Development and application of genus-specific peptide arrays for kinome analysis

Phosphorylation represents a central mechanism to regulate cell function. Protein phosphorylation is catalyzed by a class of enzymes called kinases, the cellular complement of which is referred to as its kinome. The study of the kinome has led to the development of peptide arrays as a high throughput tool. Though the approach was effective, it was limited to genera with characterized phosphoproteomes, as well as by the practice of interpreting emerging data through platforms designed for gene expression data. Within this thesis, these pivotal roadblocks are addressed through the presentation of 1) an approach for the development of custom-designed genus-specific arrays based on proteomic information and 2) a data analysis pipeline, Platform for Integrated, Intelligent Kinome Analysis (PIIKA), developed specifically for peptide array kinome data. The utility of these advances is demonstrated through the creation of the customized peptide arrays and subsequent confirmation of PIIKA-enhanced data transformation and mining. These techniques (custom array and PIIKA) were then applied to two complex host-pathogen interactions: prion diseases and Mycobacterium avium subsp. paratuberculosis (MAP) infection. Prion diseases result from the misfolding of the widely expressed and highly conserved cellular prion protein (PrPC) into an infectious and pathological scrapie-like conformation (PrPSc). Little is known about the function of PrPC in either the normal or diseased states, although a role in signal transduction has been suggested. Two PrPC protein-specific ligands, PrP 106-126 prion fragment and the prion-specific monoclonal antibody 6H4, were used to induce signalling in neuronal cells. Kinome analysis revealed distinct signalling pathways and varied signalling responses for each PrPC ligand. This observation is consistent with the emerging concept that PrPC interacts with multiple cellular proteins and plays a multifunctional role in regulating cellular responses. MAP is the causative agent of Johne’s disease in cattle. MAP establishes a persistent infection and has the ability to evade immune responses while replicating inside macrophages. Kinome analysis indicated that MAP is able to inhibit the interferon gamma (IFNγ)-induced JAK-STAT signalling pathway, eliminating the activation of downstream effectors. Further analysis indicated that the JAK-STAT pathway was blocked at the level of the IFNγ receptor, and JAK-STAT suppressor molecules known as SOCS were activated, both novel findings in the field of MAP pathogenesis. Collectively, these investigations highlight the use of custom-designed and genus-specific peptide arrays to address complex biology in distinct genera

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signalling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25 % citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlus®P]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n=23-26) and evaluated in a NE challenge model (n=3). Birds were administered 2X cocci vaccine on d14 and challenged with a cocktail of Clostridium perfringens strains (107) on d17-19. On d20-21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n=5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t-test and lesion scores analyzed using F-test two-sample for variances (P<0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P=0.004). Lesions scores were lower (P=0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P=0.19) to be heavier (848.6g) than controls (796.2g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P<0.05) in all three pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways

Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors.

Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells

Unanticipated Insights into Biomedicine from the Study of Acupuncture

Research into acupuncture has had ripple effects beyond the field of acupuncture. This paper identifies five exemplars to illustrate that there is tangible evidence of the way insights gleaned from acupuncture research have informed biomedical research, practice, or policy. The first exemplar documents how early research into acupuncture analgesia has expanded into neuroimaging research, broadening physiologic understanding and treatment of chronic pain. The second describes how the acupuncture needle has become a tool to enhance biomedical knowledge of connective tissue. The third exemplar, which illustrates use of a modified acupuncture needle as a sham device, focuses on emergent understanding of placebo effects and, in turn, on insights into therapeutic encounters in treatments unrelated to acupuncture. The fourth exemplar documents that two medical devices now in widespread use were inspired by acupuncture: transcutaneous electrical nerve stimulators for pain control and antinausea wrist bands. The final exemplar describes how pragmatic clinical trial designs applied in acupuncture research have informed current general interest in comparative effectiveness research. In conclusion, these exemplars of unanticipated outcomes of acupuncture research comprise an additional rationale for continued support of basic and clinical research evaluating acupuncture and other under-researched therapies

Human biting mosquitoes and implications for West Nile virus transmission

West Nile virus (WNV), primarily vectored by mosquitoes of the genus Culex, is the most important mosquito-borne pathogen in North America, having infected thousands of humans and countless wildlife since its arrival in the USA in 1999. In locations with dedicated mosquito control programs, surveillance methods often rely on frequent testing of mosquitoes collected in a network of gravid traps (GTs) and CO2-baited light traps (LTs). Traps specifically targeting oviposition-seeking (e.g. GTs) and host-seeking (e.g. LTs) mosquitoes are vulnerable to trap bias, and captured specimens are often damaged, making morphological identification difficult. This study leverages an alternative mosquito collection method, the human landing catch (HLC), as a means to compare sampling of potential WNV vectors to traditional trapping methods. Human collectors exposed one limb for 15 min at crepuscular periods (5:00–8:30 am and 6:00–9:30 pm daily, the time when Culex species are most actively host-seeking) at each of 55 study sites in suburban Chicago, Illinois, for two summers (2018 and 2019). A total of 223 human-seeking mosquitoes were caught by HLC, of which 46 (20.6%) were mosquitoes of genus Culex. Of these 46 collected Culex specimens, 34 (73.9%) were Cx. salinarius, a potential WNV vector species not thought to be highly abundant in upper Midwest USA. Per trapping effort, GTs and LTs collected > 7.5-fold the number of individual Culex specimens than HLC efforts. The less commonly used HLC method provides important insight into the complement of human-biting mosquitoes in a region with consistent WNV epidemics. This study underscores the value of the HLC collection method as a complementary tool for surveillance to aid in WNV vector species characterization. However, given the added risk to the collector, novel mitigation methods or alternative approaches must be explored to incorporate HLC collections safely and strategically into control programs.https://doi.org/10.1186/s13071-022-05603-

Complementary and alternative medicine use among US Navy and Marine Corps personnel

<p>Abstract</p> <p>Background</p> <p>Recently, numerous studies have revealed an increase in complementary and alternative medicine (CAM) use in US civilian populations. In contrast, few studies have examined CAM use within military populations, which have ready access to conventional medicine. Currently, the prevalence and impact of CAM use in US military populations remains unknown.</p> <p>Methods</p> <p>To investigate CAM use in US Navy and Marine Corps personnel, the authors surveyed a stratified random sample of 5,000 active duty and Reserve/National Guard members between December 2000 and July 2002. Chi-square tests and multivariable logistic regression were used to assess univariate associations and adjusted odds of CAM use in this population.</p> <p>Results and discussion</p> <p>Of 3,683 service members contacted, 1,446 (39.3%) returned a questionnaire and 1,305 gave complete demographic and survey data suitable for study. Among respondents, more than 37% reported using at least one CAM therapy during the past year. Herbal therapies were among the most commonly reported (15.9%). Most respondents (69.8%) reported their health as being very good or excellent. Modeling revealed that CAM use was most common among personnel who were women, white, and officers. Higher levels of recent physical pain and lower levels of satisfaction with conventional medical care were significantly associated with increased odds of reporting CAM use.</p> <p>Conclusion</p> <p>These data suggest that CAM use is prevalent in the US military and consistent with patterns in other US civilian populations. Because there is much to be learned about CAM use along with allopathic therapy, US military medical professionals should record CAM therapies when collecting medical history data.</p