Faculty Bibliography

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.¹.

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.

OBJECTIVE:To report on demographics, injury patterns, management strategies and outcomes of patients who sustained fractures of the tibial plateau seen at a single center over a 16-year period. DESIGN/METHODS:Prospective collection of data.Patients/ Participants: 716 patients with 725 tibia plateau fractures, were treated by one of 5 surgeons. INTERVENTION/METHODS:Treatment of tibial plateau fractures. MAIN OUTCOME MEASUREMENTS/METHODS:Outcomes were obtained at standard timepoints. Complications were recorded. Patients were stratified into 3 groups: those treated in the first 5 years, those treated in the second 5 years and those treated in the most recent 6 years. RESULTS:608 fractures were followed for a mean 13.4 months (6-120) and 82% had a minimum 1-year follow up. Patients returned to self-reported baseline function at a consistent proportion during the 3 time periods. The average knee arc was 125 degrees (75 - 135 degrees) at latest follow up and did not differ over time. The overall complication rate following surgery was 12% and did not differ between time periods. Radiographs demonstrated excellent rates of healing and low rates of PTOA and improved articular reductions at healing (0.58 mm in group 3 compared to 0.94 mm in Group 1 and 1.12 mm in Group 2) (P<0.05). CONCLUSION/CONCLUSIONS:The majority of patients regained their baseline functional status following surgical intervention and healing. Over time the ability of surgeons to achieve a more anatomic joint reduction was seen, however this did not correlate with improved functional outcomes. LEVEL OF EVIDENCE/METHODS:Prognostic Level I. See Instructions for Authors for a complete description of levels of evidence.

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.

PURPOSE/OBJECTIVE:To determine the factors associated with discharge location in patients with hip fractures and whether home discharge was associated with a lower readmission and complication rate. METHODS:Hip fracture patients who presented to our academic medical center for operative management of a hip fracture were enrolled into an IRB-approved hip fracture database. Radiographs, demographics, and injury details were recorded at the time of presentation. Patients were grouped based upon discharge disposition: home (with or without home services), acute rehabilitation facility (ARF), or sub-acute rehabilitation facility (SAR). RESULTS:The cohorts differed in marital status, with a greater proportion of patients discharged to home being married (51.7% vs. 43.8% vs. 34.1%) (P < 0.05). Patients discharged to home were less likely to require an assistive device (P < 0.05). Patients discharged to home experienced fewer post-operative complications (P < 0.05) and had lower readmission rates (P < 0.05). Being married was associated with an increased likelihood of discharge to home (OR = 1.679, CI = 1.391-2.028, P < 0.001). Being enrolled in Medicare/Medicaid was associated with decreased odds of discharge to home (OR = 0.563, CI = 0.457-0.693, P < 0.001). Use of an assistive device was associated with decreased odds of discharge to home (OR = 0.398, CI = 0.326-0.468, P < 0.001). Increases in CCI (OR = 0.903, CI = 0.846-0.964, P = 0.002) and number of inpatient complications (OR = 0.708, CI = 0.532-0.943, P = 0.018) were associated with decreased odds of home discharge. CONCLUSION/CONCLUSIONS:Hip fracture patients discharged to home were healthier and more functional at baseline, and also less likely to have had a complicated hospital course. Those discharged to home also had lower rates of readmission and post-operative complications. LEVEL OF EVIDENCE/METHODS:III.

OBJECTIVE:Rejection is common and pernicious following Vascularized Composite Allotransplantation (VCA). Current monitoring and diagnostic modalities include the clinical exam which is subjective and biopsy with dermatohistopathologic Banff grading, which is subjective and invasive. We reviewed literature exploring non- and minimally invasive modalities for diagnosing and monitoring rejection (NIMMs) in VCA. METHODS:PubMed, Cochrane, and Embase databases were queried, 3125 unique articles were reviewed, yielding 26 included studies exploring 17 distinct NIMMs. Broadly, NIMMs involved Imaging, Liquid Biomarkers, Epidermal Sampling, Clinical Grading Scales, and Introduction of Additional Donor Tissue. RESULTS:Serum biomarkers including MMP3 and donor-derived microparticles rose with rejection onset. Epidermal sampling non-invasively enabled measurement of cytokine & gene expression profiles implicated in rejection. Both hold promise for monitoring. Clinical grading scales were useful diagnostically as was reflection confocal microscopy. Introducing additional donor tissue showed promise for preemptively identifying rejection but requires additional allograft tissue burden for the recipient. CONCLUSION/CONCLUSIONS:NIMMs have the potential to dramatically improve monitoring and diagnosis in VCA. Many modalities show promise however, additional research is needed and a multimodal algorithmic approach should be explored.

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.

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.