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Globally, zoonotic diseases have been on the rise in recent years. Predictive modelling approaches have been successfully used in the literature to identify the underlying causes of these zoonotic diseases. We examine the latest research in the field of predictive modeling that verifies the growth of zoonotic pathogens and assesses the factors associated with their spread. The results of our survey indicate that popular mathematical models can successfully be used in modeling the growth rate of these pathogens under varying storage temperatures. Additionally, some of them are used for the assessment of the inactivation of these pathogens based on various conditions. Based on the results of our study, machine learning models and deep learning are commonly used to detect pathogens within food items and to predict the factors associated with the presence of the pathogens.

BackgroundNetwork studies of host-pathogen interactions (HPI) are critical in understanding the mechanisms of pathogenesis. However, accessible HPI data for agriculturally important pathogens are limited. This lack of HPI data impedes network analysis to study agricultural pathogens, for preventing and reducing the severity of diseases of relevance to agriculture.ResultsTo rapidly provide HPIs for a broad range of pathogens, we use an interolog-based approach. This approach uses sequence similarity to transfer known HPIs from better studied host-pathogen pairs and predicts 389,878 HPIs for 23 host-pathogen systems of relevance to US agriculture. Each predicted HPI is qualitatively assessed using co-localization, infection related processes, and interacting domains and this information is provided as a confidence indicator for the prediction. Evaluation of predicted HPIs demonstrates that the host proteins predicted to be involved in pathogen interactions include hubs and bottlenecks in the network, as reported in curated host proteins. Moreover, we demonstrate that the use of the predicted HPIs adds value to network analysis and recapitulates known aspects of host-pathogen biology. Access to the predicted HPIs for these agricultural host-pathogen systems is available via the Host Pathogen Interaction Database (HPIDB, hpidb.igbb.msstate.edu), and can be downloaded in standard MITAB file format for subsequent network analysis.ConclusionsThis core set of interolog-based HPIs will enable animal health researchers to incorporate network analysis into their research and help identify host-pathogen interactions that may be tested and experimentally validated. Moreover, the development of a larger set of experimentally validated HPI will inform future predictions. Our approach of transferring biologically relevant HPIs based on interologs is broadly applicable to many host-microbe systems and can be extended to support network modeling of other pathogens, as well as interactions between non-pathogenic microbes.

Binge drinking is a remarkably prevalent behavior. In 2015, 27% of U.S. residents 18 years old or older reported at least one episode of binge drinking in the previous month. Rodent models for binge drinking are widely used to study the mechanisms by which alcohol causes a variety of adverse health effects in humans. Concerns have been raised that many binge-drinking studies in rodents involve alcohol doses that would be unrealistically high in humans. Allometric dosage scaling can be used to estimate the dose of a drug or chemical in mice that would be necessary to achieve similar biological effects at a realistic dose in humans. However, it has become apparent that no single allometric conversion factor is applicable for all drugs and chemicals, so it is necessary to evaluate each compound empirically. In the present study, we compared the area under the blood alcohol concentration vs. time curve (AUC) and the peak blood alcohol concentration following oral alcohol administration at various doses in mice and humans, using data from previously published studies. The results demonstrated that the oral dose of alcohol must be larger in mice (on a g of alcohol to kg of body weight basis) than in humans to achieve similar alcohol AUC values or to achieve similar peak concentrations in the blood. The dose required in mice was about 2-fold greater than the dose required in humans to achieve similar alcohol AUC and peak concentrations. The results shown here were substantially different from the average 5–12-fold difference between mice and humans calculated in previous studies using agents other than alcohol. Results shown here demonstrate that an empirical approach using data from several independent experiments provides information needed to determine the alcohol dose in mice that produces a similar level of exposure (AUC and peak concentration) as in humans. The results indicate that a single alcohol dose in the range of 5–6 g/kg, a range often used in mouse models for binge drinking, is not excessive when modeling human binge drinking. Results presented here illustrate that in mice both alcohol AUC and peak alcohol concentration correlate well with an important biological effect – activation of the hypothalamic-pituitary-adrenal axis – as indicated by increased corticosterone AUC values.

An organism’s transcriptome is the set of all transcripts within a cell at a certain time. We often analyze the transcriptome by quantifying gene expression and performing subsequent analyses such as a differential expression or a network analysis. Such analysis helps us in understanding and interpreting the functional elements of the genome. Many challenges limit the accuracy and ability to map all the RNA-Seq correctly into its genome sequence. Some of these challenges are exemplified when mapping sequences fall at exon junctions, sequences containing polymorphisms, multiple insertions or deletions, and reads falling partially or wholly within introns. One of the most significant problems is the loss of data occurring from the inability to map sequences when they align to multiple genomic locations, sometimes called ambiguous sequence mappings. In this paper, we present a novel method to increase the accuracy of gene expression estimation by relying on a statistical approach to increase the accuracy of mapping the ambiguous reads to their proper locations within the genome. This approach allows us to better identify significantly expressed genomic locations so we can accurately map ambiguous reads to their most likely accurate genomic locations and to define more precisely which genes are expressed throughout the genome. Due to its statical nature the approach can be easily combined with other existing mapping tools and mechanisms as well.

Infections due to Streptococcus pneumoniae, a commensal in the nasopharynx, still claim a significant number of lives worldwide. Genome plasticity, antibiotic resistance, and limited serotype coverage of the available polysaccharide-based conjugate vaccines confounds therapeutic interventions to limit the spread of this pathogen. Pathogenic mechanisms that allow successful adaption and persistence in the host could be potential innovative therapeutic targets. Polyamines are ubiquitous polycationic molecules that regulate many cellular processes. We previously reported that deletion of polyamine transport operon potABCD, which encodes a putrescine/spermidine transporter (ΔpotABCD), resulted in an unencapsulated attenuated phenotype. Here, we characterize the transcriptome, metabolome, and stress responses of polyamine transport-deficient S. pneumoniae. Compared with the wild-type strain, the expression of genes involved in oxidative stress responses and the nucleotide sugar metabolism was reduced, while expression of genes involved in the Leloir, tagatose, and pentose phosphate pathways was higher in ΔpotABCD. A metabolic shift towards the pentose phosphate pathway will limit the synthesis of precursors of capsule polysaccharides. Metabolomics results show reduced levels of glutathione and pyruvate in the mutant. Our results also show that the potABCD operon protects pneumococci against hydrogen peroxide and nitrosative stress. Our findings demonstrate the importance of polyamine transport in pneumococcal physiology that could impact in vivo fitness. Thus, polyamine transport in pneumococci represents a novel target for therapeutic interventions.

Background Transcriptomics has identified at-arrival differentially expressed genes associated with bovine respiratory disease (BRD) development; however, their use as prediction molecules necessitates further evaluation. Therefore, we aimed to selectively analyze and corroborate at-arrival mRNA expression from multiple independent populations of beef cattle. In a nested case-control study, we evaluated the expression of 56 mRNA molecules from at-arrival blood samples of 234 cattle across seven populations via NanoString nCounter gene expression profiling. Analysis of mRNA was performed with nSolver Advanced Analysis software (p n = 115 Healthy; n = 119 BRD); BRD was further stratified for severity based on frequency of treatment and/or mortality (Treated_1, n = 89; Treated_2+, n = 30). Gene expression homogeneity of variance, receiver operator characteristic (ROC) curve, and decision tree analyses were performed between severity cohorts. Results Increased expression of mRNAs involved in specialized pro-resolving mediator synthesis (ALOX15, HPGD), leukocyte differentiation (LOC100297044, GCSAML, KLF17), and antimicrobial peptide production (CATHL3, GZMB, LTF) were identified in Healthy cattle. BRD cattle possessed increased expression of CFB, and mRNA related to granulocytic processes (DSG1, LRG1, MCF2L) and type-I interferon activity (HERC6, IFI6, ISG15, MX1). Healthy and Treated_1 cattle were similar in terms of gene expression, while Treated_2+ cattle were the most distinct. ROC cutoffs were used to generate an at-arrival treatment decision tree, which classified 90% of Treated_2+ individuals. Conclusions Increased expression of complement factor B, pro-inflammatory, and type I interferon-associated mRNA hallmark the at-arrival expression patterns of cattle that develop severe clinical BRD. Here, we corroborate at-arrival mRNA markers identified in previous transcriptome studies and generate a prediction model to be evaluated in future studies. Further research is necessary to evaluate these expression patterns in a prospective manner.

Bovine respiratory disease (BRD) is a multifactorial disease involving complex host immune interactions shaped by pathogenic agents and environmental factors. Advancements in RNA sequencing and associated analytical methods are improving our understanding of host response related to BRD pathophysiology. Supervised machine learning (ML) approaches present one such method for analyzing new and previously published transcriptome data to identify novel genes and mechanisms. Our objective was to apply ML models to lung and immunological tissue datasets acquired from previous clinical BRD experiments to identify genes that classify disease with high accuracy. Raw mRNA sequencing reads from 151 bovine datasets (n=123 BRD, n=28 control) were downloaded from NCBI-GEO. Quality filtered reads were assembled in a HISAT2/Stringtie2 pipeline. Raw gene counts for ML analysis were normalized, transformed, and analyzed with MLSeq, utilizing six ML models. Cross-validation parameters (5-fold, repeated 10 times) were applied in a 70:30 training/testing ratio. Downstream analysis of genes identified by the top sparse classifiers for each etiological association was performed within WebGestalt and Reactome (FDR < 0.05). Support vector machines was routinely the top non-sparse classifier for predicting etiological disease versus sham control. Nearest shrunken centroid and Poisson linear discriminant analysis with power transformation could reliably classify IBR and BRSV with 100% accuracy. Genes identified in IBR and BRSV, but not BVDV, were related to type I interferon production and IL-8 secretion, specifically in lymphoid tissue and not lung. Genes identified in Mannheimia haemolytica infections were involved in activating classical and alternative pathways of complement. Novel findings, including expression of genes related to reduced mitochondrial oxygenation and ATP synthesis in consolidated lung tissue, were discovered. Genes identified in each analysis represent distinct genomic events relevant to understanding and predicting clinical BRD. The few genes shared across analyses may be reliably associated with clinical BRD. Our analysis demonstrates the utility of ML with published datasets for discovering functional information to support prediction and understanding BRD acquisition.

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Infections due to Streptococcus pneumoniae, a commensal in the nasopharynx, still claim a significant number of lives worldwide. Genetic plasticity, antibiotic resistance, limited serotype coverage of the available polysaccharide-based conjugate vaccines confounds therapeutic interventions. Pathogenic systems that allow successful adaption and persistence in the host could be potential innovative targets for mediations. Polyamines are ubiquitous polycationic molecules and regulate many cellular processes. We previously reported that deletion of potABCD, an operon that encodes a putrescine/spermidine transporter (∆potABCD), resulted in an un-encapsulated attenuated phenotype. Here we characterize the transcriptome, metabolome, and stress responses of S. pneumoniae that is dependent on the polyamine transporter. Expression of genes involved in oxidative stress responses and the central metabolism was reduced while that of genes involved in the Leloir, tagatose, and pentose phosphate pathways was increased in ΔpotABCD. Downregulation of genes of the central metabolism will reduce production of precursors of capsule polysaccharides. Metabolomics results show reduced glutathione and pyruvate levels in the mutant. We also show that the potABCD operon protects pneumococci against hydrogen peroxide and nitrosative stress. These results show the importance of the potABCD operon and polyamine transport in pneumococcal physiology and fitness that represents a novel target for therapeutic interventions.

Streptococcus pneumoniae colonizes the human nasopharyngeal mucosa and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen across diverse biological sites and contributes significantly to colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). Supplementation of zinc, cobalt, and nickel following TPEN treatment significantly abrogated induction. BLASTP comparisons and protein topology analysis predicted this locus to encode components of ATP binding cassette (ABC) transporters involved in multidrug resistance (SP1434-1435) and energy-coupling factor (ECF) transporters (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified differences in intracellular metal content in a Δ1434-8 mutant strain compared to parental T4R. Further, analysis of the secreted metabolome of WT and Δ1434-8 strains identified significant changes in pneumococcal glycolytic and amino acid metabolic pathways, indicating a shift towards mixed acid fermentation. Additionally, proteomic analysis revealed differentially expressed proteins in the Δ1434-8 mutant strain, with nearly 20% regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the transporters encoded by SP1433-1438 are involved in regulating the central metabolism of S. pneumoniae and contributing to bacterial survival during metal stress.

Background Despite decades of extensive research, bovine respiratory disease (BRD) remains the most devastating disease in beef cattle production. Establishing a clinical diagnosis often relies upon visual detection of non-specific signs, leading to low diagnostic accuracy. Thus, post-weaned beef cattle are often metaphylactically administered antimicrobials at facility arrival, which poses concerns regarding antimicrobial stewardship and resistance. Additionally, there is a lack of high-quality research that addresses the gene-by-environment interactions that underlie why some cattle that develop BRD die while others survive. Therefore, it is necessary to decipher the underlying host genomic factors associated with BRD mortality versus survival to help determine BRD risk and severity. Using transcriptomic analysis of at-arrival whole blood samples from cattle that died of BRD, as compared to those that developed signs of BRD but lived (n = 3 DEAD, n = 3 ALIVE), we identified differentially expressed genes (DEGs) and associated pathways in cattle that died of BRD. Additionally, we evaluated unmapped reads, which are often overlooked within transcriptomic experiments. Results 69 DEGs (FDR Conclusion This study further revealed increased proinflammatory immunological mechanisms in cattle that develop BRD. DEGs upregulated in DEAD cattle were predominantly involved in innate immune pathways typically associated with antiviral defense, although no viral genes were identified within unmapped reads. Our findings provide genomic targets for further analysis in cattle at highest risk of BRD, suggesting that mechanisms related to type I interferons and antiviral defense may be indicative of viral respiratory disease at arrival and contribute to eventual BRD mortality.

The existing protein annotation in chicken is mostly limited to computational predictions based on orthology to other proteins, which often leads to a significant underestimation of the function of these proteins. Genome-scale experimental annotation can provide insight into the actual enzymatic activities of chicken proteins. Amongst post-translational modifications, ubiquitination is of interest as anomalies in ubiquitination are implicated in such diseases as inflammatory disorders, infectious diseases, or malignancies. Ubiquitination is controlled by deubiquitinases (DUBs), which remove ubiquitin from protein substrates. However, the DUBs have not been systematically annotated and quantified in chicken tissues. Here we used a chemoproteomics approach, which is based on active-site probes specific to DUBs, and identified 26 active DUBs in the chicken spleen, cecum, and liver.

Alcohol abuse increases vulnerability to infections and infection-related mortality. In previous studies, we found that acute alcohol abuse in a binge-drinking model in mice decreased resistance to bacterial sepsis when alcohol was administered near the time of bacterial challenge. In the present study, we investigated the effects of alcohol administered later in the course of sepsis (18 hr after injection of Escherichia coli). Our working hypothesis was that decreased production of cytokines caused by alcohol at this time would actually improve survival, because overproduction of pro-inflammatory mediators is thought to be the proximate cause of mortality in sepsis. Unexpectedly, administration of alcohol late in the course of sepsis led to a rapid increase in the number of viable bacteria in the peritoneal cavity. Significant increases in the concentrations of several cytokines and chemokines coincided with the increased number of bacteria in alcohol-treated mice and decreased survival time. These results demonstrated our working hypothesis to be incorrect, and reiterated the complexity of sepsis. Hypothermia is a consistent feature in this model of sepsis, and in control mice (E. coli only), body temperature was near normal by 18 hr or 21 hr after administration of E. coli, but in mice treated with alcohol 18 hr after E. coli, hypothermia was significant 3 hr later and ultimately mortality was significantly increased. However, counteracting the hypothermic effect of alcohol by external warming of mice led to earlier mortality, demonstrating that hypothermia was not the major cause of mortality. These results along with previous results from studies in which alcohol was given before initiation of sepsis suggest that decreased cytokine and chemokine production may not be the key effect of alcohol that decreases resistance to sepsis. It seems more likely that suppression of mechanisms by which macrophages and neutrophils kill bacteria is critical, and this can occur even in the presence of high levels of cytokines and chemokines.

Phosphorylation is a post-translational protein modification regulating most known cellular processes. While protein kinases constitute a large family of highly conserved enzymes, identification of active kinases is challenging due to a low abundance of some of these signaling molecules. Although chicken is the first agricultural animal to have a sequenced genome, annotation of the kinome, i.e., a complement of all protein kinases in the genome is limited. We used chemical probes consisting of ATP and ADP derivatives binding to specific lysine (Lys) residues within the ATP-binding pocket of kinases, combined with proteomics, to identify 267 peptides labeled with the ATP and ADP acyl derivatives and 188 corresponding chicken kinases in chicken spleen and liver. Our description of active chicken kinases and ATP binding sites will support future studies focused on identifying the role of this important class of enzymes in chicken health and disease. Significance Advances made in understanding chicken enzymes are critical for the improved knowledge of the regulatory pathways controlling physiological processes in chicken. Since protein phosphorylation controls multiple aspects of cell fate, it is often linked to pathological conditions, and understanding of the kinase expression in chicken is essential for future therapeutic approaches. We coupled proteomics and labeling with active-site probes binding to Lys residues within the ATP-binding pocket of kinases to identify 188 kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. Results of the present study describing catalytically active kinases is a starting point for chemoproteomic-based interrogation of kinases in chicken exposed to different conditions. Kinases identified in this study are available through the Chickspress genome browser that has previously published mRNA, miRNA, and shotgun proteomics data.

This data article contains the proteomic and transcriptomic data of the amygdala of adolescent rats involved in social play compared to non-behavioural animals. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioural testing. The protein expression was measured by analysing trypsin digested protein samples using a LTQ Orbitrap Velos mass spectrometer equipped with an Advion nanomate ESI source. The obtained tandem mass spectra were extracted by Thermo Proteome Discoverer 1.3 and the data were displayed with Scaffold v 4.5.1. The transcriptomic data were generated by llumina HiSeq 4000 system. Cuffdiff (v2.2.1) program was used to calculate RNA-seq based gene expression levels. For further interpretation of data presented in this article, please see the research article ‘Proteomic and Transcriptional Profiling of Rat Amygdala Following Social Play’ (Alugubelly et al. 2019). Keywords: Social play, Proteome, Transcriptome, Behaviour, Amygdala

Lauren A Bright,1 Wellesley Dittmar,1 Bindu Nanduri,2 Fiona M McCarthy,3 Nisma Mujahid,2 Lais RR Costa,2 Shane C Burgess,3 Cyprianna E Swiderski11Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA; 2Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA; 3School of Animal Comparative and Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USABackground: Pasture-associated severe equine asthma is a warm season, environmentally-induced respiratory disease characterized by reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronic neutrophilic airway inflammation. During seasonal exacerbation, signs vary from mild to life-threatening episodes of wheezing, coughing, and chronic debilitating labored breathing.Purpose: In human asthma, neutrophilic airway inflammation is associated with more severe and steroid-refractory asthma phenotypes, highlighting a need to decipher the mechanistic basis of this disease characteristic. We hypothesize that the collective biological activities of proteins in bronchoalveolar lavage fluid (BALF) of horses with pasture-associated severe asthma predict changes in neutrophil functions that contribute to airway neutrophilic inflammation.Methods: Using shotgun proteomics, we identified 1,003 unique proteins in cell-free BALF from six horses experiencing asthma exacerbation and six control herdmates. Contributions of each protein to ten neutrophil functions were modeled using manual biocuration to determine each protein’s net effect on the respective neutrophil functions.Results: A total of 417 proteins were unique to asthmatic horses, 472 proteins were unique to control horses (p

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics.

The purpose of this study was to seek direct physical evidence that ethanol at concentrations relevant to binge drinking alters the conformation of TLR3 and decreases ligand binding. We and others have reported that TLR3-mediated responses are significantly suppressed by concentrations of ethanol that occur in binge drinkers (J. Immunol., 2004, 173: 2715–2724). This suppression can be detected at all levels of signaling that have been tested (from MAP kinases to NF-kB), but it remains possible that ethanol acts directly on TLR3 and that its other effects are secondary to altered TLR3 conformation. Purified ectodomains of TLR3 were used in this study, because they comprise most of the TLR3 molecule, and dimerization of TLR3 ectodomains is necessary for signal transduction and activation of TLR3-mediated innate immunity. Using circular dichroism (CD) to measure changes in protein conformation, we demonstrated that ethanol concentrations from 20–100 mM substantially altered the conformation of human and mouse TLR3 ectodomains complexed with a defined dsRNA ligand. Binding of poly I:C, a dsRNA analog, was significantly inhibited by ethanol. Modeling with Autodock 4 software indicated that mouse and human TLR3 contain a putative ethanol binding site with a dissociation constant consistent with the concentration dependence of the conformational changes. These results support the hypothesis that ethanol inhibits TLR3-mediated functions by directly altering the conformation of TLR3. This work was supported by NIH R01AA09505 and SBP was supported by NIH grant P20GM103646.

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment's function. This project uses transcriptomic analysis to evaluate gene expression across early B-cell development. RNA-seq was performed with total RNA isolated from bursal B-cells at embryonic day (ED) 16 and ED 19 (n = 3). Approximately 90 million high-quality clean reads were obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in the Jak-STAT pathway, Wnt signaling pathway, MAPK signaling pathway, metabolic pathways including tyrosine metabolism, Toll-like receptor signaling pathway, and cell-adhesion molecules. The genes predicted to encode surface receptors, signal transduction proteins, and transcription factors identified in this study represent gene candidates for controlling B-cell development in response to differentiation factors in the bursal microenvironment.

Streptococcus pneumoniaecolonizes the mucosa of the human nasopharynx and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen and contributes significantly to both colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation byN,N,N’,N’-tetrakis-(2-Pyridylmethyl)ethylenediamine (TPEN). Supplementation of either zinc or cobalt following TPEN treatment drastically abrogated induction. BLAST analysis predicted this operon to encode two ABC-transporters, sharing homology to a multidrug resistance system (SP1434-1435) and an energy-coupling factor (ECF) transport system (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated changes in intracellular concentrations of iron, zinc, and manganese ions in a Δ1434-8 strain compared to parental T4R. Analysis of the secreted metabolomic profile of the T4R and Δ1434-8 strains identified significant changes in pneumococcal glycolytic pathways, indicating a shift towards increased production of acetate. Additionally, proteomic analysis revealed 41 differentially expressed proteins in the Δ1434-8 strain, with roughly 20% of them regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that theSP1433-1438operon is largely involved in the central metabolism ofS. pneumoniaeduring zinc-limitation.ImportanceMetal sequestration is a common strategy utilized by the host immune response as well as antibiotics such as vancomycin to kill invading bacterial pathogens (1). However, pneumococcus is still able to thrive under zinc-limiting conditions. This study describes a previously uncharacterized operon encoding two ABC transport systems that are strongly induced during zinc-limiting conditions. This operon was found to be regulated by a zinc-dependent regulator (SP1433) that functions independently of the overarching AdcR regulon. We have additionally utilized a 2D-NMR approach to analyze the secreted metabolome and have employed proteomic analysis to identify a role for these systems in the maintenance of cellular metabolism. This study provides new information on howStreptococcus pneumoniaeresponds and adapts to zinc-limiting conditions.

An increasing proportion of curated host-pathogen interaction (HPI) information is becoming available in interaction databases. These data represent detailed, experimentally-verified, molecular interaction data, which may be used to better understand infectious diseases. By their very nature, HPIs are context dependent, where the outcome of two proteins as interacting or not depends on the precise biological conditions studied and approaches used for identifying these interactions. The associated biology and the technical details of the experiments identifying interacting protein molecules are increasing being curated using defined curation standards but are overlooked in current HPI network modeling. Given the increase in data size and complexity, awareness of the process and variables included in HPI identification and curation, and their effect on data analysis and interpretation is crucial in understanding pathogenesis. We describe the use of HPI data for network modeling, aspects of curation that can help researchers to more accurately model specific infection conditions, and provide examples to illustrate these principles. © 2018 by John Wiley & Sons, Inc.

Bovine respiratory disease (BRD) is the most common economically important disease affecting cattle. For developing accurate diagnostics that can predict disease susceptibility/resistance and stratification, it is necessary to identify the molecular mechanisms that underlie BRD. To study the complex interactions among the bovine host and the multitude of viral and bacterial pathogens, as well as the environmental factors associated with BRD etiology, genome-scale high-throughput functional genomics methods such as microarrays, RNA-seq, and proteomics are helpful. In this review, we summarize the progress made in our understanding of BRD using functional genomics approaches. We also discuss some of the available bioinformatics resources for analyzing high-throughput data, in the context of biological pathways and molecular interactions. Although resources for studying host response to infection are available, the corresponding information is lacking for majority of BRD pathogens, impeding progress in identifying diagnostic signatures for BRD using functional genomics approaches.

Listeria monocytogenes is a Gram-positive, foodborne pathogen responsible for approximately 28% of all food-related deaths each year in the United States. L. monocytogenes infections are linked to the consumption of minimally processed ready-to-eat (RTE) products such as cheese, deli meats, and cold-smoked finfish products. L. monocytogenes is resistant to stresses commonly encountered in the food-processing environment, including low pH, high salinity, oxygen content, and various temperatures. The purpose of this study was to determine if cells habituated at low temperatures would result in cross-protective effects against osmotic stress. We found that cells exposed to refrigerated temperatures prior to a mild salt stress treatment had increased survival in NaCl concentrations of 3%. Additionally, the longer the cells were pre-exposed to cold temperatures, the greater the increase in survival in 3% NaCl. A proteomics analysis was performed in triplicate in order to elucidate mechanisms involved in cold-stress induced cross protection against osmotic stress. Proteins involved in maintenance of the cell wall and cellular processes, such as penicillin binding proteins and osmolyte transporters, and processes involving amino acid metabolism, such as osmolyte synthesis, transport, and lipid biosynthesis, had the greatest increase in expression when cells were exposed to cold temperatures prior to salt. By gaining a better understanding of how this pathogen adapts physiologically to various environmental conditions, improvements can be made in detection and mitigation strategies.

Social play is the most characteristic form of social interaction which is necessary for adolescents to develop proper cognitive, emotional, and social competency. The information available on neural substrates and the mechanism involved in social play is limited. This study characterized social play by proteomic and transcriptional profiling studies. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioral testing. The proteomic analysis led to the identification of 170 differentially expressed proteins (p ≤ 0.05) with 67 upregulated and 103 downregulated proteins. The transcriptomic analysis led to the identification of 188 genes (FDR ≤ 0.05) with 55 upregulated and 133 downregulated genes. DAVID analysis of gene/protein expression data revealed that social play altered GABAergic signaling, glutamatergic signaling, and G-protein coupled receptor (GPCR) signaling. These data suggest that the synaptic levels of GABA and glutamate increased during play. Ingenuity Pathway Analysis (IPA) confirmed these alterations. IPA also revealed that differentially expressed genes/proteins in our data had significant over representation of neurotransmitter signaling systems, including the opioid, serotonin, and dopamine systems, suggesting that play alters the systems involved in the regulation of reward. In addition, corticotropin-releasing hormone signaling was altered indicating that an increased level of stress occurs during play. Overall, our data suggest that increased inhibitory GPCR signaling in these neurotransmitter pathways occurs following social play as a physiological response to regulate the induced level of reward and stress and to maintain the excitatory-inhibitory balance in the neurotransmitter systems.

Streptococcus pneumoniae is the most common bacterial etiology of pneumococcal pneumonia in adults worldwide. Genomic plasticity, antibiotic resistance and extreme capsular antigenic variation complicates the design of effective therapeutic strategies. Polyamines are ubiquitous small cationic molecules necessary for full expression of pneumococcal virulence. Polyamine transport system is an attractive therapeutic target as it is highly conserved across pneumococcal serotypes. In this study, we compared an isogenic deletion strain of S. pneumoniae TIGR4 in polyamine transport operon (ΔpotABCD) with the wild type in a mouse model of pneumococcal pneumonia. Our results show that the wild type persists in mouse lung 24 h post infection while the mutant strain is cleared by host defense mechanisms. We show that intact potABCD is required for survival in the host by providing resistance to neutrophil killing. Comparative proteomics analysis of murine lungs infected with wild type and ΔpotABCD pneumococci identified expression of proteins that could confer protection to wild type strain and help establish infection. We identified ERM complex, PGLYRP1, PTPRC/CD45 and POSTN as new players in the pathogenesis of pneumococcal pneumonia. Additionally, we found that deficiency of polyamine transport leads to up regulation of the polyamine synthesis genes speE and cad in vitro.

Listeria monocytogenes is a Gram-positive facultative anaerobe that is the causative agent of the disease listeriosis. The infectious ability of this bacterium is dependent upon resistance to stressors encountered within the gastrointestinal tract, including bile. Previous studies have indicated bile salt hydrolase activity increases under anaerobic conditions, suggesting anaerobic conditions influence stress responses. Therefore, the goal of this study was to determine if reduced oxygen availability increased bile resistance of L. monocytogenes. Four strains representing three serovars were evaluated for changes in viability and proteome expression following exposure to bile in aerobic or anaerobic conditions. Viability for F2365 (serovar 4b), EGD-e (serovar 1/2a), and 10403S (serovar 1/2a) increased following exposure to 10% porcine bile under anaerobic conditions (P < 0.05). However, HCC23 (serovar 4a) exhibited no difference (P > 0.05) in bile resistance between aerobic and anaerobic conditions, indicating that oxygen availability does not influence resistance in this strain. The proteomic analysis indicated F2365 and EGD-e had an increased expression of proteins associated with cell envelope and membrane bioenergetics under anaerobic conditions, including thioredoxin-disulfide reductase and cell division proteins. Interestingly, HCC23 had an increase in several dehydrogenases following exposure to bile under aerobic conditions, suggesting that the NADH:NAD+ is altered and may impact bile resistance. Variations were observed in the expression of the cell shape proteins between strains, which corresponded to morphological differences observed by scanning electron microscopy. These data indicate that oxygen availability influences bile resistance. Further research is needed to decipher how these changes in metabolism impact pathogenicity in vivo and also the impact that this has on susceptibility of a host to listeriosis.

The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis) fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

Sepsis is a major cause of death worldwide. The associated risks and mortality are known to significantly increase on exposure to alcohol (chronic or acute). The underlying mechanisms of the association of acute ethanol ingestion and poor prognosis of sepsis are largely unknown. The study described here was designed to determine in detail the role of ethanol and TLR4 in the pathogenesis of the sepsis syndrome. The effects of acute ethanol exposure and TLR4 on bacterial clearance, spleen cell numbers, peritoneal macrophage numbers, and cytokine production were evaluated using wild type and TLR4 hypo-responsive mice treated with ethanol and then challenged with a non pathogenic strain of Escherichia. coli (E. coli). Ethanol treated mice exhibited a decreased clearance of bacteria and produced lesser amounts of most pro-inflammatory cytokines in both strains of mice at two hours after challenge. Neither ethanol treatment nor a hypo-responsive TLR4 had significant effects on the cell numbers in the peritoneal cavity and spleen 2 hours post infection. The suppressive effect of acute ethanol exposure on cytokine and chemokine production was more pronounced in the wild type mice, but the untreated hyporesponsive mice produced less of most cytokines than untreated wild type mice. The major conclusion of this study is that acute ethanol exposure suppresses pro-inflammatory cytokine production and that a hypo-responsive TLR4 (in C3H/HeJ mice) decreases pro-inflammatory cytokine levels but the cytokines and other mediators induced through other receptors are sufficient to ultimately clear the infection but not enough to induce lethal septic shock. In addition, results reported here demonstrate previously unknown effects of acute ethanol exposure on LIF (leukemia inhibitory factor) and eotaxin and provide the first evidence that IL-9 is induced through TLR4 in vivo.

Alcohol is the most frequently abused substance in the world. Both acute and chronic alcohol consumption have diverse and well-documented effects on the human immune system, leading to increased susceptibility to infections like bacterial pneumonia. Streptococcus pneumoniae is the most common bacterial etiology of community-acquired pneumonia worldwide. The frequency and severity of pneumococcal infections in individuals with a history of alcohol abuse is much higher than the general population. Despite this obvious epidemiological relevance, very few experimental studies have focused on the interaction of pneumococci with the immune system of a host acutely or chronically exposed to alcohol. Understanding these host-pathogen interactions is imperative for designing effective prophylactic and therapeutic interventions for such populations. Recent advances in pneumococcal research have greatly improved our understanding of pneumococcal pathogenesis and virulence mechanisms. Additionally, a large body of data is available on the effect of alcohol on the physiology of the lungs and the innate and adaptive immune system of the host. The purpose of this review is to integrate the available knowledge in these diverse areas of for a better understanding of the how the compromised immune system derived from alcohol exposure responds to pneumococcal infections.

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.