Dynamic Regulation of the Class II Transactivator by Posttranslational Modifications

The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses

Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator

The Class II Transactivator (CIITA) is essential to the regulation of Major Histocompatibility Class II (MHC II) genes transcription. As the “master regulator” of MHC II transcription, CIITA regulation is imperative and requires various posttranslational modifications (PTMs) in order to facilitate its role. Previously we identified various ubiquitination events on CIITA. Monoubiquitination is important for CIITA transactivity, while K63 linked ubiquitination is involved in crosstalk with ERK1/2 phosphorylation, where together they mediate cellular movement from the cytoplasm to nuclear region. Further, CIITA is also modified by degradative K48 polyubiquitination. However, the E3 ligase responsible for these modifications was unknown. We show CIITA ubiquitination and transactivity are enhanced with the histone acetyltransferase (HAT), p300/CBP associated factor (pCAF), and the E3 ligase region within pCAF is necessary for both. Additionally, pCAF mediated ubiquitination is independent of pCAF’s HAT domain, and acetylation deficient CIITA is K48 polyubiquitinated and degraded in the presence of pCAF. Lastly, we identify the histone acetyltransferase, pCAF, as the E3 ligase responsible for CIITA’s ubiquitination

Salmonella prevalence in pigs reared on farms with and without antimicrobials

A convenience sample of farms using antimicrobials (antimicrobial-using, AMU) post-weaning for therapy and/or growth promotion (n=21) were contrasted to a convenience sample of farms not using antimicrobials post-weaning (antimicrobial-free, AMF, n=21) distributed across three US geographic areas

Campylobacter Prevalence and Diversity in Antimicrobial Free and Conventionally Reared Market Swine

The objectives of this study were to determine the prevalence and antimicrobial resistance of Campylobacter spp. among pigs raised antimicrobial free (ABF) and those raised conventionally. Bacterial isolation was done on-farm and at slaughter using conventional methods and antimicrobial susceptibility tests were done for 12 antimicrobials using Kirby-Bauer and epsilometric test (E-test) methods. All 14 herds were positive for Campylobacter. On-farm prevalence among ABF herds was 71% and 81% among conventional herd. In contrast, the prevalence among carcass swabs was higher among ABF herds than conventional herds with 60% and 29% respectively. There was significant reduction after chilling in all groups (p\u3c0.05). On-farm frequency of antimicrobial resistance was significantly higher among isolates from conventional herds than ABF (p\u3c0.05). In contrast frequency of resistance to five of the seven antimicrobials was higher among carcass swabs of ABF herds than conventional herds

Campylobacter prevalence and antimicrobial resistance in swine reared in antimicrobial-free and conventional production systems

The objective of this study was to determine and compare the prevalence and antimicrobial resistance of Campylobacter species in swine reared in the conventional and antimicrobial free production (ABF) production systems as part of a multi-state study. To date, 19 conventional and 16 ABF groups have been followed

Prevalence and antimicrobial resistance profile of Campylobacter isolated from conventional and antibiotic free swine farms in three geographic locations

The prevalence and antimicrobial resistance of Campylobacter were examined from swine reared in conventional and antimicrobial-free (ABF) production systems in three geographical locations: North Carolina (NC), Ohio (OH) and Wisconsin (WI). Methods: A total of 1500 pigs and 1930 carcass swab samples were evaluated for the prevalence of Campylobacter. Fecal samples from 662 pigs from NC (370 conventional farms and 292 ABF farms), 379 from OH (268 conventional and 111 ABF) and 459 from WI (160 conventional and 299 ABF) were included. Antimicrobial susceptibility testing was performed using agar dilution method against a panel of six antimicrobials

Evaluating a Modular Decision Support Application For Colorectal Cancer Screening

BACKGROUND: There is a need for health information technology evaluation that goes beyond randomized controlled trials to include consideration of usability, cognition, feedback from representative users, and impact on efficiency, data quality, and clinical workflow. This article presents an evaluation illustrating one approach to this need using the Decision-Centered Design framework. OBJECTIVE: To evaluate, through a Decision-Centered Design framework, the ability of the Screening and Surveillance App to support primary care clinicians in tracking and managing colorectal cancer testing. METHODS: We leveraged two evaluation formats, online and in-person, to obtain feedback from a range primary care clinicians and obtain comparative data. Both the online and in-person evaluations used mock patient data to simulate challenging patient scenarios. Primary care clinicians responded to a series of colorectal cancer-related questions about each patient and made recommendations for screening. We collected data on performance, perceived workload, and usability. Key elements of Decision-Centered Design include evaluation in the context of realistic, challenging scenarios and measures designed to explore impact on cognitive performance. RESULTS: Comparison of means revealed increases in accuracy, efficiency, and usability and decreases in perceived mental effort and workload when using the Screening and Surveillance App. CONCLUSION: The results speak to the benefits of using the Decision-Centered Design approach in the analysis, design, and evaluation of Health Information Technology. Furthermore, the Screening and Surveillance App shows promise for filling decision support gaps in current electronic health records

Evaluation of the association between pen fecal accumulation and prevalence of Salmonella enterica shedding in swine

One of the recommended control measures for Salmonella enterica is improved or adequate fann hygiene. Although loosely defined, a component of good hygiene practice is to minimize animal contact with feces. Increased exposure to feces would he expected to increase the likelihood of transmission and fecal shedding of Salmonella enterica. There are indications in the literature that decreased animal contact with feces may result in a decreased risk for Salmonella enterica infection. Davies et al. demonstrated increased mean prevalence in open-flush gutter and dirt lot systems when compared to pigs housed on total slatted flooring systems. Paradoxically, three-site, all-in!all-out pig flow systems did not have a significantly different mean Salmonella enterica prevalence when compared to one-site continuous flow fanns. A similar incongruity is the evidence that in poultry barns, birds placed on used litter had lower risk of salmonella shedding than birds placed on clean litter.(5) Proposed mechanisms of this result are colonization with competitive flora from the used litter as well as an inhibitory environment for Salmonella enterica. The subjective nature of determining adequate hygiene as well as the presence of apparently contradictory results regarding hygiene and prevalence of Salmonella shedding in animals warrants inquiry into the question of what is good hygiene in respect to Salmonella shedding in animals

Community based service providers' perspectives on frequent and/or avoidable admission of older people with chronic disease in rural NSW: a qualitative study

Background: Frequent and potentially avoidable hospital admission amongst older patients with ambulatory care sensitive (ACS) chronic conditions is a major topic for research internationally, driven by the imperative to understand and therefore reduce hospital admissions. Research to date has mostly focused on analysis of routine data using ACS as a proxy for 'potentially avoidable'. There has been less research on the antecedents of frequent and/or avoidable admission from the perspectives of patients or those offering community based care and support for these patients. This study aimed to explore community based service providers' perspectives on the factors contributing to admission among older patients with chronic disease and a history of frequent and potentially avoidable admission. Methods. 15 semi-structured interviews with community based providers of health care and other services, and an emergency department physician were conducted. Summary documents were produced and thematic analysis undertaken. Results: A range of complex barriers which limit or inhibit access to services were reported. We classified these as external and internal barriers. Important external barriers included: complexity of provision of services, patients' limited awareness of different services and their inexperience in accessing services, patients needing a higher level or longer length of service than they currently have access to, or an actual lack of available services, patient poverty, rurality, and transport. Important internal barriers included: fear (of change for example), a 'stoic' attitude to life, and for some, the difficulty of accepting their changed health status. Conclusions: The factors underlying frequent and/or potentially avoidable admission are numerous and complex. Identifying strategies to improve services or interventions for this group requires understanding patient, carer and service providers' perspectives. Improving accessibility of services is also complex, and includes consideration of patients' social, emotional and psychological ability and willingness to use services as well as those services being available and easily accessed

Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia

BACKGROUND: Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients. METHODS: We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations. RESULTS: We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries. CONCLUSIONS: Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension