Faculty Bibliography

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.

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.

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.

Auxins are pivotal plant hormones that regulate plant growth and transmembrane polar auxin transport (PAT) direct patterns of development. The PIN-FORMED (PIN) family of membrane transporters mediate auxin export from the plant cell and play crucial roles in PAT. Here we describe the recently solved structures of PIN transporters, PIN1, PIN3, and PIN8, and also their mechanisms of substrate recognition and transport of auxin. We compare structures of PINs in both inward- and outward-facing conformations, as well as PINs with different binding configurations for auxin. By this comparative analysis, a model emerges for an elevator transport mechanism. Central structural elements necessary for function are identified, and we show that these are shared with other distantly related protein families.

Hydrogels with long-term storage stability, controllable sustained-release properties, and biocompatibility have been garnering attention as carriers for drug/growth factor delivery in tissue engineering applications. Chitosan (CS)/Graphene Oxide (GO)/Hydroxyethyl cellulose (HEC)/β-glycerol phosphate (β-GP) hydrogel is capable of forming a 3D gel network at physiological temperature (37 °C), rendering it an excellent candidate for use as an injectable biomaterial. This work focused on an injectable thermo-responsive CS/GO/HEC/β-GP hydrogel, which was designed to deliver Atsttrin, an engineered derivative of a known chondrogenic and anti-inflammatory growth factor-like molecule progranulin. The combination of the CS/GO/HEC/β-GP hydrogel and Atsttrin provides a unique biochemical and biomechanical environment to enhance fracture healing. CS/GO/HEC/β-GP hydrogels with increased amounts of GO exhibited rapid sol-gel transition, higher viscosity, and sustained release of Atsttrin. In addition, these hydrogels exhibited a porous interconnected structure. The combination of Atsttrin and hydrogel successfully promoted chondrogenesis and osteogenesis of bone marrow mesenchymal stem cells (bmMSCs) in vitro. Furthermore, the work also presented in vivo evidence that injection of Atsttrin-loaded CS/GO/HEC/β-GP hydrogel stimulated diabetic fracture healing by simultaneously inhibiting inflammatory and stimulating cartilage regeneration and endochondral bone formation signaling pathways. Collectively, the developed injectable thermo-responsive CS/GO/HEC/βG-P hydrogel yielded to be minimally invasive, as well as capable of prolonged and sustained delivery of Atsttrin, for therapeutic application in impaired fracture healing, particularly diabetic fracture healing.

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.

We present single-shot high-performance quantitative phase imaging with a physics-inspired plug-and-play denoiser for polarization differential interference contrast (PDIC) microscopy. The quantitative phase is recovered by the alternating direction method of multipliers (ADMM), balancing total variance regularization and a pre-trained dense residual U-net (DRUNet) denoiser. The custom DRUNet uses the Tanh activation function to guarantee the symmetry requirement for phase retrieval. In addition, we introduce an adaptive strategy accelerating convergence and explicitly incorporating measurement noise. After validating this deep denoiser-enhanced PDIC microscopy on simulated data and phantom experiments, we demonstrated high-performance phase imaging of histological tissue sections. The phase retrieval by the denoiser-enhanced PDIC microscopy achieves significantly higher quality and accuracy than the solution based on Fourier transforms or the iterative solution with total variance regularization alone.