Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Belantamab Mafodotin, Bortezomib, and Dexamethasone for Multiple Myeloma.

BACKGROUND: Belantamab mafodotin had single-agent activity in patients with relapsed or refractory multiple myeloma, a finding that supports further evaluation of the agent in combination with standard-care therapies. METHODS: In this phase 3, open-label, randomized trial, we evaluated belantamab mafodotin, bortezomib, and dexamethasone (BVd), as compared with daratumumab, bortezomib, and dexamethasone (DVd), in patients who had progression of multiple myeloma after at least one line of therapy. The primary end point was progression-free survival. Key secondary end points were overall survival, response duration, and minimal residual disease (MRD)-negative status. RESULTS: In total, 494 patients were randomly assigned to receive BVd (243 patients) or DVd (251 patients). At a median follow-up of 28.2 months (range, 0.1 to 40.0), median progression-free survival was 36.6 months (95% confidence interval [CI], 28.4 to not reached) in the BVd group and 13.4 months (95% CI, 11.1 to 17.5) in the DVd group (hazard ratio for disease progression or death, 0.41; 95% CI, 0.31 to 0.53; P<0.001). Overall survival at 18 months was 84% in the BVd group and 73% in the DVd group. An analysis of the restricted mean response duration favored BVd over DVd (P<0.001). A complete response or better plus MRD-negative status occurred in 25% of the patients in the BVd group and 10% of those in the DVd group. Grade 3 or higher adverse events occurred in 95% of the patients in the BVd group and 78% of those in the DVd group. Ocular events were more common in the BVd group than in the DVd group (79% vs. 29%); such events were managed with dose modifications, and events of worsening visual acuity mostly resolved. CONCLUSIONS: As compared with DVd therapy, BVd therapy conferred a significant benefit with respect to progression-free survival among patients who had relapsed or refractory multiple myeloma after at least one line of therapy. Most patients had grade 3 or higher adverse events. (Funded by GSK; DREAMM-7 ClinicalTrials.gov number, NCT04246047; EudraCT number, 2018-003993-29.).

Nitric oxide contributes to rapid sclerostin protein loss following mechanical load.

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.

The effect of Omicron breakthrough infection and extended BNT162b2 booster dosing on neutralization breadth against SARS-CoV-2 variants of concern.

COVID-19 vaccines are playing a vital role in controlling the COVID-19 pandemic. As SARS-CoV-2 variants encoding mutations in the surface glycoprotein, Spike, continue to emerge, there is increased need to identify immunogens and vaccination regimens that provide the broadest and most durable immune responses. We compared the magnitude and breadth of the neutralizing antibody response, as well as levels of Spike-reactive memory B cells, in individuals receiving a second dose of BNT162b2 at a short (3-4 week) or extended interval (8-12 weeks) and following a third vaccination approximately 6-8 months later. We show that whilst an extended interval between the first two vaccinations can greatly increase the breadth of the immune response and generate a higher proportion of Spike reactive memory B cells, a third vaccination leads to similar levels between the two groups. Furthermore, we show that the third vaccine dose enhances neutralization activity against omicron lineage members BA.1, BA.2 and BA.4/BA.5 and this is further increased following breakthrough infection during the UK omicron wave. These findings are relevant for vaccination strategies in populations where COVID-19 vaccine coverage remains low.

Efficacy, Safety, and Cost-effectiveness of Bezlotoxumab in Preventing Recurrent Clostridioides difficile Infection : Systematic Review and Meta-analysis.

INTRODUCTION: Clostridioides difficile infection (CDI) remains a global health challenge. Bezlotoxumab (BEZ) is a monoclonal antibody against C. difficile toxin B. Two randomized controlled trials (RCTs), MODIFY I and II, confirmed BEZ efficacy in preventing recurrent Clostridioides difficile infection (rCDI). However, there are safety concerns about its use in patients with a history of congestive heart failure. Observational studies have since been conducted, and it is important to explore the consistency of BEZ efficacy, cost-effectiveness, and its safety utilizing these real-world data. METHODS: We performed a systematic review and meta-analysis to pool the rate of rCDI in patients receiving BEZ and explore its efficacy and safety in preventing rCDI compared with control. We searched PubMed, EMBASE, Cochrane Library, and Google Scholar from inception through April 2023 for relevant RCTs or observational studies assessing BEZ in preventing rCDI. Single-arm studies describing experience with BEZ in preventing rCDI were also included for proportion meta-analysis. A proportion meta-analysis with a random-effects model was used to pool the rCDI rate with its corresponding 95% CI. In a meta-analysis of efficacy, we generated the relative risk (RR) to compare BEZ versus control in preventing rCDI. RESULTS: Thirteen studies including 2 RCTs and 11 observational studies totaling 2337 patients, of which 1472 received BEZ, were included in the analysis. Of the constituent studies, 5 (1734 patients) compared BEZ versus standard-of-care (SOC). Pooled rate of rCDI in patients receiving BEZ was 15.8% (95% CI: 14%-17.8%), and was 28.9% (95% CI: 24%-34.4%) in the SOC. BEZ significantly reduced rCDI risk compared with SOC [RR=0.57 (95% CI: 0.45-0.72, I2 =16%)]. There was no difference in the overall mortality or heart failure risk. Of the 9 included cost-effectiveness analyses, 8 demonstrated BEZ+SOC cost-effectiveness compared with SOC alone. DISCUSSION: Our meta-analysis comprising real-world data revealed lower rCDI in patients receiving BEZ and supported its efficacy and safety when added to SOC therapy. The results were consistent across various subgroups. Available cost-effectiveness analyses mostly support BEZ+SOC cost-effectiveness compared with SOC alone.

Effects of Social Determinants and Pharmacogenetic Medication Interactions on 90-Day Hospital Readmissions.

Context: The present study builds on our prior work that demonstrated an association between pharmacogenetic interactions and 90-day readmission. Objective: Evaluate aggregate contribution of social determinants, comorbidity, and gene-x-drug interactions to moderate 90-day hospital readmission. Study Design and Analysis: Non-concurrent cohort study; Multivariable logistic regression Setting: Hospital/integrated healthcare delivery system in northern Illinois Population Studied: 19,999 adults tracked from 2010 through 2020 who underwent testing with a 13-gene pharmacogenetic panel Outcome Measure: 90-day hospital readmission (primary outcome) Results: Univariate logistic regression analyses demonstrated that strongest associations with 90 day hospital readmissions were the number of medications prescribed within 30 days of a first hospital admission that had Clinical Pharmacogenomics Implementation Consortium (CPIC) guidance (CPIC medications) (5+ CPIC medications, odds ratio (OR) = 7.66, 95% confidence interval 5.45-10.77) (p < 0.0001), major comorbidities (5+ comorbidities, OR 3.36, 2.61-4.32) (p < 0.0001), age (65 + years, OR = 2.35, 1.77-3.12) (p < 0.0001), unemployment (OR = 2.19, 1.88-2.64) (p < 0.0001), Black/African-American race (OR 2.12, 1.47-3.07) (p < 0.0001), median household income (OR = 1.63, 1.03-2.58) (p = 0.035), male gender (OR = 1.47, 1.21-1.80) (p = 0.0001), and one or more gene-x-drug interaction (defined as a prescribed CPIC medication for a patient with a corresponding actionable pharmacogenetic variant) (OR = 1.41, 1.18-1.70). Health insurance was not associated with risk of 90-day readmission. Race, income, employment status, and gene-x-drug interactions were robust in a multivariable logistic regression model. The odds of 90-day readmission for patients with one or more identified gene-x-drug interactions after adjustment for these covariates was attenuated by 10% (OR = 1.31, 1.08-1.59) (p = 0.006). Although the interaction between race and gene-x-drug interactions was not statistically significant, White patients were more likely to have a gene-x-drug interaction (35.2%) than Black/African-American patients (25.9%) who were not readmitted (p < 0.0001). Conclusions: These results highlight the major contribution of social determinants and medical complexity to risk for hospital readmission, and that these determinants may modify the effect of gene-x-drug interactions on rehospitalization risk.

Inpatient management of epidermolysis bullosa: Consensus-based hands-on instructions for neonates and postneonates.

BACKGROUND: Epidermolysis bullosa (EB), characterized by skin fragility and blistering, often requires hospitalization. Training for inpatient management of EB is limited, with no unified recommendations available in North America. OBJECTIVE: To develop consensus-derived best practices for hands-on inpatient management of EB in both the neonatal and postneonatal period. METHODS: A modified Delphi method (expert-based input via 2 surveys and a final review) was implemented. Available guidelines from EB Clinical Research Consortium centers were analyzed to determine areas of focus and formulate statements to be voted on by EB Clinical Research Consortium members, experienced EB nurses, and select family members. Study participants evaluated statements using a Likert scale: statements with at least 70% agreement were accepted; statements with 30% or more disagreement were rejected. RESULTS: Ten areas of focus were identified. Delphi participants included 15 dermatologists, 8 nurses, and 6 nonhealth care caregivers. Consensus was established on 103/119 neonatal statements and 105/122 postneonatal statements; no statements were rejected. Most recommendations applied to both age groups. LIMITATIONS: Recommendations may require adjustment based on individual patient's clinical context. CONCLUSION: Using the Delphi method, a consensus-derived resource for hospital-based health care professionals who manage patients with EB has been developed to improve the quality of inpatient care.

An automated respiratory data pipeline for waveform characteristic analysis.

Comprehensive and accurate analysis of respiratory and metabolic data is crucial to modelling congenital, pathogenic and degenerative diseases converging on autonomic control failure. A lack of tools for high-throughput analysis of respiratory datasets remains a major challenge. We present Breathe Easy, a novel open-source pipeline for processing raw recordings and associated metadata into operative outcomes, publication-worthy graphs and robust statistical analyses including QQ and residual plots for assumption queries and data transformations. This pipeline uses a facile graphical user interface for uploading data files, setting waveform feature thresholds and defining experimental variables. Breathe Easy was validated against manual selection by experts, which represents the current standard in the field. We demonstrate Breathe Easy's utility by examining a 2-year longitudinal study of an Alzheimer's disease mouse model to assess contributions of forebrain pathology in disordered breathing. Whole body plethysmography has become an important experimental outcome measure for a variety of diseases with primary and secondary respiratory indications. Respiratory dysfunction, while not an initial symptom in many of these disorders, often drives disability or death in patient outcomes. Breathe Easy provides an open-source respiratory analysis tool for all respiratory datasets and represents a necessary improvement upon current analytical methods in the field. KEY POINTS: Respiratory dysfunction is a common endpoint for disability and mortality in many disorders throughout life. Whole body plethysmography in rodents represents a high face-value method for measuring respiratory outcomes in rodent models of these diseases and disorders. Analysis of key respiratory variables remains hindered by manual annotation and analysis that leads to low throughput results that often exclude a majority of the recorded data. Here we present a software suite, Breathe Easy, that automates the process of data selection from raw recordings derived from plethysmography experiments and the analysis of these data into operative outcomes and publication-worthy graphs with statistics. We validate Breathe Easy with a terabyte-scale Alzheimer's dataset that examines the effects of forebrain pathology on respiratory function over 2 years of degeneration.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

Differential recognition of HIV-stimulated IL-1ß and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages.

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1ß and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1ß/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.

Influence of fecal microbial transplant (FMT) between male and female rats on methamphetamine-induced hyperthermia.

OBJECTIVE: To investigate the effect of bidirectional fecal microbial transplant (FMT) between male and female rats on methamphetamine (MA)-induced hyperthermia. METHODS: FMT was performed between male and female rats prior to MA (10 mg/kg, sc) treatment. Core body temperature, plasma drug and norepinephrine (NE) levels were measured and compared between treatment groups. 16S rRNA gene sequencing of bacterial communities between male and female rats was performed. RESULTS: MA treatment resulted in significantly higher core body temperatures in male groups (control and FMT-treated) compared to MA-treated female groups (control and FMT-treated). Plasma concentrations of MA and amphetamine were higher in females than males. Whereas, plasma norepinephrine (NE) levels were not different between male and female rats 90 minutes after MA treatment. At the phyla level, the microbiome of male and female control rats were dominated by Firmicutes and Bacteroidetes. Males had a higher relative abundance of Firmicutes and lower relative abundances of Bacteroidetes than females. The FMT procedure changed the recipient group towards their donor with males getting closer to their donors than females. In the control groups following MA treatment, Firmicutes increased and Bacteroides decreased in females and males. Conversely, in the FMT treatment groups following MA treatment, Firmicutes decreased while Bacteroidetes increased in females and males. CONCLUSIONS: Although definite differences in the structure and diversity of the gut microbiome were observed using 16S rRNA gene sequencing of bacterial communities between male and female rats, these differences do not seem to contribute to the sex-based differences in MA-induced hyperthermia.

Diagnosis and management of endothelial disorders following haematopoietic stem cell transplantation.

Haematopoietic stem cell transplantation is a mainstay of therapy for numerous malignant and nonmalignant diseases. Endothelial activation and dysfunction occur after stem cell transplantation, driven by various patient- and transplant-specific factors. This can manifest as one of the relatively uncommon endothelial injury syndromes, such as sinusoidal obstruction syndrome, transplant-associated thrombotic microangiopathy, idiopathic pneumonia syndrome, capillary leak syndrome, engraftment syndrome or posterior reversible encephalopathy syndrome. This review focuses on the pathogenesis, classification and diagnosis of these disorders, as well as provides guidance on risk mitigation and treatment.

Treatment of patients with Waldenström macroglobulinaemia: clinical practice update from the Myeloma Foundation of Australia Medical and Scientific Advisory Group.

Waldenström macroglobulinaemia (WM) is an indolent B-cell malignancy characterised by the presence of IgM paraprotein, bone marrow infiltration by clonal small B lymphocytes with plasmacytic differentiation and the MYD88 L265P mutation in >90% of cases. Traditionally, WM has been treated with chemoimmunotherapy. Recent trials have demonstrated the efficacy and safety of Bruton tyrosine kinase inhibitors in WM, both as monotherapy and in combination with other drugs. There is emerging evidence on the use of other agents including B-cell lymphoma 2 inhibitors and on the treatment of rare presentations of WM. In this update, the Medical and Scientific Advisory Group of Myeloma Australia reviews the available evidence on the treatment of WM since the last publication in 2017 and provides specific recommendations to assist Australian clinicians in the management of this disease.

The importance of frailty assessment in multiple myeloma: a position statement from the Myeloma Scientific Advisory Group to Myeloma Australia.

Multiple myeloma (MM) is a disease of older people, yet factors relating to comorbidity and frailty may threaten treatment tolerability for many of this heterogenous group. There has been increasing interest in defining specific and clinically relevant frailty assessment tools within the MM population, with the goal of using these frailty scores, not just as a prognostic instrument, but also as a predictive tool to allow for a frailty-adapted treatment approach. This paper reviews the various frailty assessment frameworks used in the evaluation of patients with MM, including the International Myeloma Working Group Frailty Index (IMWG-FI), the Mayo Frailty Index and the simplified frailty scale. While the IMWG-FI remains the most widely accepted tool, the simplified frailty scale is the most user-friendly in busy day-to-day clinics based on its ease of use. This paper summarises the recommendations from the Myeloma Scientific Advisory Group (MSAG) of Myeloma Australia, on the use of frailty assessment tools in clinical practice and proposes a frailty-stratified treatment algorithm to aid clinicians in tailoring therapy for this highly heterogeneous patient population.

A prospective study of extraesophageal reflux and potential microaspiration in patients hospitalized with COVID-19 in Jordan.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung infection has represented a global challenge. Intriguingly, it has been shown that the alveolar lung epithelium expresses little Angiotensin Converting Enzyme receptor protein (ACE2), the entry receptor for SARS-CoV-2. Upper airway establishment of infection and translocation to the lung is well documented but other anatomical niches may be relevant to potentially serious lung infection. ACE2 is heavily expressed in the gastrointestinal tract and gastrointestinal symptoms support a clinical diagnosis of Coronavirus disease 2019 (COVID-19). This suggests a research question and the need to gather patient data exploring potential aerodigestive links in SARS-CoV-2 tranlocation and infection which may be relevant in the peripheral lung. This recognizes anatomical proximity and concepts of bi-directional movement between the Gastrointestinal and lung systems in normal physiology and disease. We have therefore explored the potential for gastro oesophageal reflux disease (GORD) micro aspiration and aeorodigestive pathophysiology in a novel prospective investigation of patients hospitalized with COVID-19. METHODS: This is a prospective descriptive cohort study of 210 patients who were hospitalized with a confirmed diagnosis of COVID-19. The cohort was divided into three groups of patients based on symptom severity and radiological results. The Reflux Symptom Index (RSI) was used to evaluate the presence and severity of GOR. An RSI greater than 13 is considered to be abnormal. Patients' saliva samples were tested using enzyme-linked immunosorbent assay (ELISA) to determine the level of salivary pepsin among the cohort of patients. RESULTS: A total of 210 patients with COVID-19 were enrolled in the study with 55.2% (116/210) classified as mildly ill, 31.9% (67/210) moderately ill and 12.9% (27/210) as severely ill. 34% (72/210) of the patients had an RSI score of over 13 and a median salivary pepsin value of 54 ± 29 ng/ml which suggested an incidence of extraesophageal reflux (EOR) in around a third of patients. The presence of respiratory comorbid conditions, an RSI score of over 13 and a salivary pepsin level of > 76ng/ml increased the risk of developing a more severe COVID-19 infection. CONCLUSION: The study showed a high prevalence of EOR among the study cohort and provide the first prospective evidence suggesting the potential for aerodigestive pathophysiology including microaspiration in COVID-19 disease. We believe that the results of our study support the need for more extensive research.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Rapid changes in coastal ocean microbiomes uncoupled with shifts in environmental variables.

Disturbances, here defined as events that directly alter microbial community composition, are commonly studied in host-associated and engineered systems. In spite of global change both altering environmental averages and increasing extreme events, there has been relatively little research into the causes, persistence and population-level impacts of disturbance in the dynamic coastal ocean. Here, we utilize 3 years of observations from a coastal time series to identify disturbances based on the largest week-over-week changes in the microbiome (i.e. identifying disturbance as events that alter the community composition). In general, these microbiome disturbances were not clearly linked to specific environmental factors and responsive taxa largely differed, aside from SAR11, which generally declined. However, several disturbance metagenomes identified increased phage-associated genes, suggesting that unexplained community shifts might be caused by increased mortality. Furthermore, a category 1 hurricane, the only event that would likely be classified a priori as an environmental disturbance, was not an outlier in microbiome composition, but did enhance a bloom in seasonally abundant phytoplankton. Thus, as extreme environmental changes intensify, assumptions of what constitutes a disturbance should be re-examined in the context of ecological history and microbiome responses.

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

RATIONALE: We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. OBJECTIVE: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. METHODS AND RESULTS: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. CONCLUSIONS: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Neuronal SETD2 activity links microtubule methylation to an anxiety-like phenotype in mice.

Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.