Faculty Bibliography

Down Syndrome Cell Adhesion Molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), while abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders.SIGNIFICANCE STATEMENTDscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders.

Oculocutaneous albinism (OCA) is an inherited condition characterised by significantly reduced pigment in skin, hair and eyes, visual defects and an increased risk of skin cancer. In the South African black population, 1 in 4 000 people is affected. Quality of life in children with albinism is influenced not only by health problems, but also by stigmatisation, rejection and cultural issues. This review aims to explore the latest literature available on the epidemiology, genetics, clinical characteristics, psychosocial issues and possible management strategies, focusing on affected children. The knowledge provided here is required of health professionals if a more fully informed service is to be offered to these children and their families.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.

Hepatitis B virus (HBV) remains a major global health problem with 257 million chronically infected individuals worldwide, of whom approximately 20 million are co-infected with hepatitis delta virus (HDV). Progress toward a better understanding of the complex interplay between these two viruses and the development of novel therapies have been hampered by the scarcity of suitable cell culture models that mimic the natural environment of the liver. Here, we established HBV and HBV/HDV co-infections and super-infections in self-assembling co-cultured primary human hepatocytes (SACC-PHHs) for up to 28 days in a 384-well format and highlight the suitability of this platform for high-throughput drug testing. We performed RNA sequencing at days 8 and 28 on SACC-PHHs, either HBV mono-infected or HBV/HDV co-infected. Our transcriptomic analysis demonstrates that hepatocytes in SACC-PHHs maintain a mature hepatic phenotype over time, regardless of infection condition. We confirm that HBV is a stealth virus, as it does not induce a strong innate immune response; rather, oxidative phosphorylation and extracellular matrix-receptor interactions are dysregulated to create an environment that promotes persistence. Notably, HDV co-infection also did not lead to statistically significant transcriptional changes across multiple donors and replicates. The lack of innate immune activation is not due to SACC-PHHs being impaired in their ability to induce interferon stimulated genes (ISGs). Rather, polyinosinic:polycytidylic acid exposure activates ISGs, and this stimulation significantly inhibits HBV infection, yet only minimally affects the ability of HDV to infect and persist. Conclusion: These data demonstrate that the SACC-PHH system is a versatile platform for studying HBV/HDV co-infections and holds promise for performing chemical library screens and improving our understanding of the host response to such infections.

This retrospective, matched-cohort study analyzed 1,556 patients with diabetic ulcers treated at 470 wound centers throughout the United States to determine the effectiveness of a cryopreserved bioactive split-thickness skin allograft plus standard of care when compared to standard of care alone. There were 778 patients treated with the graft in the treatment cohort, who were paired with 778 patients drawn from a pool of 126,864 candidates treated with standard of care alone (controls), by using propensity matching to create nearly identical cohorts. Both cohorts received standard wound care, including surgical debridement, moist wound care, and offloading. Logistic regression analysis of healing rates according to wound size, wound location, wound duration, volume reduction, exposed deep structures, and Wagner grade was performed. Amputation rates and recidivism at 3 months, 6 months, and 1 year after wound closure were analyzed. Diabetic ulcers were 59% more likely to close in the treatment cohort compared to the control cohort (p = .0045). The healing rate with the graft was better than standard of care across multiple subsets, but the most significant improvement was noted in the worst wounds that had a duration of 90-179 days prior to treatment (p =.0073), exposed deep structures (p = .036), and/or Wagner Grade 4 ulcers (p = 0.04). Furthermore, the decrease in recidivism was statistically significant at 3 months, 6 months, and 1 year, with and without initially exposed deep structures (p < .05). The amputation rate in the treatment cohort was 41.7% less than that of the control cohort at 20 weeks (0.9% vs 1.5%, respectively). This study demonstrated that diabetic ulcers treated with a cryopreserved bioactive split-thickness skin allograft were more likely to heal and remain closed compared to ulcers treated with standard of care alone. This article is protected by copyright. All rights reserved.

Chronic wounds affect roughly 6.5 million patients in the United States annually. Current standard of therapy entails weekly sharp debridement. However, the sharp technique is associated with significant pain, while having minimal impact on the bioburden. Our study proposes the Er:YAG laser as an alternative method of debridement that may decrease procedural pain, reduce bioburden, and potentially improve overall healing. This pilot study was performed as a prospective, randomized, controlled, crossover clinical trial, containing two groups: 1) one group underwent single laser debridement session first, followed by single sharp debridement session one week later; 2) the other group underwent single sharp debridement session first, followed by single laser debridement session one week later. Variables analyzed included pain during debridement, pre- and post-debridement wound sizes, pre- and post-debridement bacterial loads and patient preference. Twenty-two patients were enrolled (12 patients in Group 1, plus 10 patients in Group 2). The mean pain score for patients undergoing laser debridement was 3.0 ± 1.7 versus 4.8 ± 2.6 for those undergoing sharp debridement (p = 0.003). The mean percent change in wound size one-week post-laser debridement was -20.8% ± 80.1%, as compared with -36.7% ± 54.3% one-week post-sharp debridement (p = 0.6). The percentage of patients who had a bacterial load in the Low/Negative category increased from 27.3% to 59.1% immediately after laser debridement (p = 0.04), versus 54.5% to 68.2% immediately after sharp debridement (p = 0.38). Moreover, there was a sustained decrease in bacterial load one-week post-laser debridement, as compared with no sustained decrease one-week post-sharp debridement (p < 0.02). Overall, 52.9% of patients preferred laser debridement versus 35.3% for sharp debridement. We believe that Er:YAG laser serves as a promising technology in chronic wounds, functioning as a potentially superior alternative to sharp debridement, the current standard of therapy. This article is protected by copyright. All rights reserved.

PURPOSE/OBJECTIVE:To investigate the effect of a change in USMLE Step 1 timing on Step 2 Clinical Knowledge (CK) scores, the effect of lag-time on Step 2 CK performance, and the relationship of incoming MCAT score to Step 2 CK performance pre- and post-change. METHOD/METHODS:Four LCME-accredited schools that moved Step 1 after core clerkships between academic years 2008-2009 and 2017-2018 were analyzed in a pre-post format. Standard t-tests were used to examine the change in Step 2 CK scores pre- and post-change. Tests of differences in proportions were used to evaluate whether Step 2 CK failure rates differed between curricular change groups. Linear regressions were used to examine the relationships between Step 2 CK performance, lag-time and incoming MCAT score, and curricular change group. RESULTS:Step 2 CK performance did not change significantly (P = .20). Failure rates remained highly consistent (pre-change: 1.83%, post-change: 1.79%). The regression indicated that lag-time had a significant effect on Step 2 CK performance, with scores declining with increasing lag-time. The regression yielded small but significant interaction effects between MCAT and Step 2 CK scores. Students with lower incoming MCATs tended to perform better on Step 2 CK when Step 1 was after clerkships. CONCLUSIONS:Moving Step 1 after core clerkships appears to have had no significant impact on Step 2 CK scores or failure rates, supporting the argument that such a change is noninferior to the traditional model. Students with lower MCAT scores benefit most from the change.

Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, since it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are used often as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of ¹³C₆-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with ¹³C₆-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homologue of the calcium-independent phospholipase A2β, on lipid metabolism. Although ¹³C₆-glucose gave rise to a complex pattern of ¹³C incorporation, we were able to identify discrete isotopomers in which ¹³C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.

OBJECTIVES/OBJECTIVE:This study aims to investigate the role and mechanism of FGFR3 in macrophages and their biological effects on the pathology of arthritis. METHODS:Mice with conditional knockout of FGFR3 in myeloid cells (R3cKO) were generated. Gait behaviours of the mice were monitored at different ages. Spontaneous synovial joint destruction was evaluated by digital radiographic imaging and μCT analysis; changes of articular cartilage and synovitis were determined by histological analysis. The recruitment of macrophages in the synovium was examined by immunostaining and monocyte trafficking assay. RNA-seq analysis, Western blotting and chemotaxis experiment were performed on control and FGFR3-deficient macrophages. The peripheral blood from non-osteoarthritis (OA) donors and patients with OA were analysed. Mice were treated with neutralising antibody against CXCR7 to investigate the role of CXCR7 in arthritis. RESULTS:R3cKO mice but not control mice developed spontaneous cartilage destruction in multiple synovial joints at the age of 13 months. Moreover, the synovitis and macrophage accumulation were observed in the joints of 9-month-old R3cKO mice when the articular cartilage was not grossly destructed. FGFR3 deficiency in myeloid cells also aggravated joint destruction in DMM mouse model. Mechanically, FGFR3 deficiency promoted macrophage chemotaxis partly through activation of NF-κB/CXCR7 pathway. Inhibition of CXCR7 could significantly reverse FGFR3-deficiency-enhanced macrophage chemotaxis and the arthritic phenotype in R3cKO mice. CONCLUSIONS:Our study identifies the role of FGFR3 in synovial macrophage recruitment and synovitis, which provides a new insight into the pathological mechanisms of inflammation-related arthritis.