN2-ATF4 Signaling for Establishing Viral Replication Early in Infection.

Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV) infection and viral evasion by utilizing human CD4+ T cell cultures in vitro and a simian immunodeficiency virus (SIV) model of AIDS in vivo Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4+ T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR) to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4+ T cells isolated from patients receiving suppressive antiretroviral therapy (ART). In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy.IMPORTANCE Understanding how HIV overcomes host antiviral innate defense response in order to establish infection and dissemination is critical for developing prevention and treatment strategies. Most investigations focused on the viral pathogenic mechanisms leading to immune dysfunction following robust viral infection and dissemination. Less is known about mechanisms that enable HIV to establish its presence despite rapid onset of host antiviral innate response. Our novel findings provide insights into the viral strategy that hijacks the host innate response of the suppression of protein biosynthesis to restrict the virus production. The virus leverages transcription factor ATF4 expression during the GCN2-ATF4 signaling response and utilizes it to activate viral transcription through the LTR to support viral transcription and production in both HIV and SIV infections. This unique viral strategy is exploiting the innate response and is distinct from the mechanisms of immune dysfunction after the critical mass of viral loads is generated

Transcriptome Analysis in Spleen Reveals Differential Regulation of Response to Newcastle Disease Virus in Two Chicken Lines.

Enhancing genetic resistance of chickens to Newcastle Disease Virus (NDV) provides a promising way to improve poultry health, and to alleviate poverty and food insecurity in developing countries. In this study, two inbred chicken lines with different responses to NDV, Fayoumi and Leghorn, were challenged with LaSota NDV strain at 21 days of age. Through transcriptome analysis, gene expression in spleen at 2 and 6 days post-inoculation was compared between NDV-infected and control groups, as well as between chicken lines. At a false discovery rate &lt;0.05, Fayoumi chickens, which are relatively more resistant to NDV, showed fewer differentially expressed genes (DEGs) than Leghorn chickens. Several interferon-stimulated genes were identified as important DEGs regulating immune response to NDV in chicken. Pathways predicted by IPA analysis, such as "EIF-signaling", "actin cytoskeleton organization nitric oxide production" and "coagulation system" may contribute to resistance to NDV in Fayoumi chickens. The identified DEGs and predicted pathways may contribute to differential responses to NDV between the two chicken lines and provide potential targets for breeding chickens that are more resistant to NDV

Altered Gene Response to Aflatoxin B\u3csub\u3e1\u3c/sub\u3e in the Spleens of Susceptible and Resistant Turkeys

Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1exposure

Connecting gut microbiome changes with fish health conditions in juvenile Atlantic cod (Gadus morhua) exposed to dispersed crude oil

Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.publishedVersio

Network Analysis of Skeletal Muscle During Spaceflight in Male Mice

Context: The unloading associated with spaceflight results in the rapid loss of bone and muscle tissue thereby affecting functionality. These are two of the most concerning physiologic changes that occur in space and could limit long-term occupation in space. Thus, a better understanding of the mechanisms of changes to bone and muscle could lead to development of improved therapies to counteract both spaceflight and terrestrial-based bone and muscle dysfunction.Methods: Here we used a non-biased, stringent, deep sequencing (96 million paired end reads targeting 100 bp read length) assay to examine genomic networks altered by spaceflight in the quadriceps (n=4/group). Specifically, 9 week old C57BL/6 male mice were housed on the International Space Station or at Kennedy Space Center for approximately four weeks (n=10/group). Results: 14,228 genes (70% of whole mouse genome) met the cut-off criteria and the data sets were mapped to an average of ~76% of the whole mouse genome. Of these, 840 genes met the t-test criteria, p\u3c0.05. Canonical networks linked to EIF2 signaling, calcium ion signaling, and oxidative stress response were significantly enriched by the differentially expressed genes. A comprehensive energy deprivation was indicated as functions related to protein synthesis and degradation, lipid synthesis and oxidation, and ATP hydrolysis were inhibited, and mitochondrial dysfunction was activated.Conclusions: This is the first time that skeletal muscle changes have been studied in male mice during spaceflight, and these data add important new findings to changes that occur to the musculoskeletal system in male mice during spaceflight. In orthopaedic trauma, many patients spend prolonged periods non-weight bearing and can experience significant muscle atrophy as a result. The networks analyzed in this work may prove to be targets for future therapies to counter this atrophy