Faculty Bibliography

AIMS/OBJECTIVE:Physician burnout and its consequences have been recognized as increasingly prevalent and important issues for both organizations and individuals involved in healthcare delivery. The purpose of this study was to describe and compare the patterns of self-reported wellness in orthopaedic surgeons and trainees from multiple nations with varying health systems. METHODS:A cross-sectional survey of 774 orthopaedic surgeons and trainees in five countries (Australia, Canada, New Zealand, UK, and USA) was conducted in 2019. Respondents were asked to complete the Mayo Clinic Well-Being Index and the Stanford Professional Fulfillment Index in addition to 31 personal/demographic questions and 27 employment-related questions via an anonymous online survey. RESULTS:A total of 684 participants from five countries (Australia (n = 74), Canada (n = 90), New Zealand (n = 69), UK (n = 105), and USA (n = 346)) completed both of the risk assessment questionnaires (Mayo and Stanford). Of these, 42.8% (n = 293) were trainees and 57.2% (n = 391) were attending surgeons. On the Mayo Clinic Well-Being Index, 58.6% of the overall sample reported feeling burned out (n = 401). Significant differences were found between nations with regards to the proportion categorized as being at risk for poor outcomes (27.5% for New Zealand (19/69) vs 54.4% for Canada (49/90) ; p = 0.001). On the Stanford Professional Fulfillment Index, 38.9% of the respondents were classified as being burned out (266/684). Prevalence of burnout ranged from 27% for Australia (20/74 up to 47.8% for Canadian respondents (43/90; p = 0.010). Younger age groups (20 to 29: RR 2.52 (95% confidence interval (CI) 1.39 to 4.58; p = 0.002); 30 to 39: RR 2.40 (95% CI 1.36 to 4.24; p = 0.003); 40 to 49: RR 2.30 (95% CI 1.35 to 3.9; p = 0.002)) and trainee status (RR 1.53 (95% CI 1.15 to 2.03 p = 0.004)) were independently associated with increased relative risk of having a 'at-risk' or 'burnout' score. CONCLUSIONS: 2021;2(11):932-939.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

The mammalian focal adhesion proteins Pinch1/2 activate integrins and promote cell-extracellular matrix adhesion and migration; however, their roles in adipose tissue and metabolism are unclear. Here we find that high-fat diet (HFD) feeding dramatically increases expression of Pinch1/2 proteins in white adipose tissue (WAT) in mice. Furthermore, expression of Pinch1 is largely upregulated in WAT in leptin-deficient ob/ob type 2 diabetic mice and obese humans. While mice with loss of Pinch1 in adipocytes or global Pinch2 do not display any notable phenotypes, deleting Pinch1 in adipocytes and Pinch2 globally significantly decreases body weight and WAT mass, but not brown adipose tissue mass, in HFD-fed, but not normal chow diet-fed, mice. Pinch loss ameliorates HFD-induced glucose intolerance and fatty liver. After HFD challenge, Pinch loss slightly but significantly accelerates energy expenditure. While Pinch loss decreases adipocyte size and alters adipocyte size distribution, it greatly accelerates cell apoptosis primarily in epididymal WAT and to a lesser extent in subcutaneous WAT. In vitro studies demonstrate that Pinch loss accelerates adipocyte apoptosis by activating the Bim/Caspase-8 pathway. In vivo, genetic ablation of Caspase-8 expression in adipocytes essentially abolishes the ameliorating effects of Pinch deficiency on obesity, glucose intolerance, and fatty liver in mice. Thus, we demonstrate a previously unknown function of Pinch in control of adipose mass, glucose, and fat metabolism via modulation of adipocyte apoptosis. We may define a novel target for the prevention and treatment of metabolic diseases, such as obesity and diabetes.

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

Mitochondrial cristae are extraordinarily crowded with proteins, which puts stress on the bilayer organization of lipids. We tested the hypothesis that the high concentration of proteins drives the tafazzin-catalyzed remodeling of fatty acids in cardiolipin, thereby reducing bilayer stress in the membrane. Specifically, we tested whether protein crowding induces cardiolipin remodeling and whether the lack of cardiolipin remodeling prevents the membrane from accumulating proteins. In vitro, the incorporation of large amounts of proteins into liposomes altered the outcome of the remodeling reaction. In yeast, the concentration of proteins involved in oxidative phosphorylation (OXPHOS) correlated with the cardiolipin composition. Genetic ablation of either remodeling or biosynthesis of cardiolipin caused a substantial drop in the surface density of OXPHOS proteins in the inner membrane of the mouse heart and Drosophila flight muscle mitochondria. Our data suggest that OXPHOS protein crowding induces cardiolipin remodelling and that remodeled cardiolipin supports the high concentration of these proteins in the inner mitochondrial membrane.

Inflammatory Bowel Disease (IBD) is an autoimmune condition with complicated pathology and diverse clinical signs. TNFα is believed to play a crucial role in the pathogenesis of IBD. We recently identified fexofenadine, a well-known antagonist of histamine H1 receptor, as a novel inhibitor of TNFα signaling. Additionally, cytosolic phospholipase A2 (cPLA2) was isolated as a binding target of fexofenadine, and fexofenadine-mediated anti-TNF activity relied on cPLA2 in vitro. The objective of this study is to determine whether fexofenadine is therapeutic against chemically-induced murine IBD model and whether cPLA2 and/or histamine H1 receptor is important for fexofenadine's anti-inflammatory activity in vivo by leveraging various genetically modified mice and chemically induced murine IBD models. Both dextran sulfate sodium- and 2, 4, 6-trinitrobenzene sulfonic acid-induced murine IBD models revealed that orally delivered fexofenadine was therapeutic against IBD, evidenced by mitigated clinical symptoms, decreased secretions of the proinflammatory cytokine IL-6 and IL-1β, lowered intestinal inflammation, and reduced p-p65 and p-IĸBα. Intriguingly, Fexofenadine-mediated protective effects against IBD were lost in cPLA2 deficient mice but not in histamine H1 receptor-deficient mice. Collectively, these findings demonstrate the therapeutic effects of over-the-counter drug Fexofenadine in treating DSS-induced IBD murine and provide first in vivo evidence showing that cPLA2 is required for fexofenadine's therapeutic effects in murine IBD model and probably other inflammatory and autoimmune diseases as well.

Gpr125, encoded by Adgra3, is an orphan adhesion G-protein coupled receptor (aGPCR) implicated in modulating Wnt signaling and planar polarity. Here we establish both physiological and pathological roles for Gpr125. We show that mice lacking Gpr125 or its signaling domains display an ocular phenotype with many hallmarks of human dry eye syndrome. These include squinting, abnormal lacrimation, mucus accumulation, swollen eyelids and inflammatory infiltration of lacrimal and meibomian glands. Utilizing a Gpr125-β-gal reporter and scRNAseq, we identify Gpr125 expression in a discrete population of cells located at the tips of migrating embryonic lacrimal ducts. By lineage tracing we show these cells function as progenitors of the adult lacrimal myoepithelium. Beyond defining an essential role for Gpr125 in tear film and identifying its utility as a marker of lacrimal progenitors, this study implicates Gpr125 in the etiology of blepharitis and dry eye syndrome, and defines novel animal models of these common maladies

PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.