Faculty Bibliography

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.

The purpose of this study was to assess the impact of COVID-19 on long-term outcomes in the geriatric hip fracture population. We hypothesize that COVID + geriatric hip fracture patients had worse outcomes at 1-year follow-up. Between February and June 2020, 224 patients > 55 years old treated for a hip fracture were analyzed for demographics, COVID status on admission, hospital quality measures, 30- and 90-day readmission rates, 1-year functional outcomes (as measured by the EuroQol- 5 Dimension [EQ5D-3L] questionnaire), and inpatient, 30-day, and 1-year mortality rates with time to death. Comparative analyses were conducted between COVID + and COVID- patients. Twenty-four patients (11%) were COVID + on admission. No demographic differences were seen between cohorts. COVID + patients experienced a longer length of stay (8.58 ± 6.51 vs. 5.33 ± 3.09, p < 0.01) and higher rates of inpatient (20.83% vs. 1.00%, p < 0.01), 30-day (25.00% vs. 5.00%, p < 0.01), and 1-year mortality (58.33% vs. 18.50%, p < 0.01). There were no differences seen in 30- or 90-day readmission rates, or 1-year functional outcomes. While not significant, COVID + patients had a shorter average time to death post-hospital discharge (56.14 ± 54.31 vs 100.68 ± 62.12, p = 0.171). Pre-vaccine, COVID + geriatric hip fracture patients experienced significantly higher rates of mortality within 1 year post-hospital discharge. However, COVID + patients who did not die experienced a similar return of function by 1-year as the COVID- cohort.

Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.

Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNA_Ser

BACKGROUND/UNASSIGNED:Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS/UNASSIGNED: RESULTS/UNASSIGNED: CONCLUSIONS/UNASSIGNED:Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution. However, this has been hindered by the lack of direct, sensitive, and noninvasive tools. We developed a series of GRAB (G protein-coupled receptor activation‒based) sensors for detecting somatostatin (SST), corticotropin-releasing factor (CRF), cholecystokinin (CCK), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors, which enable detection of specific neuropeptide binding at nanomolar concentrations, establish a robust tool kit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

INTRODUCTION/BACKGROUND:The purpose of this study was to determine what effect, if any, concomitant deformity correction has on outcomes following femoral nonunion repair. METHODS:605 consecutive patients who presented to our center with a long bone nonunion treated by one of 3 surgeons was queried. Sixty-two patients (10 %) with complete follow up were treated for a fracture nonunion following a Type 32 femur fracture (subtrochanteric, femoral shaft or distal third metaphysis) over an 11-year period. Twenty of these patients underwent a deformity correction (DC)-angular, rotational, or a combination of both-as part of their femoral reconstruction. Patient demographics and initial injury information was reviewed and compared. Outcomes including radiographic healing, time to union, postoperative complications, patient reported pain scores, and functional outcome scores using the Short Musculoskeletal Functional Assessment (SMFA) were recorded. Patients with and without deformity correction were analyzed and compared using independent T-tests and Chi-Square tests. RESULTS:Compared to the non-deformity correction (NDC) cohort, the DC cohort demonstrated a worse complication profile. Notably, the DC cohort had longer time to union (11.6 ± 7.3 months vs 7.6 ± 8.5 months, P = 0.042), reported significantly higher VAS pain scores at 1-year post-op (4.2 ± 2.8 vs 2.3 ± 2.6, P = 0.007), experienced more complications (25 % vs 4.8 %, P = 0.019), and had a higher rate of secondary procedures (30 % vs 4.8 %, P = 0.006). The DC patients reported less improvement in functional capability as displayed by a smaller average improvement in initial and final SMFA scores (P = 0.042) There was no difference in ultimate bone healing (P = 0.585), baseline SMFA (P = 0.294), and latest SMFA (P = 0.066). CONCLUSION/CONCLUSIONS:Deformity correction, if needed as part of femoral nonunion repair, is associated with an increased time to heal, greater rate of complications and diminished improvement of functionality. Eventual healing and patient reported outcomes were similar whether a deformity correction is necessary or not. LEVEL OF EVIDENCE/METHODS:III.

Rapid and conditional protein depletion is the gold standard genetic tool for deciphering the molecular basis of developmental processes. Previously, we showed that by conditionally expressing the E3 ligase substrate adaptor ZIF-1 in Caenorhabditis elegans somatic cells, proteins tagged with the first CCCH Zn finger 1 (ZF1) domain from the germline regulator PIE-1 degrade rapidly, resulting in loss-of-function phenotypes. The described role of ZIF-1 is to clear PIE-1 and several other CCCH Zn finger proteins from early somatic cells, helping to enrich them in germline precursor cells. Here, we show that proteins tagged with the PIE-1 ZF1 domain are subsequently cleared from primordial germ cells (PGCs) in embryos and from undifferentiated germ cells in larvae and adults by ZIF-1. We harness germline ZIF-1 activity to degrade a ZF1-tagged fusion protein from PGCs and show that its depletion produces phenotypes equivalent to those of a null mutation. Our findings reveal that ZIF-1 transitions from degrading CCCH Zn finger proteins in somatic cells to clearing them from undifferentiated germ cells, and that ZIF-1 activity can be harnessed as a new genetic tool to study the early germline.