Faculty Bibliography

To determine if radiographic evidence of posttraumatic osteoarthritis (PTOA) following tibial plateau fracture correlates with poorer clinical and functional outcomes, patients with tibial plateau fractures were followed at 3, 6, and 12 months. All patients had baseline radiographs and computed tomography scan. Radiographs obtained at each follow-up were reviewed for healing, articular incongruence, hardware positional changes, and the development of postinjury arthritic change. Cohorts were determined based on the presence (PTOA) or absence (NPTOA) of radiographic evidence of PTOA. Demographics, fracture classification, complications, additional procedures, and functional status were compared between cohorts. Sixty patients had radiographic evidence of PTOA on follow-up radiographs at a mean final follow-up of 24.2 months. The NPTOA cohort was composed of 210 patients who were matched to the PTOA cohort based on age and Charlson comorbidity index. Mean time to fracture union for the overall cohort was 4.86 months. Cohorts did not differ in Schatzker classification, time to healing, injury mechanism, or baseline Short Musculoskeletal Function Assessment (SMFA). Patients with PTOA had a greater degree of initial depression and postoperative step-off, higher incidence of initial external fixator usage, higher rates of reoperation for any reason, and higher rates of wound complications. Associated soft tissue injury and meniscal repair did not coincide with the development of PTOA. Range of motion and SMFA scores were significantly worse at all time points in patients with PTOA. Although fracture patterns are similar, patients who required an initial external fixator, had a greater degree of initial depression or residual articular incongruity, underwent more procedures, and developed an infection were found to have increased incidence of PTOA. Radiographic evidence of osteoarthritis correlated with worse functional status in patients. The goal of surgery should be restoration of articular congruity and stability to mitigate the risk of PTOA, although this alone may not prevent degenerative changes. Patients with early loss of range of motion should be aggressively treated as this may precede the development of PTOA.

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering¹ to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

BACKGROUND:Developing genomic resources for a diverse range of species is an important step towards understanding the mechanisms underlying complex traits. Specifically, organisms that exhibit unique and accessible phenotypes-of-interest allow researchers to address questions that may be ill-suited to traditional model organisms. We sequenced the genome and transcriptome of Alston's singing mouse (Scotinomys teguina), an emerging model for social cognition and vocal communication. In addition to producing advertisement songs used for mate attraction and male-male competition, these rodents are diurnal, live at high-altitudes, and are obligate insectivores, providing opportunities to explore diverse physiological, ecological, and evolutionary questions. RESULTS:Using PromethION, Illumina, and PacBio sequencing, we produced an annotated genome and transcriptome, which were validated using gene expression and functional enrichment analyses. To assess the usefulness of our assemblies, we performed single nuclei sequencing on cells of the orofacial motor cortex, a brain region implicated in song coordination, identifying 12 cell types. CONCLUSIONS:These resources will provide the opportunity to identify the molecular basis of complex traits in singing mice as well as to contribute data that can be used for large-scale comparative analyses.

Atherosclerosis is a common pathology present in many cardiovascular diseases. Although the current therapies (including statins and inhibitors of the serine protease PCSK9) can effectively reduce low-density lipoprotein (LDL) cholesterol levels to guideline-recommended levels, major adverse cardiovascular events still occur frequently. Indeed, the subendothelial retention of lipoproteins in the artery wall triggers multiple events of inflammation in macrophages and is a major contributor to the pathological progression of atherosclerosis. It has been gradually recognized that modulating inflammation is, therefore, an attractive avenue to forestall and treat atherosclerosis and its complications. Unfortunately, challenges with specificity and efficacy in managing plaque inflammation have hindered progress in atherosclerosis treatment. Herein, we report an NP-mediated mRNA therapeutic approach to target atherosclerotic lesional macrophages, modulating inflammation in advanced atherosclerotic lesions for the treatment of atherosclerosis. We demonstrated that the targeted NPs containing IL-10 mRNA colocalized with M2-like macrophages and induced IL-10 production in atherosclerotic plaques following intravenous administration to Western diet (WD)-fed Ldlr

OBJECTIVE:To determine if a peri-operative pain cocktail injection improves post-operative pain, ambulation distance and long-term outcomes in hip fracture patients. DESIGN/METHODS:Prospective, single-blinded, randomized controlled trial. SETTING/METHODS:Academic Medical Center. PATIENTS/PARTICIPANTS/METHODS:Patients with OTA/AO 31A1-3 and 31B1-3 fractures undergoing operative fixation, excluding arthroplasty. INTERVENTION/METHODS:Multimodal local injection of bupivacaine (Marcaine), morphine sulfate (Duramorph), ketorolac (Toradol) given at the fracture site at the time of hip fracture surgery (Hip Fracture Injection, HiFI). MAIN OUTCOME MEASUREMENTS/METHODS:Patient-reported pain, American Pain Society Patient Outcome Questionnaire (APS-POQ), narcotic usage, length of stay, post-operative ambulation, Short Musculoskeletal Function Assessment (SMFA). RESULTS:75 patients were in the treatment group and 109 in the control group. Patients in the HiFI group had a significant reduction in pain and narcotic usage compared to the control group on post-operative day (POD) 0 (p<0.01). Based on the APS-POQ, patients in the control group had a significantly harder time falling asleep, staying asleep, and experienced increased drowsiness on POD 1 (p<0.01). Patient ambulation distance was greater on POD 2 (p<0.01) and POD 3 (p<0.05) in the HiFI group. The control group experienced more major complications (p<0.05). At six-weeks post-op, patients in the treatment group reported significantly less pain, better ambulatory function, less insomnia, less depression, and better satisfaction than the control group as measured by the APS-POQ. The SMFA bothersome index was also significantly lower for patients in the HiFI group, p<0.05. CONCLUSIONS:Intraoperative HiFI not only improved early pain management and increased ambulation in patients undergoing hip fracture surgery while in the hospital, it was also associated with early improved health related quality of life following discharge. LEVEL OF EVIDENCE/METHODS:Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

Several phospholipid (PL) molecules are intertwined with some mitochondrial complex I (CI) subunits in the membrane domain of CI, but their function is unclear. We report that when the Drosophila melanogaster ortholog of the intramitochondrial PL transporter, STARD7, is severely disrupted, assembly of the oxidative phosphorylation (OXPHOS) system is impaired, and the biogenesis of several CI subcomplexes is hampered. However, intriguingly, a restrained knockdown of STARD7 impairs the incorporation of NDUFS5 and NDUFA1 into the proximal part of the CI membrane domain without directly affecting the incorporation of subunits in the distal part of the membrane domain, OXPHOS complexes already assembled, or mitochondrial cristae integrity. Importantly, the restrained knockdown of STARD7 appears to induce a modest amount of cardiolipin remodeling, indicating that there could be some alteration in the composition of the mitochondrial phospholipidome. We conclude that PLs can regulate CI biogenesis independent of their role in maintaining mitochondrial membrane integrity.