Q Fever Knowledge, Attitudes and Vaccination Status of Australia’s Veterinary Workforce in 2014

Q fever, caused by Coxiella burnetii, is a serious zoonotic disease in humans with a worldwide distribution. Many species of animals are capable of transmitting C. burnetii, and consequently all veterinary workers are at risk for this disease. An effective Q fever vaccine has been readily available and used in Australia for many years in at-risk groups, and the European Centre for Disease Prevention and Control has recently also called for the use of this vaccine among at-risk groups in Europe. Little is known about attitudes towards this vaccine and vaccine uptake in veterinary workers. This study aimed to determine the Q fever vaccination status of veterinarians and veterinary nurses in Australia and to assess and compare the knowledge and attitudes towards Q fever disease and vaccination of each cohort. An online cross-sectional survey performed in 2014 targeted all veterinarians and veterinary nurses in Australia. Responses from 890 veterinarians and 852 veterinary nurses were obtained. Binary, ordinal and multinomial logistic regression were used to make comparisons between the two cohorts. The results showed that 74% of veterinarians had sought vaccination compared to only 29% of veterinary nurses. Barriers to vaccination among those not vaccinated did not differ between cohorts, and included a lack of perceived risk, financial expense, time constraints, and difficulty in finding a vaccine provider. Poor knowledge and awareness of Q fever disease and vaccination were additional and notable barriers for the veterinary nursing cohort, suggesting veterinary clinics and veterinarians may not be meeting their legal responsibility to educate staff about risks and risk prevention. Further evaluation is needed to identify the drivers behind seeking and recommending vaccination so that recommendations can be made to improve vaccine uptake

Biophysical Probes Reveal a “Compromise” Nature of the Methyl-lysine Binding Pocket in L3MBTL1

Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and di-methyl marks, but ignores unmodified and trimethylated lysine residues. We made use of Molecular Dynamics (MD) and Free Energy Perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyllysine recognition. Isothermal Titration Calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed “biophysical” probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a “compromise” that non-optimally fit to both mono- and di-methyl-lysine marks

Field Performance of a Rapid Test to Detect Progressive, Regressive, and Abortive Feline Leukemia Virus Infections in Domestic Cats in Australia and Germany

Different feline leukemia virus (FeLV) infection outcomes are possible in cats following natural exposure, such as progressive infections (persistent viremia), regressive infections (transient or no viremia followed by proviral persistence) and abortive infections (presence of only antibodies). Laboratory-based testing is currently required for categorization of infection outcomes in cats. The aim of this study was to evaluate the field performance of a novel, rapid, combination point-of-care (PoC) test kit commercially available in Europe (v-RetroFel®Ag/Ab; 2020–2021 version) to determine different FeLV infection outcomes by concurrent detection of FeLV antigen (p27) and antibodies against FeLV transmembrane envelope protein (p15E). A secondary aim was to evaluate the performance of the same test kit (v-RetroFel®FIV) to determine positive/negative feline immunodeficiency virus (FIV) infection status by the detection of antibodies to FIV capsid protein (p24) and transmembrane glycoprotein (gp40). Two cohorts of domestic cats were recruited and tested with v-RetroFel® using plasma or serum, including cats in Australia (n = 200) and cats in Germany (n = 170). Results from p27 antigen PoC testing, proviral DNA PCR, and neutralizing antibody testing or testing for antibodies against non-glycosylated surface unit envelope protein (p45) were used to assign cats to groups according to different FeLV infection outcomes. Testing with a laboratory-based FeLV p15E antibody ELISA was also performed for comparison. In the first cohort, v-RetroFel®Ag/Ab correctly identified 89% (109/122) FeLV-unexposed cats and 91% (21/23) progressive infections, but no regressive (0/23) or abortive (0/32) infections. In the second cohort, v-RetroFel®Ag/Ab correctly identified 94% (148/158) FeLV-unexposed cats and 100% (4/4) progressive infections, but no regressive (0/2) and only 17% (1/6) abortive infections. There was test agreement between v-RetroFel®Ab and the p15E laboratory ELISA in 58.9% of samples. As a secondary outcome of this study, the sensitivity and specificity of v-RetroFel®FIV testing in cohort 1 were 94.7% (18/19) and 98.3% (178/181), and in cohort 2, 30.0% (3/10) and 100.0% (160/160), respectively. Prior history of FIV vaccination did not produce any false-positive FIV results. In conclusion, v-RetroFel®Ag/Ab (2020–2021 version) was unable to accurately determine different FeLV infection outcomes in the field. Improvements of the test prior to application to field samples are required

Oncogenic Ha-Ras-induced Signaling Activates NF-κB Transcriptional Activity, Which Is Required for Cellular Transformation

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-kappaB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-kappaB through the induced phosphorylation and degradation of the inhibitor IkappaB followed by nuclear translocation of NF-kappaB. We show here that oncogenic forms of Ha-Ras activate NF-kappaB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-kappaB RelA/p65 subunit. Importantly, RelA/p65 -/- cells are inefficient in the activation of kappaB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IkappaBalpha expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-kappaB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential

Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MerTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MerTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket

Chromodomain Ligand Optimization via Target-Class Directed Combinatorial Repurposing

Efforts to develop strategies for small molecule chemical probe discovery against the readers of the methyl-lysine (Kme) post-translational modification have been met with limited success. Targeted disruption of these protein-protein interactions via peptidomimetic inhibitor optimization is a promising alternative to small molecule hit discovery; however, recognition of identical peptide motifs by multiple Kme reader proteins presents a unique challenge in the development of selective Kme reader chemical probes. These selectivity challenges are exemplified by the Polycomb repressive complex 1 (PRC1) chemical probe, UNC3866, which demonstrates sub-micromolar off-target affinity toward the non-PRC1 chromodomains CDYL2 and CDYL. Moreover, since peptidomimetics are challenging subjects for structure-activity relationship (SAR) studies, traditional optimization of UNC3866 would prove costly and time-consuming. Herein, we report a broadly applicable strategy for the affinity-based, target-class screening of chromodomains via the repurposing of UNC3866 in an efficient, combinatorial peptide library. A first-generation library yielded UNC4991, a UNC3866 analog that exhibits a distinct selectivity profile while maintaining sub-micromolar affinity toward the CDYL chromodomains. Additionally, in vitro pull-down experiments from HeLa nuclear lysates further demonstrate the selectivity and utility of this compound for future elucidation of CDYL protein function

The structure–activity relationships of L3MBTL3 inhibitors: flexibility of the dimer interface

We recently reported the discovery of UNC1215, a potent and selective chemical probe for the L3MBTL3 methyllysine reader domain. In this article, we describe the development of structure-activity relationships (SAR) of a second series of potent L3MBTL3 antagonists which evolved from the structure of the chemical probe UNC1215. These compounds are selective for L3MBTL3 against a panel of methyllysine reader proteins, particularly the related MBT family proteins, L3MBTL1 and MBTD1. A co-crystal structure of L3MBTL3 and one of the most potent compounds suggests that the L3MBTL3 dimer rotates about the dimer interface to accommodate ligand binding

Small-Molecule Ligands of Methyl-Lysine Binding Proteins: Optimization of Selectivity for L3MBTL3

Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells

One health in our backyard: Design and evaluation of an experiential learning experience for veterinary medical students

Background: New educational approaches are needed to improve student understanding of the wider sociological and ecological determinants of health as well as professional responsibilities in related areas. Field trips allow students to observe interaction between plant, animal and human communities, making them an ideal tool for teaching One Health concepts. Methods: Veterinary medical students participated in a field trip to a local parklands area, frequented by humans, dogs, horses, and wildlife. Students rotated through 5 learning activities (‘stations’) that focused on: (1) response to exotic animal disease incursion (equine influenza); (2) impact of cultures and belief systems on professional practice; (3) management of dangerous dogs; (4) land use change, biodiversity and emerging infectious disease; and (5) management of environmentally-acquired zoonoses (botulism). Intended learning outcomes were for students to: evaluate the various roles and responsibilities of veterinarians in society; compare the benefits and risks associated with human-animal and animal-animal interactions; and evaluate the contributions made by various professionals in safeguarding the health and welfare of animals, humans and the environment. Following the field trip, students participated in a debrief exercise and completed an online survey on their experiences. Results: Feedback from students collected in 2016/2017 (n = 211) was overwhelmingly positive. The learning experience at each station was rated as 4 (‘Good’) or 5 (‘Very Good’) out of 5 by 82–96% of students. Responses to closed- and open-ended questions − as well as outputs generated in the debrief session − indicated that students achieved the learning outcomes. Overall, 94% of students agreed or strongly agreed that they had a better understanding of One Health because of the field trip. Conclusions: Field trips to local parklands are effective in promoting learning about One Health and can be incorporated into the core curriculum to maximize student exposure at relatively low cost. Keywords: One health, Field trip, Experiential learning, Veterinary education, Cultural competence, Zoonoses, Animal behavio