Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells.

Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and ß-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and ß-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and ß-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the ß-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.

Conversational Agents in Health Care: Scoping Review of Their Behavior Change Techniques and Underpinning Theory.

BACKGROUND: Conversational agents (CAs) are increasingly used in health care to deliver behavior change interventions. Their evaluation often includes categorizing the behavior change techniques (BCTs) using a classification system of which the BCT Taxonomy v1 (BCTTv1) is one of the most common. Previous studies have presented descriptive summaries of behavior change interventions delivered by CAs, but no in-depth study reporting the use of BCTs in these interventions has been published to date. OBJECTIVE: This review aims to describe behavior change interventions delivered by CAs and to identify the BCTs and theories guiding their design. METHODS: We searched PubMed, Embase, Cochrane's Central Register of Controlled Trials, and the first 10 pages of Google and Google Scholar in April 2021. We included primary, experimental studies evaluating a behavior change intervention delivered by a CA. BCTs coding followed the BCTTv1. Two independent reviewers selected the studies and extracted the data. Descriptive analysis and frequent itemset mining to identify BCT clusters were performed. RESULTS: We included 47 studies reporting on mental health (n=19, 40%), chronic disorders (n=14, 30%), and lifestyle change (n=14, 30%) interventions. There were 20/47 embodied CAs (43%) and 27/47 CAs (57%) represented a female character. Most CAs were rule based (34/47, 72%). Experimental interventions included 63 BCTs, (mean 9 BCTs; range 2-21 BCTs), while comparisons included 32 BCTs (mean 2 BCTs; range 2-17 BCTs). Most interventions included BCTs 4.1 "Instruction on how to perform a behavior" (34/47, 72%), 3.3 "Social support" (emotional; 27/47, 57%), and 1.2 "Problem solving" (24/47, 51%). A total of 12/47 studies (26%) were informed by a behavior change theory, mainly the Transtheoretical Model and the Social Cognitive Theory. Studies using the same behavior change theory included different BCTs. CONCLUSIONS: There is a need for the more explicit use of behavior change theories and improved reporting of BCTs in CA interventions to enhance the analysis of intervention effectiveness and improve the reproducibility of research.

#EatingDisorderRecovery: a qualitative content analysis of eating disorder recovery-related posts on Instagram.

PURPOSE: Limited research has evaluated the role of Social Networking Sites (SNS) in eating disorder (ED) recovery. While research has demonstrated the deleterious effects of pro-ED SNS content, less is known regarding SNS content documenting ED recovery. This study evaluates orientation towards help-seeking, ongoing ED warning signs and recovery themes on ED recovery SNS hashtags. METHODS: 600 Instagram posts were retrieved from two popular hashtags: #EDrecovery and #EatingDisorderRecovery. They were categorized into four thematic concerns: Food, Quotes, People or Others. Posts were analysed for behavioural and psychological signs of ED based on the Mental Health First Aid Eating Disorders Guidelines, and whether they encouraged seeking professional help. Thematic qualitative analysis to evaluate themes posted on recovery hashtags was conducted. RESULTS: Of the 600 posts, 405 were used for analysis. The majority of posts were on Food (49.6%), Quotes (24.2%) and People (22.7%). Behavioural and psychological signs suggestive of EDs were present in 18.0% and 22.5% of images, respectively. Only 9.4% of posts encouraged seeking professional help. Important themes that emerged from the qualitative analysis included the recovery journey, increased awareness and stigma for EDs and the development of a supportive community. CONCLUSIONS: Despite identifying with ED recovery, posts had a high prevalence of ongoing ED behaviour and low prevalence of help-seeking. Thematic analysis emphasized the role of recovery as a journey and the role of stigma and community in recovery. These findings suggest that SNSs could potentially be leveraged as a platform to improve help-seeking and encourage recovery for users with eating disorders. LEVEL OF EVIDENCE: Level V, descriptive study.

Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation.

Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

Assembling the RNA therapeutics toolbox.

From the approval of COVID-19 mRNA vaccines to the 2023 Nobel Prize awarded for nucleoside base modifications, RNA therapeutics have entered the spotlight and are transforming drug development. While the term "RNA therapeutics" has been used in various contexts, this review focuses on treatments that utilize RNA as a component or target RNA for therapeutic effects. We summarize the latest advances in RNA-targeting tools and RNA-based technologies, including but not limited to mRNA, antisense oligos, siRNAs, small molecules and RNA editors. We focus on the mechanisms of current FDA-approved therapeutics but also provide a discussion on the upcoming workforces. The clinical utility of RNA-based therapeutics is enabled not only by the advances in RNA technologies but in conjunction with the significant improvements in chemical modifications and delivery platforms, which are also briefly discussed in the review. We summarize the latest RNA therapeutics based on their mechanisms and therapeutic effects, which include expressing proteins for vaccination and protein replacement therapies, degrading deleterious RNA, modulating transcription and translation efficiency, targeting noncoding RNAs, binding and modulating protein activity and editing RNA sequences and modifications. This review emphasizes the concept of an RNA therapeutic toolbox, pinpointing the readers to all the tools available for their desired research and clinical goals. As the field advances, the catalog of RNA therapeutic tools continues to grow, further allowing researchers to combine appropriate RNA technologies with suitable chemical modifications and delivery platforms to develop therapeutics tailored to their specific clinical challenges.

Genome-wide studies in prostate cancer poised liquid biopsy as a molecular discovery tool.

Liquid biopsy is emerging as an intriguing tool in clinical disease detection and monitoring. Compared to a standard tissue biopsy, performing a liquid biopsy incurs minimal invasiveness, captures comprehensive disease representation, and can be more sensitive at an early stage. Recent genome-wide liquid biopsy studies in prostate cancer analyzing plasma samples have provided insights into the genome and epigenome dynamics during disease progression. In-depth genomic sequencing can offer a comprehensive understanding of cancer evolution, enabling more accurate clinical decision-making. Furthermore, exploring beyond the DNA sequence itself provides opportunities to investigate the regulatory mechanisms underlying various disease phenotypes. Here, we summarize these advances and offer prospects for their future application.

The Basis and Promise of Programmable RNA Editing and Modification.

One key advantage of RNA over genomic editing is its temporary effects. Aside from current use of DNA-targeting CRISPR-Cas9, the more recently discovered CRISPR-Cas13 has been explored as a means of editing due to its RNA-targeting capabilities. Specifically, there has been a recent interest in identifying and functionally characterizing biochemical RNA modifications, which has spurred a new field of research known as "epitranscriptomics". As one of the most frequently occurring transcriptome modifications, N6-methyladenosine (m6A) has generated much interest. The presence of m6A modifications is under the tight control of a series of regulators, and the ability of fusing these proteins or demethylases to catalytically inactive CRISPR proteins have resulted in a new wave of programmable RNA methylation tools. In addition, studies have been conducted to develop different CRISPR/Cas and base editor systems capable of more efficient editing, and some have explored the effects of in vivo editing for certain diseases. As well, the application of CRISPR and base editors for screening shows promise in revealing the phenotypic outcomes from m6A modification, many of which are linked to physiological, and pathological effects. Thus, the therapeutic potential of CRISPR/Cas and base editors for not only m6A related, but other RNA and DNA related disease has also garnered insight. In this review, we summarize/discuss the recent findings on RNA editing with CRISPR, base editors and non-CRISPR related tools and offer a perspective regarding future applications for basic and clinical research.

A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-ß signaling.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19.

Choice of Behavioral Change Techniques in Health Care Conversational Agents: Protocol for a Scoping Review.

BACKGROUND: Conversational agents or chatbots are computer programs that simulate conversations with users. Conversational agents are increasingly used for delivery of behavior change interventions in health care. Behavior change is complex and comprises the use of one or several components collectively known as behavioral change techniques (BCTs). OBJECTIVE: The objective of this scoping review is to identify the BCTs that are used in behavior change-focused interventions delivered via conversational agents in health care. METHODS: This scoping review will be performed in line with the Joanna Briggs Institute methodology and will be reported according to the PRISMA extension for scoping reviews guidelines. We will perform a comprehensive search of electronic databases and grey literature sources, and will check the reference lists of included studies for additional relevant studies. The screening and data extraction will be performed independently and in parallel by two review authors. Discrepancies will be resolved through consensus or discussion with a third review author. We will use a data extraction form congruent with the key themes and aims of this scoping review. BCTs employed in the included studies will be coded in line with BCT Taxonomy v1. We will analyze the data qualitatively and present it in diagrammatic or tabular form, alongside a narrative summary. RESULTS: To date, we have designed the search strategy and performed the search on April 26, 2021. The first round of screening of retrieved articles is planned to begin soon. CONCLUSIONS: Using appropriate BCTs in the design and delivery of health care interventions via conversational agents is essential to improve long-term outcomes. Our findings will serve to inform the development of future interventions in this area. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/30166.

The relationship between variability in baseball pitching kinematics and consistency in pitch location.

The purpose of this study was to explore the relationship between variability in pitching kinematics and consistency in pitch location. Data were collected for 47 healthy baseball pitchers throwing ten full-effort fastballs to the centre of the strike zone. For each pitch, 20 kinematic parameters were calculated with an automated motion capture system while pitch location was measured with a PITCHf/x system. Variability of each kinematic parameter was defined for each pitcher as the standard deviation among his fastballs thrown. For calculating consistency, each pitcher's mean pitch location was first calculated. The distances from each individual pitch to the mean pitch location were then found for each pitcher tested. A consistency metric was then calculated for each pitcher by averaging these distances. A multiple linear regression model was developed using stepwise regression with backwards elimination. The resulting model explained 58% of the variance in the consistency metric and included five parameters, three at foot contact (upper trunk tilt, shoulder abduction, and shoulder horizontal abduction) and two at time of maximum shoulder external rotation (shoulder external rotation and shoulder horizontal adduction). Reducing variability at the shoulder during the early portions of the pitching motion may improve consistency of ball location.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-ß signaling.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

The influence of mound height on baseball movement and pitching biomechanics.

OBJECTIVES: To determine whether mound height is associated with baseball movement (velocity, spin and break) and baseball pitching biomechanics (kinematics and kinetics). DESIGN: Controlled laboratory study. METHODS: Twenty collegiate baseball pitchers threw five fastballs and five curveballs from four different mound heights (15cm, 20cm, 25cm, 30cm) in a randomized order. Ball movement was computed by a ball tracking system, while pitching biomechanics were calculated with an 11-camera optical motion capture system. Repeated measures analysis of variance was utilized to detect significant differences among the four different mound heights (p<0.05) for the fastball and curveball pitches. RESULTS: There were no significant differences observed for ball movement. There were seven significant kinematic differences for fastballs and eight kinematic differences for curveballs. Although these differences were statistically significant, the magnitudes were small, with most joint angles changing by less than 2°. There were no significant kinetic differences for curveballs, but five kinetic parameters (elbow varus torque, elbow flexion torque, elbow proximal force, shoulder internal rotation torque, and shoulder anterior force) varied with mound height for fastballs. In general, fastball kinetics were 1%-2% less from the lowered (15cm, 20cm) mounds than from the standard (25cm) or raised (30cm) mounds. CONCLUSIONS: Lowering the mound may not affect a pitcher's ball movement, but may slightly reduce shoulder and elbow kinetics, possibly reducing the risk of injury.