Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron.

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

IRS2 Signaling Protects Against Stress-Induced Arrhythmia by Maintaining Ca2+ Homeostasis.

BACKGROUND: The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. METHODS: A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. RESULTS: The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. CONCLUSIONS: Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells.

The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2.

The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins without evidence.

Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

A comprehensive exploration of the druggable conformational space of protein kinases using AI-predicted structures.

Protein kinase function and interactions with drugs are controlled in part by the movement of the DFG and ɑC-Helix motifs that are related to the catalytic activity of the kinase. Small molecule ligands elicit therapeutic effects with distinct selectivity profiles and residence times that often depend on the active or inactive kinase conformation(s) they bind. Modern AI-based structural modeling methods have the potential to expand upon the limited availability of experimentally determined kinase structures in inactive states. Here, we first explored the conformational space of kinases in the PDB and models generated by AlphaFold2 (AF2) and ESMFold, two prominent AI-based protein structure prediction methods. Our investigation of AF2's ability to explore the conformational diversity of the kinome at various multiple sequence alignment (MSA) depths showed a bias within the predicted structures of kinases in DFG-in conformations, particularly those controlled by the DFG motif, based on their overabundance in the PDB. We demonstrate that predicting kinase structures using AF2 at lower MSA depths explored these alternative conformations more extensively, including identifying previously unobserved conformations for 398 kinases. Ligand enrichment analyses for 23 kinases showed that, on average, docked models distinguished between active molecules and decoys better than random (average AUC (avgAUC) of 64.58), but select models perform well (e.g., avgAUCs for PTK2 and JAK2 were 79.28 and 80.16, respectively). Further analysis explained the ligand enrichment discrepancy between low- and high-performing kinase models as binding site occlusions that would preclude docking. The overall results of our analyses suggested that, although AF2 explored previously uncharted regions of the kinase conformational space and select models exhibited enrichment scores suitable for rational drug discovery, rigorous refinement of AF2 models is likely still necessary for drug discovery campaigns.

Dynamics of the most common pathogenic mtDNA variant m.3243A > G demonstrate frequency-dependency in blood and positive selection in the germline.

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A > G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A > G levels decrease in blood with age, and an age correction representing ~ 2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate that the dynamics of m.3243A > G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate 'on average', creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A > G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A > G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A > G. Germline selection of m.3243A > G shows an 'arching' profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

Feasibility of structuring electronic health record data to facilitate real-world data research: ICAREdata methods applied to multicenter cancer clinical trials.

BACKGROUND: The use of electronic health record (EHR) data for research is limited by a lack of structure and a standard data model. The objective of the ICAREdata (Integrating Clinical Trials and Real-World Endpoints Data) project was to structure key research data elements in EHRs using a minimal Common Oncology Data Elements (mCODE) data model to extract and transmit data. METHODS: The ICAREdata project captured two EHR data elements essential to clinical trials: cancer disease status and treatment plan change. The project was implemented in clinical sites participating in Alliance for Clinical Trials in Oncology trials. Data were extracted from EHRs and sent by secure Fast Healthcare Interoperability Resource messaging (a standard for exchanging EHRs) to a database. Selected elements were compared with corresponding data from the trial's electronic data capture (EDC) system, Medidata Rave. RESULTS: By December 2023, data were extracted and transmitted from 10 sites for 35 patients, involving 367 clinical encounters across 15 clinical trials. Data through March 2023 demonstrated that concordance for the elements treatment plan change and cancer disease status was 79% and 34%, respectively. When disease evaluation was reported by both EHR and EDC (n = 15), there was 87% agreement on cancer disease status. CONCLUSIONS: Documentation, extraction, and aggregation of structured data elements in EHRs using mCODE and ICAREdata methods is feasible in multi-institutional cancer clinical trials. EDC as a reference data set allowed assessment of the completeness of EHR data capture. Future initiatives will focus on elements with shared definitions in clinical and research environments and efficient workflows. PLAIN LANGUAGE SUMMARY: Clinical trials use electronic case report forms to report data, and data must be manually entered on these forms, which is costly and time consuming. ICAREdata methods use structured, organized data from clinical trials that can be more easily shared instead having to enter free text into electronic health records.

Identification of discriminative gene-level and protein-level features associated with pathogenic gain-of-function and loss-of-function variants.

Identifying whether a given genetic mutation results in a gene product with increased (gain-of-function; GOF) or diminished (loss-of-function; LOF) activity is an important step toward understanding disease mechanisms because they may result in markedly different clinical phenotypes. Here, we generated an extensive database of documented germline GOF and LOF pathogenic variants by employing natural language processing (NLP) on the available abstracts in the Human Gene Mutation Database. We then investigated various gene- and protein-level features of GOF and LOF variants and applied machine learning and statistical analyses to identify discriminative features. We found that GOF variants were enriched in essential genes, for autosomal-dominant inheritance, and in protein binding and interaction domains, whereas LOF variants were enriched in singleton genes, for protein-truncating variants, and in protein core regions. We developed a user-friendly web-based interface that enables the extraction of selected subsets from the GOF/LOF database by a broad set of annotated features and downloading of up-to-date versions. These results improve our understanding of how variants affect gene/protein function and may ultimately guide future treatment options.

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

The Influence of Social Determinants on Receiving Outpatient Treatment with Monoclonal Antibodies, Disease Risk, and Effectiveness for COVID-19.

BACKGROUND: Limited research has studied the influence of social determinants of health (SDoH) on the receipt, disease risk, and subsequent effectiveness of neutralizing monoclonal antibodies (nMAbs) for outpatient treatment of COVID-19. OBJECTIVE: To examine the influence of SDoH variables on receiving nMAb treatments and the risk of a poor COVID-19 outcome, as well as nMAb treatment effectiveness across SDoH subgroups. DESIGN: Retrospective observational study utilizing electronic health record data from four health systems. SDoH variables analyzed included race, ethnicity, insurance, marital status, Area Deprivation Index, and population density. PARTICIPANTS: COVID-19 patients who met at least one emergency use authorization criterion for nMAb treatment. MAIN MEASURE: We used binary logistic regression to examine the influence of SDoH variables on receiving nMAb treatments and risk of a poor outcome from COVID-19 and marginal structural models to study treatment effectiveness. RESULTS: The study population included 25,241 (15.1%) nMAb-treated and 141,942 (84.9%) non-treated patients. Black or African American patients were less likely to receive treatment than white non-Hispanic patients (adjusted odds ratio (OR) = 0.86; 95% CI = 0.82-0.91). Patients who were on Medicaid, divorced or widowed, living in rural areas, or living in areas with the highest Area Deprivation Index (most vulnerable) had lower odds of receiving nMAb treatment, but a higher risk of a poor outcome. For example, compared to patients on private insurance, Medicaid patients had 0.89 (95% CI = 0.84-0.93) times the odds of receiving nMAb treatment, but 1.18 (95% CI = 1.13-1.24) times the odds of a poor COVID-19 outcome. Age, comorbidities, and COVID-19 vaccination status had a stronger influence on risk of a poor outcome than SDoH variables. nMAb treatment benefited all SDoH subgroups with lower rates of 14-day hospitalization and 30-day mortality. CONCLUSION: Disparities existed in receiving nMAbs within SDoH subgroups despite the benefit of treatment across subgroups.

Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response.

Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

Compassionate Use of REGEN-COV® in Patients With Coronavirus Disease 2019 (COVID-19) and Immunodeficiency-Associated Antibody Disorders.

BACKGROUND: Patients with immunodeficiency-associated antibody disorders are at a higher risk of prolonged/persistent COVID-19 infection, having no viable treatment options. METHODS: A retrospective analysis of patients with primary and/or secondary immunodeficiency-associated antibody disorders who received casirivimab and imdevimab (REGEN-COV®) under emergency compassionate use. Objective were to describe safety and response to REGEN-COV, focusing on the subset of patients who had COVID-19 duration ≥21 days before treatment. RESULTS: Quantitative (change in oxygenation status and/or viral load) and/or qualitative (physician-reported clinical status) outcomes data are reported from 64 patients. Improvement in ≥1 outcome was observed in 90.6% of the overall patient group. Thirty-seven of these had COVID-19 duration ≥21 days before treatment; median time from diagnosis to REGEN-COV treatment was 60.5 days. Of the 29 patients with COVID-19 duration ≥21 days before treatment and available outcome data, 96.6% showed improvement in ≥1 outcome. In the 14 patients with post-treatment reverse transcription-polymerase chain reaction (RT-PCR) results available, 11 (78.6%) reported a negative RT-PCR following treatment, with 5 (45.5%) and 8 (72.7%) patients reporting a negative RT-PCR within 5 days and 21 days of treatment, respectively. Ten of 85 patients (11.8%) experienced serious adverse events; only one was an infusion-related reaction, possibly related to REGEN-COV. Two deaths were reported; neither were attributed to REGEN-COV. CONCLUSIONS: In this retrospective analysis of immunodeficient patients granted REGEN-COV under emergency compassionate use, REGEN-COV treatment was associated with rapid viral clearance and clinical improvement in patients with longstanding COVID-19. Adverse events were consistent with COVID-19 and its associated complications, and due to patients' concurrent medical conditions.

Efficacy and Safety of Sarilumab in Hospitalized Patients With Coronavirus Disease 2019: A Randomized Clinical Trial.

BACKGROUND: Open-label platform trials and a prospective meta-analysis suggest efficacy of anti-interleukin (IL)-6R therapies in hospitalized patients with coronavirus disease 2019 (COVID-19) receiving corticosteroids. This study evaluated the efficacy and safety of sarilumab, an anti-IL-6R monoclonal antibody, in the treatment of hospitalized patients with COVID-19. METHODS: In this adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial, adults hospitalized with COVID-19 received intravenous sarilumab 400 mg or placebo. The phase 3 primary analysis population included patients with critical COVID-19 receiving mechanical ventilation (MV). The primary outcome was proportion of patients with ≥1-point improvement in clinical status from baseline to day 22. RESULTS: There were 457 and 1365 patients randomized and treated in phases 2 and 3, respectively. In phase 3, patients with critical COVID-19 receiving MV (n = 298; 28.2% on corticosteroids), the proportion with ≥1-point improvement in clinical status (alive, not receiving MV) at day 22 was 43.2% for sarilumab and 35.5% for placebo (risk difference, +7.5%; 95% confidence interval [CI], -7.4 to 21.3; P =.3261), a relative risk improvement of 21.7%. In post hoc analyses pooling phase 2 and 3 critical patients receiving MV, the hazard ratio for death for sarilumab vs placebo was 0.76 (95% CI, .51 to 1.13) overall and 0.49 (95% CI, .25 to .94) in patients receiving corticosteroids at baseline. CONCLUSIONS: This study did not establish the efficacy of sarilumab in hospitalized patients with severe/critical COVID-19. Post hoc analyses were consistent with other studies that found a benefit of sarilumab in patients receiving corticosteroids. CLINICAL TRIALS REGISTRATION: NCT04315298.

Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells.

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Prognosis of Chronically Ventilated Patients in a Long-Term Ventilation Facility: Association with Age, Consciousness and Cognitive State.

Background: The number of adults requiring prolonged mechanical ventilation (PMV) including those with cognitive impairment or disorders of consciousness is escalating. We aimed to compare in a long-term acute care hospital (LTACH) mortality and length of stay (LOS) among three age groups (40-59y, 60-79y, ≥80y) of hospitalized PMV patients, and according to consciousness and cognitive state at admission. Methods: We obtained data from the health records of 308 adults aged ≥40 years requiring PMV hospitalized at a Chronic Ventilator Dependent Unit in a LTACH between 01/01/2015 to 06/30/2019 and followed-up until discharge or death or until 12/31/2019. Results: At admission to LTACH, 42.2% of PMV patients were in a vegetative state/ minimally conscious state (VS/MCS); 32.5% were severely cognitively impaired, 11.0% were mildly to moderately cognitively impaired, 12.3% had no cognitive impairment, and 1.9% had intellectual disability/psychiatric disorder. In-LTACH LOS (months) decreased from 34.6 ± 42.6 at age 40-59y, 19.1 ± 22.3 at 60-79y to 14.4 ± 19.3 at age ≥80y (p = .006). In-LTACH mortality was 30.6% for 40-59y, 41.1% for 60-79y and 54.8% for age ≥80y. In-LTACH LOS (months) was 23.8 ± 30.7 for VS/MCS, 15.1 ± 19.5 for the severely cognitively impaired, 10.0 ± 12.8 for mild to moderate cognitive impairment and 18.9 ± 21.9 for those without cognitive impairment (p = .02). In-LTACH mortality was 50.8% for VS/MCS, 58.0% for the severely cognitively impaired, 26.5% for mild to moderate cognitive impairment and 13.2% for those without cognitive impairment (p < .001). Conclusion: In this population requiring PMV, mortality and in-LTACH LOS worsened with age. In-LTACH LOS was longest for VS/MCS patients, who had a mean survival of about two years, followed by those without cognitive impairment and then those with severe cognitive impairment. Mortality was associated with worse consciousness and cognitive state. These findings highlight the importance of discussing end-of-life decisions with patients and family members regarding resuscitation/intubation and the long-term management of these patients.

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers.

BACKGROUND: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. METHODS: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time. RESULTS: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner. CONCLUSIONS: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

Swirling Instability of the Microtubule Cytoskeleton.

In the cellular phenomena of cytoplasmic streaming, molecular motors carrying cargo along a network of microtubules entrain the surrounding fluid. The piconewton forces produced by individual motors are sufficient to deform long microtubules, as are the collective fluid flows generated by many moving motors. Studies of streaming during oocyte development in the fruit fly Drosophila melanogaster have shown a transition from a spatially disordered cytoskeleton, supporting flows with only short-ranged correlations, to an ordered state with a cell-spanning vortical flow. To test the hypothesis that this transition is driven by fluid-structure interactions, we study a discrete-filament model and a coarse-grained continuum theory for motors moving on a deformable cytoskeleton, both of which are shown to exhibit a swirling instability to spontaneous large-scale rotational motion, as observed.

Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., marine, cellulolytic endosymbiotic bacteria isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter.

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12T, purple; others beige to brown), marine salt requirement (Bs12T, Bsc2T and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T).

Malate-aspartate shuttle promotes l-lactate oxidation in mitochondria.

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD+ /NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.

Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torso-like in receptor activation.

Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase Torso. Both Torso and Torso's presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torso-like, which is expressed by follicle cells adjacent to the ends of the developing oocyte. We find that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torso-like or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torso-like were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torso-like were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torso-like alone were ineffective but acted synergistically to stimulate Torso signaling. Our results suggest that Torso interacts with both Trunk and Torso-like, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo.