Probing conformational dynamics of DNA binding by CO-sensing transcription factor, CooA.

The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.

Surveyed veterinary students in Australia find ChatGPT practical and relevant while expressing no concern about artificial intelligence replacing veterinarians.

Background: Chat Generative Pre-trained Transformer (ChatGPT) is a freely available online artificial intelligence (AI) program capable of understanding and generating human-like language. This study assessed veterinary students' perceptions about ChatGPT in education and practice. It compared perceptions about ChatGPT between students who had completed a critical analysis task and those who had not. Methods: This cross-sectional study surveyed 498 Doctor of Veterinary Medicine (DVM) students at The University of Sydney, Australia. Second-year DVM students researched a veterinary pathogen and then completed a critical analysis of ChatGPT (version 3.5) output for the same pathogen. A survey based on the Technology Acceptance Model was then delivered to all DVM students from all years of the programme, collecting data using Likert-style, categorical and free-text items. Results: Over 75% of the 100 respondents reported having used ChatGPT. The students found ChatGPT's output relevant and practical for their use but perceived it as inaccurate. They perceived ChatGPT output to be more useful for veterinary students than for pet owners or veterinarians. Those who had completed the critical analysis assignment had a more positive view of ChatGPT's practicality for veterinary students but noted its authoritative tone even when delivering inaccurate information. Over 50% of the students agreed that information about tools such as ChatGPT should be included in the veterinary curriculum. Students agreed that veterinarians should embrace AI but disagreed that AI would eventually replace the need for veterinarians. Conclusions: A critical appraisal of outputs from AI tools such as ChatGPT may help prepare future veterinarians for the effective use of these tools.

Carbon Monoxide-Sensing Transcription Factors: Regulators of Microbial Carbon Monoxide Oxidation Pathway Gene Expression.

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate. In this review, we examine two known heme-dependent transcription factors, CooA and RcoM, that regulate inducible CO metabolism pathways in anaerobic and aerobic microorganisms. We provide an analysis of the known physiological and genomic contexts of these sensors and employ this analysis to contextualize known biochemical properties. In addition, we describe a growing list of putative transcription factors associated with CO metabolism that potentially use cofactors other than heme to sense CO.

Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator PxRcoM.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.

Resting and postprandial serum bile acid concentrations in dogs with liver disease.

BACKGROUND: Serum bile acids (SBAs) are frequently measured in dogs. However, there is limited data comparing SBAs in different liver diseases diagnosed according to standardized histological criteria. OBJECTIVES: To compare resting and postprandial SBAs, and determine their sensitivity and specificity, for various liver diseases in dogs. ANIMALS: Three hundred and forty-one client-owned dogs with suspected liver disease that had a liver biopsy and SBAs measured. METHODS: Multicenter retrospective study. Cases were classified according to standardized histological criteria. The sensitivity and specificity of resting and postprandial SBAs for the diagnosis of each liver disease, and all liver diseases combined, were calculated. RESULTS: The median resting SBAs were highest in dogs with cirrhosis (98.8 µmol/L; range, 6-135) and congenital circulatory anomalies (CCa; 79.45 µmol/L; 0.3-705). The highest median postprandial concentrations were found in CCa (126 µmol/L; 0-726) and chronic hepatitis (CH; 54.3 µmol/L; 0-260). Using the cut-off value of 10 µmol/L, the highest sensitivities of resting SBAs were recorded in dogs with CCa (87.5%; 95% confidence interval, 76.8-94.4) and CH (81.1%; 71.5-88.6). The sensitivities of postprandial SBAs were the highest in cholangitis (100%; 47.8-100.0) and CCa (91.1%; 78.8-97.5). The specificities of resting and postprandial SBAs for all diseases were 49.3% (37.6-61.1) and 29.7% (15.9-47.0), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Postprandial SBAs are more sensitive but less specific than resting SBAs for the diagnosis of liver disease. There were dogs in all categories of liver disease with resting SBAs <10 and >90 µmol/L. Therefore, careful interpretation of both normal and elevated values is required.

Prevalence of bacteria and changes in trends in antimicrobial resistance of Escherichia coli isolated from positive canine urinary samples from an Australian referral hospital over a 5-year period (2013-2017).

Lower urinary tract disease is common in dogs with approximately 14% developing a bacterial lower urinary tract infection (UTI) during their lifetime. Empirical antimicrobials are often prescribed while waiting urine culture and susceptibility results. Regional knowledge of bacterial prevalence and antimicrobial resistance patterns aids veterinarians in antimicrobial choice. This study aimed to identify the prevalence of uropathogens in canine urine tract isolates and to assess for changes in antimicrobial resistance of Escherichia coli (E. coli) over a 5-year study period at a large multidisciplinary private referral hospital in Australia (January 2013-December 2017). The proportion of resistant isolates was compared across 5 years (Fisher's exact test and Cochran Armitage test for trend) for select antimicrobials towards E. coli. A total of 246 positive urine cultures were included. E. coli was the most prevalent uropathogen at 64%, followed by Proteus sp., Staphylococcus sp. and Enterococcus sp., respectively (9%, 8% and 7%). E. coli was most commonly resistant to amoxicillin at 41%. There was no statistically significant difference, nor trend, in resistance of E. coli isolates towards the selected antimicrobials over the 5 years. Resistance towards trimethoprim-sulfonamide was lower at 15%. This information will aid local veterinarians in selecting empirical antimicrobials pending culture results for the treatment of UTIs in dogs.

Persuasive system features in computer-mediated lifestyle modification interventions for physical activity.

Objective: Increasing physical activity has been identified as one of the most important factors in lifestyle modification. Previous studies have reported the effectiveness of using the Internet in motivating behavioral modifications of physical activities. The aim of this study is to identify the persuasive system features most frequently used in computer-mediated physical activities in the current literature.Materials and Methods: In this review, intervention studies were identified through a structured computerized search of PubMed, PsychInfo, and Web of Science. The results of the search were analyzed using the persuasive systems design (PSD) features identified by Oinas-Kukkonen and Harjumaa (2009).Results: Thirty-eight articles were reviewed, and the features of the physical activity interventions described were mapped to the identified facets of PSD. The PSD features used most often by researchers in the studies considered in this research included tailoring, tunneling, reminders, trustworthiness, and expertise. The effectiveness of the interventions described in the studies was also compared. The stage of change theory was applied in several intervention studies, and the importance of stage of change has been identified in effectiveness of persuasion toward physical activity.

Caroli's-type ductal plate malformation and a portosystemic shunt in a 4-month-old kitten.

CASE SUMMARY: A 4-month-old neutered male Russian Blue kitten had a 4 week history of hypersalivation and failure to thrive. In addition, there was a 2 week history of soft tissue swelling on the ventral abdomen, which had failed to improve with antimicrobial therapy. There were no significant physical examination or neurological deficits on examination; however, the cat had a quiet demeanour for its age. Postprandial bile acids were increased (32 µmol/l; reference interval <25 µmol/l). An abdominal CT scan revealed changes consistent with an extrahepatic portosystemic shunt and inflammation of fat of the ventral abdominal body wall. Surgical biopsy and culture of the subcutaneous swelling identified non-infectious steatitis. Ten weeks following initial presentation, surgical exploration, liver biopsy and ligation of the portosystemic shunt were performed. Liver biopsy was submitted to the Anatomical Pathology Laboratory of Cornell University Animal Health Diagnostic Center, New York, USA. Histopathology revealed a ductal plate malformation (Caroli's type), as well as changes consistent with a portosystemic shunt. RELEVANCE AND NOVEL INFORMATION: Ductal plate malformations are rarely described in the veterinary literature. To our knowledge this is the first reported case of Caroli's-type malformation in a cat. There are no biochemical changes that allow for differentiation of ductal plate malformations from other hepatopathies. Liver biopsy is required for a definitive diagnosis.

Effect of age and surgical approach on perioperative wound complication following ovariohysterectomy in shelter-housed cats in Australia.

OBJECTIVES: The aim of the study was to investigate the effects of age and/or surgical approach on perioperative wound complication following ovariohysterectomy (OHE). METHODS: A retrospective search of perioperative monitoring records from a shelter desexing program was conducted to identify cats that underwent OHE between 1 June 2010 and 31 December 2012 inclusive. A wound complication was defined as gross observation of inflammation or wound dehiscence at the surgical site in the 5 day postoperative period. Cases were grouped according to age (≤12 weeks or >12 weeks) and surgical approach (flank or midline). Stratified analyses were conducted to evaluate the association between surgical approach and wound complications, after adjusting for age. Mantel-Haenszel adjusted risk ratio, Cochran-Mantel-Haenszel test statistic and their 95% confidence intervals were presented. RESULTS: A total of 312 cases met the study criteria. The overall wound complication risk was low (6.09%) and was not related to age. A midline approach was associated with a 4.59-times increased risk of wound complication, compared with a flank approach in cats up to 12 weeks of age (P = 0.015) but not in older cats. CONCLUSIONS AND RELEVANCE: These findings support the practice of prepubertal desexing for cats.