Faculty Bibliography

Objective: DMSO alters the epigenetic state of mouse oocytes and human cultured cells. The effect of vitrification with DMSO containing cryoprotectant on gene and TE expression in human oocytes is unknown.
Material(s) and Method(s): A prospective paired controlled cohort laboratory study was performed from February - June 2021. Twenty-four discarded oocytes in the germinal vesical (GV) stage were donated from four patients. All oocytes were paired such that half of the oocytes from each patient were vitrified with DMSO-containing cryoprotectant, while the other half were frozen, unexposed to DMSO. All oocytes underwent RNA sequencing via Switching Mechanism At the end of the 5'-end of the RNA Transcript sequencing 2 (SMARTseq2). Reads containing adapters, bases that could not be determined >10% and low quality reads were excluded. Gene mapping to the human reference genome, followed by gene quantification, were performed. Next, differential gene expression analysis between the two cohorts was performed. Then functional enrichment analyses of dysregulated gene sets were performed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Human Disease Ontology (DO). Raw data obtained from RNA sequencing was used to analyze TE transcripts using the BonaFide-TEseq method, which were mapped to specific TE loci using Software for Quantifying Interspersed Repeat Expression (SQuIRE), to identify differentially expressed TEs. Real time-qPCR validated results of selected genes and TEs.
Result(s): Of the 27,837 genes identified by SMARTseq2, 7,331 (26.3%) were differentially expressed (p<0.05). Specifically, 3,987 genes were upregulated and 3,344 genes were downregulated in oocytes exposed to DMSO. Genes involved in chromatin and histone modification, and mitochondrial function, as well as WNT, insulin, MTOR, HIPPO and MAPK signaling pathways were affected by DMSO. There was no significant over expression of human disease ontology terms within our data set. Expression of a number of TEs was also affected by exposure to DMSO, including Alu, endogenous retrovirus family members 1 and K (ERV1, ERVK) and long interspersed nuclear elements 1 (LINE-1). Notably, the effects of DMSO on TE expression were most pronounced in the oldest patient. The expression of TEs was negatively correlated with age, and positively correlated with the expression of PIWI-like protein 2 (PIWIL2), DNA Methyltransferase (DNMT) 3A and 3B.
Conclusion(s): Vitrification with DMSO exposure leads to significant changes in gene and TE expression in human GV oocytes. Future experiments should determine whether MII oocytes respond similarly. Impact Statement: Oocyte vitrification with DMSO containing cryoprotectant induces significant transcriptome changes, including those involving TEs, in human GV oocytes. Further studies are needed to evaluate the clinical significance of these findings. Support: This study was supported by the Stanley H Kaplan Fund.
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BACKGROUND:MT1-MMP (membrane-type 1 matrix metalloproteinase, MMP-14) is a transmembrane-anchored protein with an extracellular proteinase domain and a cytoplasmic tail devoid of proteolytic functions but capable of mediating intracellular signaling that regulates tissue homeostasis. MT1-MMP extracellular proteolytic activity has been shown to regulate pathological remodeling in aortic aneurysm and atherosclerosis. However, the role of the nonproteolytic intracellular domain of MT1-MMP in vascular remodeling in abdominal aortic aneurysms (AAA) is unknown. METHODS:We generated a mutant mouse that harbors a point mutation (Y573D) in the MT1-MMP cytoplasmic domain that abrogates the MT1-MMP signaling function without affecting its proteolytic activity. These mice and their control wild-type littermates were subjected to experimental AAA modeled by angiotensin II infusion combined with PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression and high-cholesterol feeding. RESULTS:The mutant mice developed more severe AAA than the control mice, with concomitant generation of intraaneurysmal atherosclerotic lesions and dramatically increased macrophage infiltration and elastin degradation. Aortic lesion-associated and bone marrow-derived macrophages from the mutant mice exhibited an enhanced inflammatory state and expressed elevated levels of proinflammatory Netrin-1, a protein previously demonstrated to promote both atherosclerosis and AAA. CONCLUSIONS:Our findings show that the cytoplasmic domain of MT1-MMP safeguards from AAA and atherosclerotic plaque development through a proteolysis-independent signaling mechanism associated with Netrin-1 expression. This unexpected function of MT1-MMP unveils a novel mechanism of synchronous onset of AAA and atherogenesis and highlights its importance in the control of vascular wall homeostasis.

Introduction: Nearly a quarter of older adults with inflammatory bowel disease (IBD) require surgery. Patients with IBD are at risk for complications postoperatively and this risk is increased in older adults. However, little is known about the risk factors leading to these complications.We assessed risk factors associated with adverse postoperative outcomes among older adults who underwent IBD-related surgery, as well as evaluated trends in emergency vs. elective surgery in this population.
Method(s): Using the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database, we identified adults >=60 years of age who underwent an IBD-related intestinal resection from 2005-2019. Our primary outcome included a 30-day composite of mortality, readmission, reoperation, and/or what we identified as serious complications listed in NSQIP.
Result(s): In total, 9,640 intestinal resections were performed among older adults with IBD from 2005-2019, with 48.3% having undergone resection for Crohn's disease (CD), and 51.7% for ulcerative colitis (UC). Nearly 37% experienced an adverse outcome, with the most common complication being infection (20.21%). From 2005 to 2015, there was no decrease in the number of emergent cases among older adults. On univariate analysis, higher rates of adverse postoperative outcomes were seen with increasing age (p< 0.001), with nearly 50% of those >=80 years of age having an adverse outcome. Patients who underwent an emergency surgery had a higher likelihood of postoperative complications (66.86%; p< 0.001). On multivariable analysis, albumin <=3 (aOR 1.99; 95%CI 1.69-2.33), the presence of two or more comorbidities (aOR, 1.50; 95%CI 1.27-1.76), totally dependent functional status as compared to those partially dependent or independent (aOR, 7.28; 95%CI 3.14-21.2), and emergency surgery (aOR, 1.70; 95% CI 1.36-2.11) significantly increased the odds of an adverse outcome. (Figure)
Conclusion(s): Overall 37% of older adults with IBD experienced an adverse outcome as a result of IBD-related surgery. Limited functional health status, low preoperative serum albumin levels, and those undergoing emergent surgery were associated with a significantly higher risk. This is particularly important as the number of older adults with IBD is increasing, with a persisting number of emergency cases over time. Given the high rate of surgery in this population, future research should focus on preoperative rehabilitation, nutritional optimization, and timely surgery to improve outcomes. (Table Presented)

To characterize the dysregulation of chromatin accessibility in Alzheimer's disease (AD), we generated 636 ATAC-seq libraries from neuronal and nonneuronal nuclei isolated from the superior temporal gyrus and entorhinal cortex of 153 AD cases and 56 controls. By analyzing a total of ~20 billion read pairs, we expanded the repertoire of known open chromatin regions (OCRs) in the human brain and identified cell-type-specific enhancer-promoter interactions. We show that interindividual variability in OCRs can be leveraged to identify cis-regulatory domains (CRDs) that capture the three-dimensional structure of the genome (3D genome). We identified AD-associated effects on chromatin accessibility, the 3D genome and transcription factor (TF) regulatory networks. For one of the most AD-perturbed TFs, USF2, we validated its regulatory effect on lysosomal genes. Overall, we applied a systematic approach to understanding the role of the 3D genome in AD. We provide all data as an online resource for widespread community-based analysis.

Skin wounds in adult mammals typically heal with a fibrotic scar and fail to restore ectodermal appendages, such as hair follicles or adipose tissue. Intriguingly, new hair follicles regenerate in the center of large full-thickness wounds of mice in a process called wound-induced hair neogenesis (WIHN). WIHN is followed by neogenesis of dermal adipose tissue. Both neogenic events reactivate embryonic-like cellular and molecular programs. The WIHN model provides a platform for studying mammalian regeneration, and findings from this model could instruct future regenerative medicine interventions for treating wounds and alopecia. Since Ito et al. rediscovered WIHN 15 years ago, numerous investigators have worked on the WIHN model using varying wounding protocols and model interpretations. Because a variety of factors, including environmental variables and choice of mouse strains, can affect the outcomes of a WIHN study, the purpose of this article is to provide an overview of the experimental variables that impact WIHN so that experiments between laboratories can be compared in a meaningful manner.

OBJECTIVE:To assess the effectiveness of a multimodal analgesic regimen containing "safer" opioid and non-narcotic pain medications in decreasing opioid prescriptions following surgical fixation in orthopedic trauma. DESIGN/METHODS:Retrospective cohort study. SETTING/METHODS:One urban, academic medical center. SUBJECTS/METHODS:Traumatic fracture patients from 2018 (848) and 2019 (931). METHODS:In 2019 our orthopedic trauma division began a standardized protocol of post-operative pain medications that included: 50 mg of tramadol four times daily, 15 mg of meloxicam once daily, 200 mg gabapentin twice daily, and 1 g of acetaminophen every 6 hours as needed. This multimodal regimen was dubbed the "Lopioid" protocol. We compared this protocol to all patients from the prior year who followed a standard protocol that included Schedule II narcotics. RESULTS:Greater mean MME were prescribed at discharge from fracture surgery under the standard protocol compared to the Lopioid protocol (252.3 vs 150.0; p < 0.001) and there was a difference in the type of opioid medication prescribed (p < 0.001). There was a difference in the number of refills filled for patients discharged with opioids after surgical treatment between standard and Lopioid cohorts (0.31 vs 0.21; p = 0.002). There was no difference in the types of medication-related complications (p = 0.710) or the need for formal pain management consults (p = 0.199), but patients in the Lopioid cohort had lower pain scores at discharge (2.2 vs 2.7; p = 0.001). CONCLUSIONS:The Lopioid protocol was effective in decreasing the amount of Schedule II narcotics prescribed at discharge and the number of opioid refills following orthopedic surgery for fractures.

A disproportionate tall stature is the most evident manifestation in Marfan syndrome (MFS), a multisystem condition caused by mutations in the extracellular protein and TGFβ modulator, fibrillin-1. Unlike cardiovascular manifestations, there has been little effort devoted to unravel the molecular mechanism responsible for long bone overgrowth in MFS. By combining the Cre-LoxP recombination system with metatarsal bone cultures, here we identify the outer layer of the perichondrium as the tissue responsible for long bone overgrowth in MFS mice. Analyses of differentially expressed genes in the fibrillin-1 deficient perichondrium predicted that loss of TGFβ signaling may influence chondrogenesis in the neighboring epiphyseal growth plate (GP). Immunohistochemistry revealed that fibrillin-1 deficiency in the outer perichondrium is associated with decreased accumulation of latent TGFβ-binding proteins (LTBPs)-3 and - 4, and reduced levels of phosphorylated (activated) Smad2. Consistent with these findings, mutant metatarsal bones grown in vitro were longer and released less TGFβ than the wild type counterparts. Moreover, addition of recombinant TGFβ1 normalized linear growth of mutant metatarsal bones. We conclude that longitudinal bone overgrowth in MFS is accounted for by diminished sequestration of LTBP-3 and LTBP-4 into the fibrillin-1 deficient matrix of the outer perichondrium, which results in less TGFβ signaling locally and improper GP differentiation distally.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.