Faculty Bibliography

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C₂-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

OBJECTIVE:The purpose of this study was to perform a network meta-analysis of level I and II evidence comparing different management techniques to define the optimum treatment method for humeral shaft fractures (HSFs). DATA SOURCES/METHODS:A systematic review of the literature using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines of MEDLINE, Embase, and Cochrane Library was screened from 2010 to 2023. STUDY SELECTION/METHODS:Inclusion criteria were evidence level I or II studies comparing nonoperative and/or operative repair techniques including open reduction internal fixation plate osteosynthesis (ORIF-Plate), minimally invasive percutaneous plating (MIPO), and intramedullary nail (IMN) fixation for the management of HSFs (OTA/AO 12A, B, C). DATA EXTRACTION/METHODS:The risk of bias and methodologic quality of evidence were assessed according to the guidelines designed by the Cochrane Statistical Methods Group and Cochrane Methods Bias Group. DATA SYNTHESIS/RESULTS:Network meta-analysis was conducted with a frequentist approach with a random-effects model using the netmeta package version 0.9-6 in R. RESULTS:A total of 25 studies (1908 patients) were included. MIPO resulted in the lowest complication rate (2.1%) when compared with ORIF-Plate (16.1%) [odds ratio (OR), 0.13; 95% confidence interval (CI), 0.04-0.49]. MIPO resulted in the lowest nonunion rate (0.65%) compared with all management techniques (OR, 0.28; 95% CI, 0.08-0.98), whereas Non-Op resulted in the highest (15.87%) (OR, 3.48; 95% CI, 1.98-6.11). MIPO demonstrated the lowest rate of postoperative radial nerve palsy overall (2.2%) and demonstrated a significantly lower rate compared with ORIF-Plate (OR, 0.22; 95% CI, 0.07-0.71, P = 0.02). IMN resulted in the lowest rate of deep infection (1.1%) when compared with ORIF-Plate (8.6%; P = 0.013). MIPO resulted in a significantly lower Disabilities of the Arm, Shoulder, and Hand score (3.86 ± 5.2) and higher American Shoulder and Elbow Surgeons score (98.2 ± 1.4) than ORIF-Plate (19.5 ± 9.0 and 60.0 ± 5.4, P < 0.05). CONCLUSION/CONCLUSIONS:The results from this study support that surgical management results in better postoperative functional outcomes, leads to higher union rates, reduces fracture healing time, reduces revision rate, and decreases malunion rates in patients with HSFs. In addition, MIPO resulted in statistically higher union rates, lowest complication rate, lowest rate of postoperative radial nerve palsy, and lower intraoperative time while resulting in better postoperative Disabilities of the Arm, Shoulder, and Hand and American Shoulder and Elbow Surgeons scores when compared with nonoperative and operative (ORIF and IMN) treatment modalities. LEVEL OF EVIDENCE/METHODS:Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

The hair follicle and nail unit develop and regenerate through epithelial-mesenchymal interactions. Here, we review some of the key signals and molecular interactions that regulate mammalian hair follicle and nail formation during embryonic development and how these interactions are reutilized to promote their regeneration during adult homeostasis and in response to skin wounding. Finally, we highlight the role of some of these signals in mediating human hair follicle and nail conditions.

Infants are highly susceptible to invasive respiratory and gastrointestinal infections. To elucidate the age-dependent mechanism(s) that drive bacterial spread from the mucosa, we developed an infant mouse model using the prevalent pediatric respiratory pathogen, Streptococcus pneumoniae (Spn). Despite similar upper respiratory tract (URT) colonization levels, the survival rate of Spn-infected infant mice was significantly decreased compared to adults and corresponded with Spn dissemination to the bloodstream. An increased rate of pneumococcal bacteremia in early life beyond the newborn period was attributed to increased bacterial translocation across the URT barrier. Bacterial dissemination in infant mice was independent of URT monocyte or neutrophil infiltration, phagocyte-derived ROS or RNS, inflammation mediated by toll-like receptor 2 or interleukin 1 receptor signaling, or the pore-forming toxin pneumolysin. Using molecular barcoding of Spn, we found that only a minority of bacterial clones in the nasopharynx disseminated to the blood in infant mice, indicating the absence of robust URT barrier breakdown. Rather, transcriptional profiling of the URT epithelium revealed a failure of infant mice to upregulate genes involved in the tight junction pathway. Expression of many such genes was also decreased in early life in humans. Infant mice also showed increased URT barrier permeability and delayed mucociliary clearance during the first two weeks of life, which corresponded with tighter attachment of bacteria to the respiratory epithelium. Together, these results demonstrate a window of vulnerability during postnatal development when altered mucosal barrier function facilitates bacterial dissemination.

GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca²⁺-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca²⁺-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca²⁺ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca²⁺, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.