Faculty Bibliography

Microsporidia are divergent fungal pathogens that employ a unique harpoon-like apparatus called the polar tube (PT) to invade host cells. The long PT is fired out of the microsporidian spore over the course of just a few hundred milliseconds. Once fired, the PT is thought to pierce the plasma membrane of a target cell and act as a conduit for the transfer of the parasite into the host cell, which initiates infection. The PT architecture and its association with neighboring organelles within the parasite cell remain poorly understood. Here, we use cryoelectron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis. Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane layer and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryoelectron tomography with cellular modeling, we propose a model for the three-dimensional organization of the polaroplast, an organelle that surrounds the PT and is continuous with the outermost, membranous layer of the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ, laying the foundation for understanding infection mechanisms.

Glaucomatous optic neuropathy, or glaucoma, is the world's primary cause of irreversible blindness. Glaucoma is comorbid with other neurodegenerative diseases, but how it might impact the environment of the full central nervous system to increase neurodegenerative vulnerability is unknown. Two neurodegenerative events occur early in the optic nerve, the structural link between the retina and brain: loss of anterograde transport in retinal ganglion cell (RGC) axons and early alterations in astrocyte structure and function. Here, we used whole-mount tissue clearing of full mouse brains to image RGC anterograde transport function and astrocyte responses across retinorecipient regions early in a unilateral microbead occlusion model of glaucoma. Using light sheet imaging, we found that RGC projections terminating specifically in the accessory optic tract are the first to lose transport function. Although degeneration was induced in one retina, astrocytes in both brain hemispheres responded to transport loss in a retinotopic pattern that mirrored the degenerating RGCs. A subpopulation of these astrocytes in contact with large descending blood vessels were immunopositive for LCN2, a marker associated with astrocyte reactivity. Together, these data suggest that even early stages of unilateral glaucoma have broad impacts on the health of astrocytes across both hemispheres of the brain, implying a glial mechanism behind neurodegenerative comorbidity in glaucoma.

INTRODUCTION/UNASSIGNED:Alkyl nitrites ("poppers") are used recreationally for sexual enhancement, muscle relaxation, and euphoria. However, they can be toxic and cause adverse reactions such as methemoglobinemia. While inhalation is the typical route of usage, the New York City Poison Center has noted an increase in calls related to ingestion. Given the uncertainty of usage instructions at the point of sale, our study aimed to assess instructions provided to consumers about alkyl nitrite use and to evaluate the proximity and co-sale of alkyl nitrites with similarly appearing energy drink shots. METHODS/UNASSIGNED:We conducted a cross-sectional convenience sample survey of smoke shops, cannabis dispensaries, and exotic shops within the catchment area of an urban poison center. Plain clothes "investigators" (i.e., the researchers) visited these retailers and followed a predetermined protocol and script to request information regarding the availability and usage of alkyl nitrites. Additionally, the researchers attempted to visually assess the proximity of alkyl nitrites to similarly appearing energy drink shots during their visit. RESULTS/UNASSIGNED:drinks and alkyl nitrites were located near these energy drink shots in twenty (39%) of these fifty-one stores. DISCUSSION/UNASSIGNED:Many commercial alkyl nitrite retailers in our survey area lack knowledge or provide potentially inaccurate information regarding the use of alkyl nitrites. Additionally, alkyl nitrites are often sold alongside commercial energy drinks, potentially increasing the risk of incidental ingestion. CONCLUSIONS/UNASSIGNED:Further research is necessary to determine the impact of these patterns of sale and potential misinformation. Discussing preliminary results of our survey with the New York City Department of Health led to the rapid creation of an informational poster and local outreach. Clinicians should report cases of alkyl nitrite use to their regional poison center to allow for more targeted and timely public health intervention.

Embryonic development in many species, including case reports in humans, can be temporarily halted before implantation during a process called diapause. Facultative diapause occurs under conditions of maternal metabolic stress such as nursing. While molecular mechanisms of diapause have been studied, a natural inducing factor has yet to be identified. Here, we show that oxytocin induces embryonic diapause in mice. We show that gestational delays were triggered during nursing or optogenetic stimulation of oxytocin neurons simulating nursing patterns. Mouse blastocysts express oxytocin receptors, and oxytocin induced delayed implantation-like dispersion in cultured embryos. Last, oxytocin receptor-knockout embryos transferred into wild-type surrogates had low survival rates during diapause. Our results indicate that oxytocin coordinates timing of embryonic development with uterine progression through pregnancy, providing an evolutionarily conserved mechanism for ensuring successful reproduction.

The human high-affinity sodium-dicarboxylate cotransporter (NaDC3) imports various substrates into the cell as tricarboxylate acid cycle intermediates, lipid biosynthesis precursors and signaling molecules. Understanding the cellular signaling process and developing inhibitors require knowledge of the structural basis of the dicarboxylate specificity and inhibition mechanism of NaDC3. To this end, we determined the cryo-electron microscopy structures of NaDC3 in various dimers, revealing the protomer in three conformations: outward-open C_o, outward-occluded C_oo and inward-open C_i. A dicarboxylate is first bound and recognized in C_o and how the substrate interacts with NaDC3 in C_oo likely helps to further determine the substrate specificity. A phenylalanine from the scaffold domain interacts with the bound dicarboxylate in the C_oo state and modulates the kinetic barrier to the transport domain movement. Structural comparison of an inhibitor-bound structure of NaDC3 to that of the sodium-dependent citrate transporter suggests ways for making an inhibitor that is specific for NaDC3.

High-dimensional multiplexed imaging can reveal the spatial organization of tumour tissues at the molecular level. However, owing to the scale and information complexity of the imaging data, it is challenging to discover and thoroughly characterize the heterogeneity of tumour microenvironments. Here we show that self-supervised representation learning on data from imaging mass cytometry can be leveraged to distinguish morphological differences in tumour microenvironments and to precisely characterize distinct microenvironment signatures. We used self-supervised masked image modelling to train a vision transformer that directly takes high-dimensional multiplexed mass-cytometry images. In contrast with traditional spatial analyses relying on cellular segmentation, the vision transformer is segmentation-free, uses pixel-level information, and retains information on the local morphology and biomarker distribution. By applying the vision transformer to a lung-tumour dataset, we identified and validated a monocytic signature that is associated with poor prognosis.

The field of cardio-oncology has traditionally focused on the impact of cancer and its therapies on cardiovascular health. Mounting clinical and preclinical evidence, however, indicates that the reverse may also be true: cardiovascular disease can itself influence tumor growth and metastasis. Numerous epidemiological studies have reported that individuals with prevalent cardiovascular disease have an increased incidence of cancer. In parallel, studies using preclinical mouse models of myocardial infarction, heart failure, and cardiac remodeling support the notion that cardiovascular disorders accelerate the growth of solid tumors and metastases. These findings have ushered in a new and burgeoning field termed reverse cardio-oncology that investigates the impact of cardiovascular disease pathophysiology on cancer emergence and progression. Recent studies have begun to illuminate the mechanisms driving this relationship, including shared risk factors, reprogramming of immune responses, changes in gene expression, and the release of cardiac factors that result in selective advantages for tumor cells or their local milieu, thus exacerbating cancer pathology. Here, we review the evidence supporting the relationship between cardiovascular disease and cancer, the mechanistic pathways enabling this connection, and the implications of these findings for patient care.

The discovery of long non-coding RNAs (lncRNAs) has provided a new perspective on the centrality of RNA in gene regulation and genome organization. Here, we screened for lncRNAs with putative functions in the host response to single-stranded RNA respiratory viruses. We identify CARINH as a conserved cis-acting lncRNA up-regulated in three respiratory diseases to control the expression of its antisense gene IRF1, a key transcriptional regulator of the antiviral response. CARINH and IRF1 are coordinately increased in the circulation of patients infected with human metapneumovirus, influenza A virus, or SARS-CoV-2, and in macrophages in response to viral infection or TLR3 agonist treatment. Targeted depletion of CARINH or its mouse ortholog Carinh in macrophages reduces the expression of IRF1/Irf1 and their associated target gene networks, increasing susceptibility to viral infection. Accordingly, CRISPR-mediated deletion of Carinh in mice reduces antiviral immunity, increasing viral burden upon sublethal challenge with influenza A virus. Together, these findings identify a conserved role of lncRNA CARINH in coordinating interferon-stimulated genes and antiviral immune responses.

Cytokinins are adenine-based hormones that have been well-characterized in plants but are also made by bacteria, including the human-exclusive pathogen Mycobacterium tuberculosis. Like plants, M. tuberculosis uses cytokinins to regulate gene expression. We previously established that cytokinin overaccumulation in M. tuberculosis results in a buildup of aldehydes produced during cytokinin breakdown. In plants, dedicated enzymes called cytokinin oxidases convert cytokinins into adenine and various aldehydes. Proteasome degradation-deficient M. tuberculosis, which cannot degrade the cytokinin-producing enzyme Log, accumulates several cytokinins and at least one cytokinin-associated aldehyde, resulting in increased sensitivity to nitric oxide and copper. We therefore hypothesized that M. tuberculosis encodes one or more cytokinin oxidases, and disruption of this enzyme might restore resistance to nitric oxide and copper in a proteasome-defective strain. Using a homology-based search, we identified Rv3719 as a protein with high similarity to a plant cytokinin oxidase. Deletion of this gene, however, did not restore nitric oxide or copper resistance to a degradation-defective mutant. Instead, we observed increased copper sensitivity when Rv3719 was deleted from either wild-type or proteasome-defective strains. Finally, we characterized Rv3718c, a protein encoded adjacent to Rv3719, and found that it bound a cytokinin with high specificity. Collectively, these data support a role for cytokinin activity in M. tuberculosis physiology that remains to be further elucidated.IMPORTANCENumerous bacterial species encode cytokinin-producing enzymes, the functions of which are almost completely unknown. This work contributes new knowledge to the cytokinin field for bacteria and reveals further conservation of cytokinin-associated proteins between plants and prokaryotes.

OBJECTIVE:The purpose of this study was to determine the association of hypoalbuminemia with adverse outcomes in patients undergoing surgical repair of nonunions or malunions of upper and lower extremity long bones. METHODS:DESIGN: Retrospective. SETTING/METHODS:Hospitals participating in American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) from 2005 to 2019. PATIENTS/METHODS:Patients in the ACS-NSQIP database with upper extremity and lower extremity fractures who underwent nonunion or malunion repairs and had preoperative serum albumin levels. Outcome Measures and Comparisons: Demographic variables, comorbidities and postoperative complications were collected and compared using t tests and chi squared tests. Multivariate linear regression models were used to assess complications, adjusting for variables such as age, sex, BMI, hospital length of stay, and operation time. RESULTS:Univariate analysis of 1640 total patients (338 [20.6%] with hypoalbuminemia and 1302 [79.4%] with normal albumin) showed patients with hypoalbuminemia had significantly increased 30-day mortality rates, increased lengths of stay, and returns to the operating room. Multivariate analysis showed patients with hypoalbuminemia had significantly greater odds for any complication (OR: 2.62; 95% CI [1.77, 3.84]; p < 0.001), surgical site infections (OR: 2.62; 95% CI [1.34, 4.99]; p = 0.004) and transfusions (OR: 2.77; 95% CI: [1.62, 4.69]; p < 0.001) compared to the normal albumin group. CONCLUSIONS:There was a significant difference in 30-day postoperative complications between patients with normal albumin levels and those who were hypoalbuminemic after surgical repairs of nonunions or malunions. Albumin level is a risk factor that should be monitored and counseled upon prior to surgical intervention for nonunion or malunion correction. LEVEL OF EVIDENCE/METHODS:Level III Retrospective Comparative Study.