Faculty Bibliography

The improvements accomplished in assisted reproductive technology have emphasized more than ever the role played by chronological age, notably for predicting oocyte quality. Studies in cellular aging have directed research on telomere length measurements as possible markers of functional aging and, notably, female reproductive outcomes. Although further research is still needed, encouraging results are already available on the possibility that leucocyte telomere length may be a useful parameter for assessing reproductive potential in aging women.

The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include the accumulation of oxidative damage, mitochondrial dysfunction, and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult-derived from the last oogonia to exit mitotic replication in the fetus. On the basis of this, we proposed a two-hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit was that oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit was that oocytes accumulated more environmental and endogenous oxidative damage throughout the life of the woman. Telomeres (Ts) could mediate both of these aspects of oocyte aging. Telomeres provide a "mitotic clock," with T attrition an inevitable consequence of cell division because of the end replication problem. Telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in postmitotic cells. Telomerase, the reverse transcriptase that restores Ts, is better at maintaining than elongating T. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short Ts, on the brink of viability. In support of this theory, mice with induced T attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development, and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest Ts in the body. In mice, cleavage-stage embryos elongate Ts via "alternative lengthening of telomeres," a recombination-based mechanism rarely encountered outside of telomerase-deficient cancers. Many questions about Ts and reproduction are raised by these findings: does the "normal" T attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic nondisjunction? Does recombination-based T elongation render embryos susceptible to mitotic nondisjunction (and mosaicism)? Can some features of Ts serve as markers of oocyte quality?

OBJECTIVE:To compare the outcomes of patients with segmental bone loss who underwent repair with the induced membrane technique (IMT) with a matched cohort of nonunion fractures without bone loss. DESIGN/METHODS:Retrospective analysis on prospectively collected data. SETTING/METHODS:Academic medical center. PATIENTS/METHODS:Two cohorts of patients, those with upper and lower extremity diaphyseal large segmental bone loss and those with ununited fractures, were enrolled prospectively between 2013 and 2020. Sixteen patients who underwent repair of 17 extremities with segmental diaphyseal or meta-diaphyseal bone defects treated with the induced membrane technique were identified, and matched with 17 patients who were treated for 17 fracture nonunions treated without an induced membrane. Sixteen of the bone defects treated with the induced membrane technique were due to acute bone loss, and the other was a chronic aseptic nonunion. MAIN OUTCOME MEASUREMENTS/METHODS:Healing rate, time to union, functional outcome scores using the Short Musculoskeletal Functional Assessment (SMFA) and pain assessed by the Visual Analog Scale (VAS). RESULTS:The initial average defect size for patients treated with the induced membrane technique was 8.85 cm. Mean follow-up times were similar with 17.06 ± 10.13 months for patients treated with the IMT, and 20.35 ± 16.68. months for patients treated without the technique. Complete union was achieved in 15/17 (88.2%) of segmental bone loss cases treated with the IMT and 17/17 (100%) of cases repaired without the technique at the latest follow up visit. The average time to union for patients treated with the induced membrane technique was 13.0 ± 8.4 months and 9.64 ± 4.7 months for the matched cohort. There were no significant differences in reported outcomes measured by the SMFA or VAS. Patients treated with the induced membrane technique required more revision surgeries than those not treated with an induced membrane. CONCLUSION/CONCLUSIONS:Outcomes following treatment of acute bone loss from the diaphysis of long bones with the induced membrane technique produces clinical and radiographic outcomes similar to those of long bone fracture nonunions without bone loss that go on to heal. LEVEL OF EVIDENCE/METHODS:III.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain¹. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes², their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Na_v1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Na_v1.7 channels, with a density of 0.1 to 0.15 channels per µm² and 350 to 525 channels per cell. Serial genetic ablation of Na_v1.7 in multiple mouse models demonstrates that Na_v1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Na_v1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Na_v1.7 with selective or clinically used pan-Na_v channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Na_v1.7 blockers regulate intracellular Ca²⁺ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Na_v1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

BACKGROUND:Essential patterning processes transform the heart tube into a compartmentalized organ with distinct chambers separated by an atrioventricular canal (AVC). This transition involves the refinement of expression of genes that are first found broadly throughout the heart tube and then become restricted to the AVC. Despite the importance of cardiac patterning, we do not fully understand the mechanisms that limit gene expression to the AVC. RESULTS:We show that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, is critical for cardiac patterning. In smarcc1a mutants, myocardial differentiation and heart tube assembly appear to proceed normally. Subsequently, the smarcc1a mutant heart fails to exhibit refinement of gene expression patterns to the AVC, and the persistence of broad gene expression is accompanied by failure of chamber expansion. In addition to their cardiac defects, smarcc1a mutants lack pectoral fins, indicating similarity to tbx5a mutants. However, comparison of smarcc1a and tbx5a mutants suggests that perturbation of tbx5a function is not sufficient to cause the smarcc1a mutant phenotype. CONCLUSIONS:Our data indicate an important role for Smarcc1a-containing chromatin remodeling complexes in regulating the changes in gene expression and morphology that distinguish the AVC from the cardiac chambers. This article is protected by copyright. All rights reserved.

There has been a paradigm shift towards fixing the posterior malleolus in trimalleolar ankle fractures. This study evaluated whether a surgeon's preference to intraoperatively flip or not flip patients from prone to supine for medial malleolar fixation following repair of fibular and posterior malleoli impacted surgical outcomes. A retrospective patient cohort treated at a large urban academic center and level 1 trauma center was reviewed to identify all operative trimalleolar ankle fractures initially positioned prone. One hundred and forty-seven patients with mean 12-month follow up were included and divided based on positioning for medial malleolar fixation, prone or supine (following closure, flip and re-prep and drape). Data was collected on patient demographics, injury mechanism, perioperative variables, and complication rates. Postoperative reduction films were reviewed by orthopedic traumatologists to grade the accuracy of anatomic fracture reduction. Overall, 74 (50.3%) had the medial malleolus fixed prone, while 73 (49.7%) were flipped and fixed supine. No differences in demographics, injury details, and fracture type existed between the groups. The supine group had a higher rate of initial external fixation (p=0.047), longer operative time in minutes (p<0.001), and a higher use of plate and screw constructs for medial malleolar fixation (p=0.019). There were no differences in clinical and radiographic outcomes and complication rates. This study demonstrated that intraoperative change in positioning for improved medial malleolar visualization in trimalleolar ankle fractures results in longer operative times but similar radiographic and clinical results. The decision of operative position should be based on surgeon comfort.

The gastrointestinal ecosystem has received the most attention when examining the contributions of the human microbiome to health and disease. This concentration of effort is logical due to the overwhelming abundance of microbes in the gut coupled with the relative ease of sampling compared with other organs. However, the intestines are intimately connected to multiple extraintestinal organs, providing an opportunity for homeostatic microbial colonisation and pathogenesis in organs traditionally thought to be sterile or only transiently harbouring microbiota. These habitats are challenging to sample, and their low microbial biomass among large amounts of host tissue can make study challenging. Nevertheless, recent findings have shown that many extraintestinal organs that are intimately linked to the gut harbour stable microbiomes, which are colonised from the gut in selective manners and have highlighted not just the influence of the bacteriome but that of the mycobiome and virome on oncogenesis and health.

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.