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Influence of MAP of IFNγ signaling. JAK-STAT signaling networks characterized by kinome analysis in bovine monocytes under different conditions of (A) uninfected and (B) MAP infected. Relative degrees of phosphorylation are shown with genes showing increased phosphorylation in red, decreased phosphorylation in green, and insignificant change in blue. (C) Mechanisms of network silencing was determined by qRT-PCR. Shortly after MAP infection, SOCS1 and SOCS3 levels increased and remained so 18 h post-infection. While 3 h post-infection, no change in IFNγR1 and IFNγR2 expression were observed, both genes were down-regulated 18 h post-infection. These mechanisms together, explain the decrease in JAK-STAT signaling observed by kinome analysis 24 h post-infection.
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Reversible protein phosphorylation is a central mechanism for both the transfer of intracellular information and the initiation of cellular responses. Within human medicine, considerable emphasis is placed on understanding and controlling the enzymes (kinases) that are responsible for catalyzing these modifications. This is evident in the prominent...
Citations
... Herein, we compared the effects of feed additives on the immune and metabolic health of birds that have been challenged with IBV and environmental cold stress, using performance metrics, the kinome peptide array technique and gene expression. The kinome peptide array uses species-specific and process-specific kinase target peptides printed on a glass array; these target peptides can be phosphorylated by active kinases in the biological samples (Arsenault et al., 2011;Daigle et al., 2014;Arsenault and Kogut, 2015). The phosphorylation of these peptides can be measured and visualized to determine changes in signaling cascades in a sample which may alter biological functions and activities (Arsenault and Kogut, 2015). ...
The aim of this study was to evaluate the effectiveness and mechanism of action of two feed additives in reducing the impacts of virus and temperature stressors. We determined the effects of protected biofactors and antioxidants (P(BF+AOx)), and protected biofactors and antioxidants with protected organic acids and essential oils (P(BF+AOx)+P(OA+EO)) on the immune and metabolic health of Ross 308 broiler chickens. These biofactors and antioxidants were derived from vitamins, and Aspergillus niger, Aspergillus oryzae and Bacillus subtilis fermentation extracts. All Ross 308 chickens were exposed to a double-dose of live bronchitis vaccine at day 0 and environmentally challenged by reducing the temperature from 32°C to 20°C at day 3 for 48 hours. Control birds were fed without feed additives in the diet. Performance data and jejunum samples were collected to evaluate the effects of these treatments on growth, cytokine expression and protein phosphorylation via kinome peptide array. ANOVA was used for statistical analysis of the performance and gene expression data (p-value of 0.05), and PIIKA2 was used for statistical evaluation and comparison of the kinome peptide array data. The P(BF+AOx) and P(BF+AOx)+P(OA+EO) treatments significantly increased bird weight gain and decreased feed conversion. The kinome peptide array data analysis showed increased activity of cytoskeletal, cell growth and proliferation proteins, and metabolic signaling in the jejunum of P(BF+AOx)+P(OA+EO) treated chickens. There was a significant decrease in IL-6 gene expression in the jejunum of P(BF+AOx)+P(OA+EO) samples compared to control at day 15. P(BF+AOx)+P(OA+EO) treatments in the jejunum showed strong immunomodulatory effects, perhaps to control inflammation. P(BF+AOx)+P(OA+EO) improves gut health via growth and metabolic signaling in the jejunum while inducing stronger immunomodulation.
... With this technique, digested and separated sample fragments are run through a tandem mass spectrometer and the fragment spectrum is analyzed to identify metabolites or proteins containing specific modifications. Finally, the species-specific kinome peptide array has been used extensively in the past several years to analyze the kinome of chicken, and other agricultural species (Daigle et al., 2014). The technique involves printing kinase enzyme target sequences on an array, when exposed to a biological sample the active kinases will recognize and phosphorylate their target on the array. ...
Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its microbiome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the Symposium by leading international researchers in this field. The Symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system was presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new -omics platforms and data management tools to more completely understand host microbiomes was stressed.
... The different philosophical and technological basis of these approaches have been reviewed elsewhere (19). Each approach is associated with unique challenges and opportunities for application to livestock species (20). ...
Within human health research, the remarkable utility of kinase inhibitors as therapeutics has motivated efforts to understand biology at the level of global cellular kinase activity (the kinome). In contrast, the diminished potential for using kinase inhibitors in food animals has dampened efforts to translate this research approach to livestock species. This, in our opinion, was a lost opportunity for livestock researchers given the unique potential of kinome analysis to offer insight into complex biology. To remedy this situation, our lab developed user-friendly, cost-effective approaches for kinome analysis that can be readily incorporated into most research programs but with a specific priority to enable the technology to livestock researchers. These contributions include the development of custom software programs for the creation of species-specific kinome arrays as well as comprehensive deconvolution and analysis of kinome array data. Presented in this review are examples of the application of kinome analysis to highlight the utility of the technology to further our understanding of two key complex biological events of priority to the livestock industry: host immune responses to infectious diseases and animal stress responses. These advances and examples of application aim to provide both mechanisms and motivation for researchers, particularly livestock researchers, to incorporate kinome analysis into their research programs.
... As protein phosphorylation often serves as a central mechanism for direct regulation of cellular processes, characterization of cellular responses at the level of kinase-mediated phosphorylation (kinome analysis) has the potential to offer unobstructed insight into complex biology 32 . That phosphorylation events can be represented by short peptides, enabling the creation of arrays for high throughput analysis of global cellular kinase activity, in particular in a manner that can be customized for species of interest 33 . ...
The mite Varroa destructor is a serious threat to honeybee populations. Selective breeding for Varroa mite tolerance could be accelerated by biomarkers within individual bees that could be applied to evaluate a colony phenotype. Previously, we demonstrated differences in kinase-mediated signaling between bees from colonies of extreme phenotypes of mite susceptibility. We expand these findings by defining a panel of 19 phosphorylation events that differ significantly between individual pupae from multiple colonies with distinct Varroa mite tolerant phenotypes. The predictive capacity of these biomarkers was evaluated by analyzing uninfested pupae from eight colonies representing a spectrum of mite tolerance. The pool of biomarkers effectively discriminated individual pupae on the basis of colony susceptibility to mite infestation. Kinome analysis of uninfested pupae from mite tolerant colonies highlighted an increased innate immune response capacity. The implication that differences in innate immunity contribute to mite susceptibility is supported by the observation that induction of innate immune signaling responses to infestation is compromised in pupae of the susceptible colonies. Collectively, biomarkers within individual pupae that are predictive of the susceptibility of colonies to mite infestation could provide a molecular tool for selective breeding of tolerant colonies.
... These studies do not mimic the wide variety of conditions and challenges of a normal production situation. Our approach has been to determine the molecular mechanism of action of feed interventions by studying the relevant host tissue itself from a proteomic perspective [11]. This molecular proteomic approach has two major advantages. ...
With the reemergence of poultry diseases such as necrotic enteritis following the restriction of in-feed antibiotics, the search for antibiotic alternatives has become critically important. Postbiotics are non-viable bacterial products or metabolic byproducts from probiotic microorganisms that have positive effects on the host or microbiota. These are a promising alternative to antibiotics. Here, we describe the mechanism of action of a postbiotic in the context of a Clostridium perfringens (C. perfringens) challenge model. By using performance measurements and a peptide array kinome analysis, we describe the kinotypes and signal transduction changes elicited by the postbiotic with and without C. perfringens challenge. The postbiotic improves lesion scores, C. perfringens counts and mortality compared to challenge groups without the postbiotic, and it improves weight gain in the most severely challenged birds. The postbiotic predominantly affects the innate immune response and appears immunomodulatory. In the context of infection, it reduces the proinflammatory responses and generates a homeostatic-like response. This postbiotic is a viable alternative to antibiotics to improve poultry health in the context of C. perfringens pathogen challenge.
... These arrays contain short (15 amino acid) peptides that represent the target site for specific kinases (see Daigle et al., 2014 for an example array). These sites have been shown to be acceptable substrates for kinases, with similar enzyme kinetics to the full protein (Zhu et al., 2000). ...
... Strings of amino acids containing kinase target sites (colored dots) are exposed to kinases, which recognize and phosphorylate the sites (red squares). After staining and imaging, phosphorylated sites appear as fluorescent dots, which can then be quantified and compared to a control or other treatments to find significant differences and active biological pathways kinome profile (Shakiba et al., 2009) by utilizing a species-specific peptide array, which measures phosphorylation changes at the protein level, thus characterizing cellular signaling events (Daigle et al., 2014). The peptide array has been in use in biomedical research for nearly two decades (Cahill, 2001;Cretich, Damin, Pirri, & Chiari, 2006;Davies et al., 2010) where it was applied to humans and to the mouse, a principal model species, but has only recently been available for animal research per se. ...
Here, we describe the design of an Anas‐specific kinome peptide array that can be used to study the immunometabolic responses of mallard and American black duck to pathogens, contaminants, and environmental stress. The peptide arrays contain 2,642 unique phosphorylate‐able peptide sequences representing 1,900 proteins. These proteins cover a wide array of metabolic and immunological processes, and 758 Gene Ontology Biological processes are statistically significantly represented on the duck peptide array of those 164 contain the term “metabolic” and 25 “immune.” In addition, we conducted a comparison of mallard to American black duck at a genetic and proteomic level. Our results show a significant genomic and proteomic overlap between these two duck species, so that we have designed a cross‐reactive peptide array capable of studying both species. This is the first reported development of a wildlife species‐specific kinome peptide array. We describe the design and creation of a new molecular biology tool that characterizes immunometabolism of two sympatric, congeneric duck species (American black duck and mallard duck) that have shown markedly contrasting population trends in recent decades. This tool delineates cellular signaling mechanisms and can be used to characterize species’ differences in physiological tolerance to experimental stimuli.
... By comparing the relative signals of experimental cell or tissue samples, one can identify changes in signal transduction pathways and phosphorylation-regulated events. A detailed description of this technique can be found in Daigle et al. [28]. ...
All cellular functions, ranging from regular cell maintenance and homeostasis, specialized functions specific to cellular types, or generating responses due to external stimulus, are mediated by proteins within the cell. Regulation of these proteins allows the cell to alter its behavior under different circumstances. A major mechanism of protein regulation is utilizing protein kinases and phosphatases; enzymes that catalyze the transfer of phosphates between substrates [1]. Proteins involved in phosphate signaling are well studied and include kinases and phosphatases that catalyze opposing reactions regulating both structure and function of the cell. Kinomics is the study of kinases, phosphatases and their targets, and has been used to study the functional changes in numerous diseases and infectious diseases with aims to delineate the cellular functions affected. Identifying the phosphate signaling pathways changed by certain diseases or infections can lead to novel therapeutic targets. However, a daunting 518 putative protein kinase genes have been identified [2], indicating that this protein family is very large and complex. Identifying which enzymes are specific to a particular disease can be a laborious task. In this review, we will provide information on large-scale systems biology methodologies that allow global screening of the kinome to more efficiently identify which kinase pathways are pertinent for further study.
... Through extensive research and development, a methodology for designing and using species-specific kinome arrays was developed (41)(42)(43). The use of this technology in agricultural species has been reviewed extensively elsewhere (44) and has been used to design peptide arrays to study important biological questions in a number of agriculturally important species, including bovine (45) and poultry (46). ...
Immunometabolism is a relatively new research perspective, focusing on both metabolism and immunology and the cross-talk between these biological processes. Immunometabolism can be considered from two perspectives; 1) the role that immune cells play in organ metabolism and metabolic disease, and 2) the metabolic processes that occur within immune cells and how they affect overall immunity. The gut may be the prototypical organ of immunometabolism. The gut is the site of nutrient absorption and is a major, if not the major, immune organ. We also describe the integration of kinomics and the species-specific peptide array to the study of the gut. This unique immunometabolic tool combined with the unique immunometabolic nature of the gut provides significant research potential to many animal health applications.
... In contrast, the yeast (Saccharomyces cerevisiae) genome has only 130 kinases. Similarly, mouse (Mus musculus) and human (Homo sapiens) genomes also encodes about 500 PKs (Daigle et al., 2014;Sadowski et al., 2013). ...
Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events in numerous signal transduction pathways in eukaryotes. Type 2C PPs (PP2Cs) represent the major group of PPs in plants, and recent discovery of novel abscisic acid (ABA) receptors (ABARs) has placed the PP2Cs at the center stage of the major signaling pathway regulating plant responses to stresses and plant development. Several studies have provided deep insight into vital roles of the PP2Cs in various plant processes. Global analyses of the PP2C gene family in model plants have contributed to our understanding of their genomic diversity and conservation, across plant species. In this review, we will discuss the genomic and structural accounts of PP2Cs in plants. Recent advancements in their interaction paradigm with ABARs and sucrose non-fermenting related kinases 2 (SnRK2s) in ABA signaling are also highlighted. In addition, expression analyses and important roles of PP2Cs in regulation of biotic and abiotic stress responses, potassium (K+) deficiency signaling, plant immunity and development are elaborated. Knowledge of functional roles of specific PP2Cs could be exploited for the genetic manipulation of crop plants. Genetic engineering using PP2C genes could provide great impetus in the agricultural biotechnology sector in terms of imparting desired traits, including higher degree of stress tolerance and productivity without yield penalty.
Introduction: The cellular response to infection by bacterial pathogens involves a complex and highly regulated series of pathways that carry messages to various parts of the cell. These messages are transferred using post-translational modifications including phosphorylation by kinases. Understanding the host’s signaling pathways is valuable in identifying potential treatment targets, but the bacterial signaling pathways and host-pathogen crosstalk are equally important to the development of therapeutics.
Areas covered: This review summarizes some of the recent findings related to the bacterial phosphoproteome and especially serine/threonine/tyrosine sites, including methods and considerations for identifying novel phosphosites. We also consider the bioinformatics tools that have been developed to sift through the large volume of data in these studies and connect them to biologically relevant knowledge about pathways and function. Literature databases used include PubMed and Google Scholar from April 2018 to December 2018.
Expert opinion: While the field has developed significantly in the past decade of research, high-quality experimental sequence data remains the limiting factor to future research into bacterial phosphoproteomics. As more proteomes are explored, it will be easier to tailor tools and techniques to prokaryotes. It will be necessary to consider the phosphoproteome in the broader biological context, through interdisciplinary collaborations.
