The rockefeller university

Retrotransposons are genomic DNA sequences that copy them-selves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expres-sion. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Background: Transbronchial microwave ablation (MWA) is a promising novel therapy. Despite advances in bronchoscopy and virtual navigation, real time image guidance of probe delivery is lacking, and distal maneuverability is limited. Cone-beam computed tomography (CBCT) based augmented fluoroscopy guidance using steerable sheaths may help overcome these shortcomings. The aim of this study was to evaluate feasibility and accuracy of augmented fluoroscopy guided transbronchial MWA with a steerable sheath and without a bronchoscope. Methods: In this prospective study, procedures were performed under general anesthesia. Extra-bronchial lung synthetic targets were placed percutaneously. Target and airways extracted from CBCT, with planned bronchial parking point close to the target were overlaid on live fluoroscopy. Endobronchial navigation was solely performed under augmented fluoroscopy guidance. A 6.5 Fr steerable sheath was parked in the bronchus per plan, and a flexible MWA probe was inserted coaxially then advanced through the bronchus wall towards the target. Final in-target position was confirmed by CBCT. Only one ablation of 100 W-5 min was performed per target. Animals were euthanized and pathology analysis of the lungs was performed. Results: Eighteen targets with a median largest diameter of 9 mm (interquartile range, 7-11 mm) were ablated in 9 pigs. Median needle-target center distance was 2 mm (interquartile range, 0-4 mm), and was higher for lower/middle than for upper lobes [0 mm (interquartile range, 0-4 mm) vs. 4 mm (interquartile range, 3-8 mm), P=0.04]. No severe complications or pneumothorax occurred. Two cases of rib fractures in the ablation zone resolved after medical treatment. Median longest axis of the ablation zone on post-ablation computed tomography was 38 mm (interquartile range, 30-40 mm). Histology showed coagulation necrosis of ablated tissue. Conclusions: Transbronchial MWA under augmented fluoroscopy guidance using a steerable sheath is feasible and accurate.

Author summaryAlthough genetic diversity in the olfactory receptor repertoire contributes to variation in odor perception, we have few explicit predictions relating variation in a specific OR to perception. Here, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). We identified novel receptors for musk and human body odor that have implications for how structurally different molecules can have similar odors. Summarizing all the published genetic variation that associates with odor perception, we found that individuals with ancestral versions of the receptors tend to rate the corresponding odor as more intense, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study of olfactory genetic and perceptual variation will improve our understanding of how the olfactory system encodes odor properties. The olfactory system combines input from multiple receptor types to represent odor information, but there are few explicit examples relating olfactory receptor (OR) activity patterns to odor perception. To uncover these relationships, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). In both populations, consistent with previous studies, we replicated three previously reported associations (beta-ionone/OR5A1, androstenone/OR7D4, cis-3-hexen-1-ol/OR2J3 LD-band), but not for odors containing aldehydes, suggesting that olfactory phenotype/genotype studies are robust across populations. Two novel associations between an OR and odor perception contribute to our understanding of olfactory coding. First, we found a SNP in OR51B2 that associated with trans-3-methyl-2-hexenoic acid, a key component of human underarm odor. Second, we found two linked SNPs associated with the musk Galaxolide in a novel musk receptor, OR4D6, which is also the first human OR shown to drive specific anosmia to a musk compound. We noticed that SNPs detected for odor intensity were enriched with amino acid substitutions, implying functional changes of odor receptors. Furthermore, we also found that the derived alleles of the SNPs tend to be associated with reduced odor intensity, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study provides information about coding for human body odor, and gives us insight into broader mechanisms of olfactory coding, such as how differential OR activation can converge on a similar percept.

Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to similar to 4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1's MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

The export of antimicrobial peptides is mediated by diverse mechanisms in bacterial quorum sensing pathways. One such binary system employed by gram-positive bacteria is the PCAT1 ABC transporter coupled to a cysteine protease. The focus of this study is the N-terminal C39 peptidase (PEP) domain from Clostridium thermocellum PCAT1 that processes its natural substrate CtA by cleaving a conserved -GG- motif to separate the cargo from the leader peptide prior to secretion. In this study, we are primarily interested in elucidating the dynamic and structural determinants of CtA binding and how it is coupled to cleavage efficiency in the PCAT1 PEP domain. To this end, we have characterized CtA interactions with PEP domain and PCAT1 transporter in detergent micelles using solution nuclear magnetic resonance spectroscopy. The bound CtA structure revealed the disordered C-terminal cargo peptide is linked by a sterically hindered cleavage site to a helix docked within a hydrophobic cavity in the PEP domain. The wide range of internal motions detected by amide nitrogen (N-15) relaxation measurements in the free enzyme and substrate-bound complex suggests the binding site is relatively floppy. This flexibility plays a key role in the structural rearrangement necessary to relax steric inhibition in the bound substrate. In conjunction with previously reported PCAT1 structures, we offer fresh insight into the ATP-mediated association between PEP and transmembrane domains as a putative mechanism to optimize peptide cleavage by regulating the width and flexibility of the enzyme active site.

In this paper, the hepatitis C virus inhibitors Pibrentasvir and Ombitasvir are found to inhibit the SARS-CoV-2 exonuclease and are shown to have therapeutic potential when combined with SARS-CoV-2 polymerase inhibitors in viral cell cultures. SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Public Health Significance Persons with dual severe opioid and cocaine use disorders are at particular risk of morbidity and mortality. This study shows that in persons with such dual diagnoses, there is a shared vulnerability to escalation of both heroin and cocaine. Persons with dual severe opioid and cocaine use disorders are at risk of considerable morbidity, and the bidirectional relationship of escalation of mu-opioid agonists and cocaine use is not well understood. The aim of this study was to examine the bidirectional relationship between escalation of heroin and cocaine use in volunteers dually diagnosed with opioid and cocaine dependence (OD + CD). Volunteers from New York with OD + CD (total n = 295; male = 182, female = 113; age >= 18 years) were interviewed with the Structured Clinical Interview for the DSM-IV Axis I Disorders and Kreek-McHugh-Schluger-Kellogg scales for dimensional measures of drug exposure, which also collect ages of 1st use and onset of heaviest use. Time of escalation was defined as age of onset of heaviest use minus age of 1st use in whole years. Times of escalation of heroin and cocaine were positively correlated in both men (Spearman r = .34, 95% confidence interval [CI: .17, .48], p < .0001) and women (Spearman r = .51, [.27, .50], p < .0001) volunteers. After we adjusted for demographic variables, a Cox regression showed that time of cocaine escalation was a predictor of time of heroin escalation (hazard ratio [HR] = 0.97, 95% CI [0.95, 0.99], p = .003). Another Cox regression showed that this relationship is bidirectional, because time of heroin escalation was also a predictor of time of cocaine escalation (HR = 0.98, [0.96-0.99], p = .016). In these adjusted models, gender was not a significant predictor of time of escalation of either heroin or cocaine. Therefore, escalation did not differ robustly by gender when adjusting for demographics and other major variables. Overall, rapid escalation of cocaine use was a predictor of rapid escalation of heroin use, and vice versa, in persons with dual severe opioid and cocaine use disorders. These findings suggest a shared vulnerability to rapid escalation of these 2 drugs in persons with dual severe opioid and cocaine use disorders.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

A sarcomere is the contractile unit of the myofibril in striated muscles such as cardiac and skeletal muscles. The assembly of sarcomeres depends on multiple molecules that serve as raw materials and participate in the assembly process. However, the mechanism of this critical assembly process remains largely unknown. Here, we found that the cell fate determinant Numb and its homolog Numblike regulated sarcomere assembly and maintenance in striated muscles. We discovered that Numb and Numblike are sarcomeric molecules that were gradually confined to the Z-disc during striated muscle development. Conditional knockout of Numb and Numblike severely compromised sarcomere assembly and its integrity and thus caused organelle dysfunction. Notably, we identified that Numb and Numblike served as sarcomeric alpha-Actin-binding proteins (ABPs) and shared a conserved domain that can bind to the barbed end of sarcomeric alpha-Actin. In vitro fluorometric alpha-Actin polymerization assay showed that Numb and Numblike also played a role in the sarcomeric alpha-Actin polymerization process. Last, we demonstrate that Numb and Numblike regulate sarcomeric alpha-Actinin-dependent (ACTN-dependent) Z-disc consolidation in the sarcomere assembly and maintenance. In summary, our studies show that Numb and its homolog Numblike regulate sarcomere assembly and maintenance in striated muscles, and demonstrate a molecular mechanism by which Numb/Numblike, sarcomeric alpha-Actin, and ACTN cooperate to control thin filament formation and Z-disc consolidation.

Background: Although hidradenitis suppurativa (HS) shares some transcriptomic and cellular infiltrate features with psoriasis, their skin proteome remains unknown. Objective: To define and compare inflammatory protein biomarkers of HS and psoriasis skin. Methods: We assessed 92 inflammatory biomarkers in HS (n = 13), psoriasis (n = 11), and control skin (n = 11) using Olink high-throughput proteomics. We also correlated HS skin and blood biomarkers using proteomics and RNA sequencing. Results: We identified 57 differentially expressed proteins (DEPs) in lesional psoriasis and 64 DEPs in lesional HS skin, compared to healthy controls. Both HS and psoriasis lesional skin demonstrated a significant upregulation of T helper 1 and T helper 17 proteins. Healthy-appearing perilesional HS skin had 63 DEPs compared to healthy controls. Nonlesional HS and psoriasis skin had 24 and 7 DEPs, respectively, compared to healthy controls. Tumor necrosis factor and 8 other proteins were significantly correlated with clinical severity in perilesional HS skin (2 cm from a nodule). Limitations: Inclusion of only moderate-to-severe patients and the cohort size. Conclusion: HS has a greater inflammatory profile and is more diffusely distributed compared with psoriasis. Proteins correlated with disease severity are potential disease mediators. Perilesional skin is comparably inflamed to lesional skin, suggesting the need to treat beyond skin nodules. ( J Am Acad Dermatol 2022;86:322-30.)