Liver Transcriptome Response to Hyperthermia Stress in Three Distinct Chicken Lines
ABSTRACT: We investigated the effects of heat stress on the liver transcriptome of 3wk-old chicks of a broiler line, the Fayoumi and an advanced intercross line (AIL). Transcriptome sequencing of 48 male chickens using Illumina HiSeq 2500 technology yielded an average of 3.4 million, 100-base -pair single-end, reads per sample.
Project description:Background In broilers, heat stress can result in reduced feed consumption, digestive inefficiency, impaired metabolism, and even death. The broiler sector of the U.S. poultry industry incurs approximately $52 million in heat stress-related losses annually. The objective of this study is to characterize the effects of chronic, cyclic heat stress on the transcriptome of a metabolically active organ, the liver. Characterizing the liver transcriptome of heat-stressed broilers will help clarify the effects of heat stress on metabolism. This information will provide a platform for future investigations that further elucidate physiologic responses to heat stress and seek methods to ameliorate the negative impacts of heat. Results Transcriptome sequencing of the livers of 8 broiler males using Illumina HiSeq 2000 technology, resulted in a total of 138 million, 100 base pair single end reads, yielding 13.8 gigabases of sequence. Forty genes were differentially expressed at a significance level of P-value < 0.05 and a fold change ≥ 2 in response to chronic, cyclic heat stress (mid-point of the last day of a 7-day cyclic heat stress of 7 hours per day), with 27 down-regulated and 13 up-regulated. Two gene networks were created from the function-based Ingenuity Pathway Analysis (IPA) of the differentially expressed genes; “Cell Signaling, Molecular Transport, Small Molecule Biochemistry” and “Endocrine System Development and Function, Small Molecule Biochemistry Cell Signaling”. Members of the MAPK signaling pathway and differentially expressed genes that are associated with MAPK-related functions were prominent in the networks. Cellular proliferation and differentiation, inflammationand stress-related signaling, and apoptosis-associated genes were down-regulated in response to heat stress. Genes responsible for inhibiting feed intake and sphingolipidrelated signaling were up-regulated. Genes involved with the regulation of inflammation, stress, thyroid hormone level, and body temperature were both up- and down-regulated. Conclusions Chronic, cyclic heat stress of broilers results in metabolic changes that can be characterized through RNA-seq analysis of the liver transcriptome. The primary affected pathways included cell signaling, molecular transport, endocrine system development and signaling, and small molecule biochemistry. Examination of 2 heat treatments. Four heat stressed liver samples and 4 control liver samples analyzed.
Project description:The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ~50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30-50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.
Project description:Anti-androgen therapies including the new androgen receptor (AR) antagonist MDV3100 are the first therapeutic approach in treating prostate cancer (PCa). However, tumors frequently become castration resistant through multiple mechanisms including alternative expression of AR splice variants. To identify new inhibitors which block both full-length AR (AR-FL) and constitutively-active truncated AR splice variants (AR-Vs), a library of natural compounds was screened for molecules that could inhibit AR-driven transcription in PCa cells expressing AR-FL or AR-Vs. We identified the small-molecule Ailanthone (AIL) as a potent inhibitor of both AR-FL and AR-Vs. AIL down-regulates both AR itself and its target genes in PCa cell lines as well as orthotopic animal tumors. Moreover，AIL directly binds to the co-chaperone protein p23 and prevents AR’s interaction with HSP90, which results in the disruption of the AR-chaperone complex followed by Ubiquitin/proteasome-mediated degradation of AR as well as other p23 clients including AKT and Cdk4. Meanwhile, overexpression of p23 dose-dependently rescued AIL-mediated cell proliferation inhibition, suggesting that p23 might be a critical target of AIL. Furthermore, treatment of MDV3100-resistant 22RV1 xenografts with AIL reduced tumor volume by 77.5% (2 mg/kg/day AIL, intraperitoneal) and 79.2% (5 mg/kg/day AIL, oral) compared with the control group. Finally, AIL possesses favorable drug-like properties such as good bioavailability (25.7% F), high solubility, lack of CYP inhibition, and low hepatotoxicity, although signs of gastrointestinal toxicity were observed at high doses. In conclusion, AIL overcomes MDV3100 resistance in castration resistant prostate cancer (CRPC) with excellent absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, thus suggesting that AIL is a potential candidate for the clinical treatment of CRPC. Overall design: Retinal mRNA profiles of LNCaP cells treated with or without AIL in the absence or presence of AR1-651 were generated by deep sequencing using Illumina HiSeq2000
Project description:Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b-binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co-sedimentation assays, we demonstrate that two surface-exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail-F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000-fold more serum resistance than a ?ail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site-directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early-stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.
Project description:We demonstrated in this work the use of affinity ionic liquids, AIL 1 and AIL 2, for chemoselective detection of amine and alcohol gases on a quartz crystal microbalance (QCM). These detections of gaseous amines and alcohols were achieved by nucleophilic aromatic substitution reactions with the electrophilic 1,3,5-triazine-based AIL 1 thin-coated on quartz chips. Starting with inexpensive reagents, bicyclic imidazolium ionic liquids AIL 1 and AIL 2 were readily synthesized in six and four synthetic steps with high isolated yields: 51% and 63%, respectively. The QCM platform developed in this work is readily applicable and highly sensitive to low molecular weight amine gases: for isobutylamine gas (a bacterial volatile) at 10 Hz decrease in resonance frequency (i.e., ?F = -10 Hz), the detectability using AIL 1 was 6.3 ppb. Our preliminary investigation on detection of the much less nucleophilic alcohol gas by AIL 1 was also promising. To our knowledge, no example to date of reports based on nucleophilic aromatic substitution reactions demonstrating sensitive gas detection in these triazine ionic liquids on a QCM has been reported.
Project description:The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.
Project description:The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. Examination of mRNA profiles in two-tail samples of WRR and XH strains.
Project description:Ail is an outer membrane protein and virulence factor of Yersinia pestis, an extremely pathogenic, category A biothreat agent, responsible for precipitating massive human plague pandemics throughout history. Due to its key role in bacterial adhesion to host cells and bacterial resistance to host defense, Ail is a key target for anti-plague therapy. However, little information is available about the molecular aspects of its function and interactions with the human host, and the structure of Ail is not known. Here we describe the recombinant expression, purification, refolding, and sample preparation of Ail for solution and solid-state NMR structural studies in lipid micelles and lipid bilayers. The initial NMR and CD spectra show that Ail adopts a well-defined transmembrane β-sheet conformation in lipids.
Project description:The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail's biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40-0.60 ppm for 13C, 0.11-0.15 ppm for 1H, and 0.46-0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.
Project description:Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.