Search

Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Murashige-Skoog medium solutions have been used in a variety of plant plate growth assays, yet most research uses Arabidopsis thaliana as the study organism. For larger seeds such as maize ( Zea mays ), most protocols employ a paper towel roll method for experiments, which often involves wrapping maize seedlings in wet, sterile germination paper. What the paper towel roll method lacks, however, is the ability to image the roots over time without risk of contamination. Here, we describe a sterile plate growth assay that contains Murashige-Skoog medium to grow seedlings starting two days after germination. This protocol uses a section of a paper towel roll method to achieve uniform germination of maize seedlings, which are sterilely transferred onto large acrylic plates for the duration of the experiment. The media can undergo modification to include an assortment of plant hormones, exogenous sugars, and other chemicals. The acrylic plates allow researchers to freely image the plate without disturbing the seedlings and control the environment in which the seedlings are grown, such as modifications in temperature and light. Additionally, the protocol is widely adaptable for use with other cereal crops. Key features • Builds upon plate growth methods routinely used for Arabidopsis seedlings but that are inadequate for maize. • Real-time photographic analysis of seedlings up to two weeks following germination. • Allows for testing of various growth conditions involving an assortment of additives and/or modification of environmental conditions. • Samples are able to be collected for genotype screening., Competing Interests: Competing interestsNo competing interests to declare., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

SARS-CoV-2 infection can result in a range of outcomes from asymptomatic/mild disease to severe COVID-19/fatality. In this study, we investigated the differential expression of small noncoding RNAs (sncRNAs) between patient cohorts defined by disease severity. We collected plasma samples, stratified these based on clinical outcomes, and sequenced their circulating sncRNAs. Excitingly, we found YRNA HY4 displays significant differential expression (p=0.025) between patients experiencing mild and severe disease. In agreement with recent reports identifying plasma YRNAs as indicators of influenza infection severity, our results strongly suggest that circulating HY4 levels represent a powerful prognostic indicator of likely SARS-CoV-2 patient infection outcome., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

Purpose: Gemcitabine plus cisplatin (GC) and methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) were compared in patients with locally advanced or metastatic transitional-cell carcinoma (TCC) of the urothelium., Patients and Methods: Patients with stage IV TCC and no prior systemic chemotherapy were randomized to GC (gemcitabine 1,000 mg/m 2 days 1, 8, and 15; cisplatin 70 mg/m 2 day 2) or standard MVAC every 28 days for a maximum of six cycles., Results: Four hundred five patients were randomized (GC, n = 203; MVAC, n = 202). The groups were well-balanced with respect to prognostic factors. Overall survival was similar on both arms (hazards ratio [HR], 1.04; 95% confidence interval [CI], 0.82 to 1.32; P = .75), as were time to progressive disease (HR, 1.05; 95% CI, 0.85 to 1.30), time to treatment failure (HR, 0.89; 95% CI, 0.72 to 1.10), and response rate (GC, 49%; MVAC, 46%). More GC patients completed six cycles of therapy, with fewer dose adjustments. The toxic death rate was 1% on the GC arm and 3% on the MVAC arm. More GC than MVAC patients had grade 3/4 anemia (27% v 18%, respectively) and thrombocytopenia (57% v 21%, respectively). On both arms, the RBC transfusion rate was 13 of 100 cycles and grade 3/4 hemorrhage or hematuria was 2%; the platelet transfusion rate was four patients per 100 cycles and two patients per 100 cycles on GC and MVAC, respectively. More MVAC patients, compared with GC patients, had grade 3/4 neutropenia (82% v 71%, respectively), neutropenic fever (14% v 2%, respectively), neutropenic sepsis (12% v 1%, respectively), and grade 3/4 mucositis (22% v 1%, respectively) and alopecia (55% v 11%, respectively). Quality of life was maintained during treatment on both arms; however, more patients on GC fared better regarding weight, performance status, and fatigue., Conclusion: GC provides a similar survival advantage to MVAC with a better safety profile and tolerability. This better-risk benefit ratio should change the standard of care for patients with locally advanced and metastatic TCC from MVAC to GC.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. The androgen receptor (AR) is expressed in up to 50% of TNBCs, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by the AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by the AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in an AR + -TNBC HCI-009 patient-derived xenograft (PDX) tumors ( p < 0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells ( p < 0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow-growing CSC-like populations that resist chemotherapy which lead to metastatic disease.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.M.J., A.M.P., and J.T.R are listed as co-inventors on a patent application related to this work (U.S. 63/187,835)., (Copyright: © 2022 Porman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We have identified 38 specifically excised, differentially expressed snoRNA fragments (sdRNAs) in TCGA prostate cancer (PCa) patient samples as compared to normal prostate controls. SnoRNA-derived fragments sdRNA-D19b and -A24 emerged among the most differentially expressed and were selected for further experimentation. We found that the overexpression of either sdRNA significantly increased PC3 (a well-established model of castration-resistant prostate cancer (CRPC)) cell proliferation, and that sdRNA-D19b overexpression also markedly increased the rate of PC3 cell migration. In addition, both sdRNAs provided drug-specific resistances with sdRNA-D19b levels correlating with paclitaxel resistance and sdRNA-24A conferring dasatinib resistance. In silico and in vitro analyses revealed that two established PCa tumor suppressor genes, CD44 and CDK12, represent targets for sdRNA-D19b and sdRNA-A24, respectively. This outlines a biologically coherent mechanism by which sdRNAs downregulate tumor suppressors in AR-PCa to enhance proliferative and metastatic capabilities and to encourage chemotherapeutic resistance. Aggressive proliferation, rampant metastasis, and recalcitrance to chemotherapy are core characteristics of CRPC that synergize to produce a pathology that ranks second in cancer-related deaths for men. This study defines sdRNA-D19b and -A24 as contributors to AR-PCa, potentially providing novel biomarkers and therapeutic targets of use in PCa clinical intervention.

Many U.S. states have taken significant action on climate change in recent years, demonstrating their commitment despite federal policy gridlock and rollbacks. Yet, there is still much we do not know about the agents, discourses, and strategies of those seeking to delay or obstruct state-level climate action. We first ask,  what are the obstacles to strong and effective climate policy within U.S. states? We review the political structures and interest groups that slow action, and we examine emerging tensions between climate justice and the technocratic and/or market-oriented approaches traditionally taken by many mainstream environmental groups. Second, what are potential solutions for overcoming these obstacles? We suggest strategies for overcoming opposition to climate action that may advance more effective and inclusive state policy, focusing on political strategies, media framing, collaboration, and leveraging the efforts of ambitious local governments., (© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2022.)

Background: Limb length discrepancy (LLD) is a known complication of total hip arthroplasty (THA), leading to decreased patient function and satisfaction. It remains unknown how a patient's perception of LLD evolves over time. The aim of this study is to evaluate the relationship between measured and perceived LLD, and to assess whether perceived LLD resolved with time in most patients., Methods: This study retrospectively reviewed radiographs of 140 consecutive patients undergoing primary THA by a single surgeon via a direct anterior approach, calculating postoperative change in limb length (ΔL). Patient perceptions of LLD were recorded at standard postoperative visit intervals. A P-value of .05 was used to determine statistical significance., Results: Of 130 patients (mean ΔL = +7.9 mm), 22 patients endorsed perceived postoperative LLD and the remainder were asymptomatic (mean ΔL +11.1 mm vs +7.3 mm, P = .03). Seventeen patients reported mild symptoms and 5 reported severe symptoms (mean ΔL +10.2 mm vs +13.8 mm, P = .4). After 1 year, 45% (10) patients reported complete resolution of perceived LLD (mean follow-up 364 days), 18% (4) reported notable improvement, and 36% (8) reported no improvement. Four excluded patients endorsed perceived LLD (2 mild, 2 severe), which resolved after contralateral THA., Conclusion: This study noted a correlation between increasing postoperative ΔL and perceived LLD. A majority of patients (63%) experienced either improvement or full resolution of symptoms during the follow-up period. This data may have a role in reassuring the orthopedic surgeon and the patient regarding the natural course of postoperative LLD. Further investigation is needed to help identify risk factors for persistent LLD., Level of Evidence: Level III (Prognostic)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Methylation at the N 6 position of adenosine (m 6 A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m 6 A nucleotides within transcripts has been historically challenging due to m 6 A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m 6 A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m 6 A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m 6 A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m 6 A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m 6 A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m 6 A sites at true single-nucleotide resolution. As the effort to accurately identify m 6 As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m 6 A epitranscriptome., (© 2021 Roberts et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Triple-negative breast cancer (TNBC) is an aggressive subtype with peak recurrence as metastatic disease within the first few years of diagnosis. Androgen receptor (AR) expression is increased in anchorage-independent cells in TNBC preclinical models. Both AR knockdown and inhibition lead to reduced TNBC invasion in vitro, reduced tumorigenicity, and less recurrence in vivo in preclinical models. Transforming growth factor β (TGFβ) pathway gene signatures also increased during anchorage-independent survival both in vitro and in vivo in preclinical models and in circulating tumor cells (CTCs) from patients during emergence of chemo resistant disease. We hypothesized that a positive loop between AR and TGFβ signaling facilitates TNBC anchorage-independent survival. We find that multiple components of the TGFβ pathway, including TGFβ1 and 3, as well as pathway activity measured by nuclear localization and transcriptional activity of phosphorylated Smad3, are enhanced in anchorage-independent conditions. Further, exogenous TGFβ increased AR protein while TGFβ inhibition decreased AR and TNBC viability, particularly under anchorage-independent culture conditions. ChIP-seq experiments revealed AR binding to TGFB1 and SMAD3 regulatory regions in MDA-MB-453 cells. In clinical datasets, TGFB3 and AR positively correlate and high expression of both genes together corresponded to significantly worse recurrence-free and overall survival in both ER-negative and basal-like breast cancer. Finally, inhibiting both AR and TGFβ decreased cell survival, particularly under anchorage-independent conditions. These findings warrant further investigations into whether combined inhibition of AR and TGFβ pathways might decrease metastatic recurrence rates and mortality from TNBC., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous. In this study, we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. A mass spectrometry analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from data collected on donors expressing only the predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here, we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ∼20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145)., (© 2020 Roberts et al.)

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)