A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Inflammatory pathways in patients with post-acute sequelae of COVID-19: The role of the clinical immunologist.

As the SARS-CoV-2 pandemic progressed, some survivors noted prolonged symptoms after acute infection, termed post-acute sequelae of COVID-19 (PASC) or "long COVID." PASC is a significant clinical and public health concern that adversely affects patients' quality of life, income, and health care expenses. Moreover, PASC symptoms are highly heterogeneous, the most common being fatigue and cognitive impairment, and they likely reflect a spectrum of clinical phenotypes. The proposed role of persistent inflammation is one of leading pathophysiological theories. This review article addresses these proposed mechanisms of persistent and aberrant inflammation, their clinical evaluation, and theoretical approaches to management. A review of public databases was used to collect literature for the review. The literature supports a prominent role of persistent and aberrant inflammation as a major contributor to the symptoms of PASC. Proposed mechanisms for persistent inflammation include reactivation of latent viruses, viral persistence, loss of immunoregulatory pathways, autoimmune mechanisms, and/or mast cell dysregulation. Persistent inflammation may result in constitutional symptoms such as fatigue, brain fog, body aches, and/or organ-specific dysfunction, such as gastrointestinal dysregulation and myocardial inflammation. There are no approved or even proven therapies for PASC at this time, but some studies have identified therapeutic options that may either reduce the risk for progression to PASC or decrease symptom burden. Laboratory evaluation and therapeutic options are limited and require further investigation to establish their clinical value. A more refined definition of PASC is needed to address the wide variety of clinical presentations, pathophysiology, and therapeutic options.

PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.

RATIONALE: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. OBJECTIVE: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. METHODS AND RESULTS: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. CONCLUSIONS: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.

'No-One Can Tell a Story Better than the One Who Lived It': Reworking Constructions of Childhood and Trauma Through the Arts in Rwanda.

The intergenerational legacies of conflict and violence for children and young people are typically approached within research and interventions through the lens of trauma. Understandings of childhood and trauma are based on bio-psychological frameworks emanating from the Global North, often at odds with the historical, political, economic, social and cultural contexts in which interventions are enacted, and neglect the diversity of knowledge, experiences and practices. Within this paper we explore these concerns in the context of Rwanda and the aftermath of the 1994 Genocide Against the Tutsi. We reflect on two qualitative case studies: Connective Memories and Mobile Arts for Peace which both used arts-based approaches drawing on the richness of Rwandan cultural forms, such as proverbs and storytelling practices, to explore knowledge and processes of meaning-making about trauma, memory, and everyday forms of conflict from the perspectives of children and young people. We draw on these findings to argue that there is a need to refine and elaborate understandings of intergenerational transmission of trauma in Rwanda informed by: the historical and cultural context; intersections of structural and 'everyday' forms of conflict and social trauma embedded in intergenerational relations; and a reworking of notions of trauma 'transmission' to encompass the multiple connectivities between generations, temporalities and expressions of trauma.

Fisheries management impacts on target species status.

Fisheries management systems around the world are highly diverse in their design, operation, and effectiveness at meeting objectives. A variety of management institutions, strategies, and tactics are used across disparate regions, fishing fleets, and taxonomic groups. At a global level, it is unclear which particular management attributes have greatest influence on the status of fished populations, and also unclear which external factors affect the overall success of fisheries management systems. We used expert surveys to characterize the management systems by species of 28 major fishing nations and examined influences of economic, geographic, and fishery-related factors. A Fisheries Management Index, which integrated research, management, enforcement, and socioeconomic attributes, showed wide variation among countries and was strongly affected by per capita gross domestic product (positively) and capacity-enhancing subsidies (negatively). Among 13 management attributes considered, three were particularly influential in whether stock size and fishing mortality are currently in or trending toward desirable states: extensiveness of stock assessments, strength of fishing pressure limits, and comprehensiveness of enforcement programs. These results support arguments that the key to successful fisheries management is the implementation and enforcement of science-based catch or effort limits, and that monetary investment into fisheries can help achieve management objectives if used to limit fishing pressure rather than enhance fishing capacity. Countries with currently less-effective management systems have the greatest potential for improving long-term stock status outcomes and should be the focus of efforts to improve fisheries management globally.

A prospective observational study of ethnic and racial differences in neuraxial labor analgesia request and pain relief.

BACKGROUND: As ethnic and racial diversity increases, it is important that anesthesia providers understand the expectations and concerns of this changing population regarding labor analgesia. Our objective was to evaluate ethnic/racial differences in labor analgesia characteristics with regard to the timing of request for neuraxial analgesia. METHODS: Three hundred ninety-seven parturients were enrolled in this prospective observational cohort study. Term laboring parturients who planned vaginal delivery and requested neuraxial labor analgesia were eligible for inclusion. Data collected included cervical dilation at the time of neuraxial analgesia request, self-identified ethnicity/race, parity, education, insurance status, pain score before and after the initiation of neuraxial analgesia, and mode of delivery. The primary outcome was cervical dilation at the time of neuraxial analgesia request. Ethnicity/race classification was determined by asking the patient, "How would you define your ethnicity?" Patients were categorized into the ethnic/racial groups of non-Hispanic White, African American, Hispanic, or other. Univariate associations between cervical dilation and categorical variables were examined. Multivariate analysis was performed for the primary outcome of cervical dilation at the time of initiation of neuraxial analgesia. RESULTS: At the time of neuraxial analgesia placement, the mean difference in cervical dilation of Hispanic parturients was 0.8 cm compared to non-Hispanic Whites (95% confidence interval [CI], 0.1-1.4; P = 0.047). After controlling for education, reason for placement, labor augmentation, and mode of delivery in a multivariate model, Hispanic parturients had 0.5 cm greater cervical dilation compared to non-Hispanic Whites, which was not significant (95% confidence interval, -0.1 to 1.1; P = 0.089). CONCLUSIONS: Our data indicate that ethnicity/race plays a small role in acceptance and request for neuraxial labor analgesia.

Contexts of urgency may go beyond emotion.

Introduction: Urgency has been defined as the tendency towards rash speech and behavior in the context of emotion. Measures of Urgency have been found to have robust predictive power for psychopathologies and problematic behaviors. In the current study, we question whether emotions are unique drivers of urgency, or if emotions are potent exemplars of contexts that lead to rash speech and behavior. The Emotion Specific model and the Broader Contexts model correspond with these two conceptualizations of urgency, and they frame our pre-registered hypotheses. Methods: Participants from two well-powered samples (n = 600,n = 588) completed 9 modified items from the Urgency and Positive Urgency scales to assess rash responses in each of four contexts - "Upset," "Excited," "Tired," and "Hungry" - and a fifth "In General" set. After data cleaning, we used principal components analysis to construct a unidimensional, 4-item set that was applied to capture impulsive behavior across the five contexts. Results: We found that this research tool, called the Contexts of Impulsive Behaviors (CIBS), replicated in the second dataset, and it had adequate internal reliability in both samples. Although the Emotion Specific model was supported by the fact that the Upset context had a greater mean and greater variance than the Tired and Hungry contexts, most results supported the Broader Contexts model. That is, CIBS contexts were highly intercorrelated, and bivariate correlations with psychopathology were not significantly different across contexts. In partial correlations, effects of the Upset and Excited contexts were partially or fully statistically mediated by the Tired and Hungry contexts. Discussion: These findings suggest that emotions are potent contexts for impulsive behaviors. At the same time, those with high urgency are vulnerable to impulsivity in other contexts, such as fatigue and hunger, that challenge the regulatory functions of the prefrontal cortex. Limitations, future directions, and clinical implications are discussed.

Emotion-related impulsivity is related to orbitofrontal cortical sulcation.

Background: Emotion-related impulsivity (ERI) describes the trait-like tendency toward poor self-control when experiencing strong emotions. ERI has been shown to be elevated across psychiatric disorders and predictive of the onset and worsening of psychiatric syndromes. Recent work has correlated ERI scores with the neuroanatomy of the orbitofrontal cortex (OFC). Informed by a growing body of research indicating that the morphology of cortical folds (sulci) can produce insights into behavioral outcomes, the present study modeled the association between ERI and the sulcal morphology of OFC at a finer scale than previously conducted. Methods: Analyses were conducted in a transdiagnostic sample of 118 individuals with a broad range of psychiatric syndromes. We first manually defined over 2000 sulci across the 118 participants. We then implemented a model-based LASSO regression to relate OFC sulcal morphology to ERI and test whether effects were specific to ERI as compared to non-emotion-related impulsivity. Results: The LASSO regression revealed bilateral associations of ERI with the depth of eight OFC sulci. These effects were specific to ERI and were not observed in non-emotion-related impulsivity. In addition, we identified a new transverse component of the olfactory sulcus in every hemisphere that is dissociable from the longitudinal component based on anatomical features and correlation with behavior, which could serve as a new transdiagnostic biomarker. Conclusions: The results of this data-driven investigation provide greater neuroanatomical and neurodevelopmental specificity on how OFC is related to ERI. As such, findings link neuroanatomical characteristics to a trait that is highly predictive of psychopathology.

Variable Presence of an Evolutionarily New Brain Structure is Related to Trait Impulsivity.

Background: Impulsivity is a multidimensional construct reflecting poor constraint over one's behaviors. Clinical psychology research identifies separable impulsivity dimensions that are each unique transdiagnostic indicators for psychopathology. Yet, despite this apparent clinical importance, the shared and unique neuroanatomical correlates of these factors remain largely unknown. Concomitantly, neuroimaging research identifies variably present human brain structures implicated in cognition and disorder: the folds (sulci) of the cerebral cortex located in the latest developing and most evolutionarily expanded hominoid-specific association cortices. Methods: We tethered these two fields to test whether variability in one such structure in anterior cingulate cortex (ACC)-the paracingulate sulcus (PCGS)-was related to individual differences in trait impulsivity. 120 adult participants with internalizing or externalizing psychopathology completed a magnetic resonance imaging scan and the Three-Factor Impulsivity Index. Using precision imaging techniques, we manually identified the PCGS, when present, and acquired quantitative folding metrics (PCGS length and ACC local gyrification index). Results: Neuroanatomical-behavioral analyses revealed that participants with leftward or symmetrical PCGS patterns had greater severity of Lack of Follow Through (LFT)-which captures inattention and lack of perseverance-than those with rightward asymmetry. Neuroanatomical-functional analyses identified that the PCGS co-localized with a focal locus found in a neuroimaging meta-analysis on a feature underlying LFT. Both quantitative folding metrics did not relate to any impulsivity dimension. Conclusions: This study advances understanding of the neuroanatomical correlates of impulsivity and establishes the notion that the topographical organization of distinct, hominoid-specific cortical expanses underlie separable impulsivity dimensions with robust, transdiagnostic implications for psychopathology.

A feedback-driven IoT microfluidic, electrophysiology, and imaging platform for brain organoid studies.

The analysis of tissue cultures, particularly brain organoids, takes a high degree of coordination, measurement, and monitoring. We have developed an automated research platform enabling independent devices to achieve collaborative objectives for feedback-driven cell culture studies. Unified by an Internet of Things (IoT) architecture, our approach enables continuous, communicative interactions among various sensing and actuation devices, achieving precisely timed control of in vitro biological experiments. The framework integrates microfluidics, electrophysiology, and imaging devices to maintain cerebral cortex organoids and monitor their neuronal activity. The organoids are cultured in custom, 3D-printed chambers attached to commercial microelectrode arrays for electrophysiology monitoring. Periodic feeding is achieved using programmable microfluidic pumps. We developed computer vision fluid volume estimations of aspirated media, achieving high accuracy, and used feedback to rectify deviations in microfluidic perfusion during media feeding/aspiration cycles. We validated the system with a 7-day study of mouse cerebral cortex organoids, comparing manual and automated protocols. The automated experimental samples maintained robust neural activity throughout the experiment, comparable with the control samples. The automated system enabled hourly electrophysiology recordings that revealed dramatic temporal changes in neuron firing rates not observed in once-a-day recordings.

Safety and efficacy of arimoclomol in patients with early amyotrophic lateral sclerosis (ORARIALS-01): a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial.

BACKGROUND: Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder leading to muscle weakness and respiratory failure. Arimoclomol, a heat-shock protein-70 (HSP70) co-inducer, is neuroprotective in animal models of amyotrophic lateral sclerosis, with multiple mechanisms of action, including clearance of protein aggregates, a pathological hallmark of sporadic and familial amyotrophic lateral sclerosis. We aimed to evaluate the safety and efficacy of arimoclomol in patients with amyotrophic lateral sclerosis. METHODS: ORARIALS-01 was a multinational, randomised, double-blind, placebo-controlled, parallel-group trial done at 29 centres in 12 countries in Europe and North America. Patients were eligible if they were aged 18 years or older and met El Escorial criteria for clinically possible, probable, probable laboratory-supported, definite, or familial amyotrophic lateral sclerosis; had an ALS Functional Rating Scale-Revised score of 35 or more; and had slow vital capacity at 70% or more of the value predicted on the basis of the participant's age, height, and sex. Patients were randomly assigned (2:1) in blocks of 6, stratified by use of a stable dose of riluzole or no riluzole use, to receive oral arimoclomol citrate 1200 mg/day (400 mg three times per day) or placebo. The Randomisation sequence was computer generated centrally. Investigators, study personnel, and study participants were masked to treatment allocation. The primary outcome was the Combined Assessment of Function and Survival (CAFS) rank score over 76 weeks of treatment. The primary outcome and safety were analysed in the modified intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT03491462, and is completed. FINDINGS: Between July 31, 2018, and July 17, 2019, 287 patients were screened, 245 of whom were enrolled in the trial and randomly assigned. The modified intention-to-treat population comprised 239 patients (160 in the arimoclomol group and 79 in the placebo group): 151 (63%) were male and 88 (37%) were female; mean age was 57·6 years (SD 10·9). CAFS score over 76 weeks did not differ between groups (mean 0·51 [SD 0·29] in the arimoclomol group vs 0·49 [0·28] in the placebo group; p=0·62). Cliff's delta comparing the two groups was 0·039 (95% CI -0·116 to 0·194). Proportions of participants who died were similar between the treatment groups: 29 (18%) of 160 patients in the arimoclomol group and 18 (23%) of 79 patients in the placebo group. Most deaths were due to disease progression. The most common adverse events were gastrointestinal. Adverse events were more often deemed treatment-related in the arimoclomol group (104 [65%]) than in the placebo group (41 [52%]) and more often led to treatment discontinuation in the arimoclomol group (26 [16%]) than in the placebo group (four [5%]). INTERPRETATION: Arimoclomol did not improve efficacy outcomes compared with placebo. Although available biomarker data are insufficient to preclude future strategies that target the HSP response, safety data suggest that a higher dose of arimoclomol would not have been tolerated. FUNDING: Orphazyme.

Labile hypertension: characteristics of a referred cohort.

From 88 subjects with labile high blood pressure (LHBP), we collected blood pressure variability (BPV) and assessed relationships with age, medications, and nocturnal pattern via ambulatory blood pressure monitoring. The average age of the subjects was 64 ± 13 years and they were on 1.5 ± 1.3 antihypertensives. BPV did not differ diurnally and was not influenced by medication. Aging associates with increasing daytime systolic but not diastolic BPV, with increasing nighttime systolic BP, and decreasing diastolic BP diurnally. Subjects had widened pulse pressure and abnormal diurnal pattern with age. Further studies are needed to stratify an individual's risk associated with LHBP.

Outreach surgical consulting services in North East Victoria.

OBJECTIVE: There is a paucity of data regarding the provision of consultative outreach specialist surgical services to rural areas. This paper aims to describe a model of outreach consultative practice to deliver specialist surgical services to rural communities. DESIGN: Analysis of prospectively collected data for consultations in a three month period for two surgeons based in Wangaratta. SETTING: Two surgeons in regional Victoria based in Wangaratta, North East Victoria, conducting outreach consultations to Beechworth, Benalla, Bright and Mansfield. PARTICIPANTS: All patients seen in consultations over a 3-month period. MAIN OUTCOME MEASURES: Patient workload, casemix of presenting complaint, consultation outcome including plan for surgical procedure. RESULTS: Outreach surgical consulting was associated with a higher proportion of new consultations, and there was trend towards being more likely to result in a surgical procedure than consultations in the base rural centre. CONCLUSIONS: Outreach surgical consulting provides surgeons with a larger referral base and provides communities with better access to local specialists. Outreach practice should be encouraged for surgeons in regional centres.

Emotion-related impulsivity moderates the role of arousal on reflection impulsivity.

Emotion-related impulsivity is an important behavioural phenotype in clinical psychology and public health. Here, we test the hypothesis that emotion-related impulsivity moderates the effects of arousal on cognition using pharmacological manipulation. Participants completed a measure of emotion-related impulsivity, four cognitive tasks tapping onto different facets of impulsive behaviours, and a blinded arousal manipulation using yohimbine hydrochloride, which acts on noradrenergic receptors. Our findings suggest that emotion-related impulsivity moderates the role of arousal on impulsive performance on the Information Sampling Task. As expected, more severe emotion-related impulsivity was related to more impulsive decisions in the yohimbine but not in the placebo group. Results provide some of the first experimental evidence that emotion-related impulsivity is related to differential behavioural responses in the face of high arousal. Despite this preliminary support, we discuss findings for one task that did not fit hypotheses, and provide suggestions for replication and extension.

An Update on Monoclonal Antibody Therapy to Treat Moderate-to-Severe Asthma: Benefits, Choices, and Limitations.

Moderate or severe asthma is a complex disease process clinically manifesting as at least partially reversible airway obstruction due to airway hyperresponsiveness. Asthma therapy was based primarily on symptom control until recent studies of its mechanisms have led to a host of new targeted, safe, and effective therapies. These biologic therapies directly attack culprit inflammatory mediators at the molecular level. In this article we review currently available biologic agents for the treatment of moderate-to-severe asthma. We provide information deemed necessary to optimally consult with an asthma specialist to choose, assist in financial arrangements for, and coordinate the use of these new, promising, Food and Drug Administration-approved biologic agents. We will also briefly review the molecular pathways targeted with each class of biologic to provide a more in-depth understanding of why these targeted therapies are effective. These biologics are the first of many to come that modify newly discovered components of the immune system with which many physicians are unfamiliar.

Emotion-related impulsivity and risky decision-making: A systematic review and meta-regression.

Emotion-related impulsivity, the trait-like tendency toward regrettable behavior during states of high emotion, is a robust predictor of internalizing and externalizing psychopathology. Despite substantial evidence that emotion-related impulsivity is important transdiagnostically, relatively little is known about its cognitive correlates. This systematic review and meta-regression investigates one such candidate, risky decision-making. We analyzed 195 effect sizes from 51 studies of 14,957 total participants, including 105 newly calculated effect sizes that were not reported in the original publications. The meta-regression demonstrated evidence for a small, positive relationship of emotion-related impulsivity with behavioral indices of risky decision-making (ß = 0.086). Effects generalized across sample age, gender, Positive versus Negative Urgency, and clinical versus nonclinical samples. The average effect size varied by task type, with stronger effects for the Iowa Gambling Task and Delay Discounting Task. Experimental arousal manipulation was nearly a significant moderator, with stress and pharmacological manipulations yielding significant effect sizes. Analyses indicated that publication bias did not skew the current findings. Notwithstanding limitations, the data suggest that risky decision-making is a cognitive domain that relates to emotion-related impulsivity. We conclude with recommendations regarding the specific types of tasks and arousal inductions that will best capture emotion-related impulsivity in future experimental research.

Neuroanatomical Correlates of Emotion-Related Impulsivity.

BACKGROUND: Emotion-related impulsivity (ERI) refers to chronically poor self-control during periods of strong emotion. ERI robustly predicts psychiatric disorders and related problems, yet its neuroanatomical correlates are largely unknown. We tested whether local brain morphometry in targeted brain regions that integrate emotion and control could explain ERI severity. METHODS: One hundred twenty-two adults (ages 18-55 years) with internalizing or externalizing psychopathology completed a structural magnetic resonance imaging (MRI) scan, the Three-Factor Impulsivity Index, and the Structured Clinical Interview for DSM-5. The Three-Factor Impulsivity Index measures two types of ERI and a third type of impulsivity not linked to emotion. Cortical reconstruction yielded cortical thickness and local gyrification measurements. We evaluated whether morphometry in the orbitofrontal cortex (OFC), insula, amygdala, and nucleus accumbens was associated with ERI severity. Hypotheses and analyses were preregistered. RESULTS: Lower cortical gyrification in the right lateral OFC was associated with high ERI severity in a full, preregistered model. Separate examinations of local gyrification and cortical thickness also showed a positive association between gyrification in the left lateral OFC and ERI. An integrated measure of hemispheric imbalance in lateral OFC gyrification (right < left) correlated with ERI severity. These findings were specific to ERI and did not appear with non-emotion-related impulsivity. CONCLUSIONS: Local gyrification in the lateral OFC is associated with ERI severity. The current findings fit with existing theories of OFC function, strengthen the connections between the transdiagnostic literature in psychiatry and neuroscience, and may guide future treatment development.