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Abstract Genome-wide association studies (GWAS) identify genetic variants underlying complex traits but are limited by stringent genome-wide significance thresholds. We present GRIN (Gene set Refinement through Interacting Networks), which increases confidence in the expanded gene set by retaining genes strongly connected by biological networks when GWAS thresholds are relaxed. GRIN was validated on both simulated interrelated gene sets as well as multiple GWAS traits. From multiple GWAS summary statistics of suicide attempt, a complex phenotype, GRIN identified additional genes that replicated across independent cohorts and retained biologically interrelated genes despite a relaxed significance threshold. We present a conceptual model of how these retained genes interact through neurobiological pathways that may influence suicidal behavior, and identify existing drugs associated with these pathways that would not have been identified under traditional GWAS thresholds. We demonstrate GRIN’s utility in boosting GWAS results by increasing the number of true positive genes identified from GWAS results.

Previous studies have demonstrated an association between low personal protective equipment (PPE) availability and high stress and anxiety among frontline healthcare workers during the COVID-19 pandemic. It is unclear how other factors, such as infection prevention and control (IPC) training and IPC policy support, correlate with workers’ distress. The current study explores these relationships. We conducted a secondary analysis of a public survey dataset from Statistics Canada. Acute care workers’ survey responses (n = 7379) were analyzed using structural equation modeling to examine relationships between features of the IPC work environment and acute care workers’ ratings of their stress and mental health. We found that PPE availability (β = −0.16), workplace supports (i.e., training, IPC policy compliance, and enforcement) (β = −0.16), and support for staying home when sick (β = −0.19) were all negatively correlated with distress. Together, these features explained 18.4% of the overall variability in workers’ distress. Among surveyed acute care workers, PPE availability was related to their distress; however, having workplace support and an emphasis on staying home when sick was also relevant. Overall, the results highlight that, in addition to PPE availability, workplace supports and emphasis on staying home are important. IPC professionals and healthcare leaders should consider these multiple features as they support acute care workers during future infectious disease outbreaks.

Abstract Pulmonary vascular dysfunction in the absence of pulmonary hypertension (PH) has been observed in patients with idiopathic pulmonary fibrosis (IPF). We describe the prevalence and etiology of elevated pulmonary vascular resistance (PVR) without PH among patients with IPF. Hemodynamic, echocardiographic, and functional respiratory imaging (FRI) data was compared between patients with IPF without PH with normal (

ABSTRACT Francisella tularensis is a highly infectious Gram-negative bacterial pathogen capable of animal-to-human transmission. Due to its remarkable pathogenicity and potential for widespread public health impact, F. tularensis is classified as a high-priority pathogen by the Centers for Disease Control and Prevention. The historical development of Francisella as a bioweapon, coupled with the ease of engineering antibiotic-resistant strains, has spurred interest in the discovery of novel antibiotics. A library of drug-like compounds was screened for their ability to inhibit the growth of Francisella novicida, a biosafety level-2 surrogate for F. tularensis. Tolfenpyrad, a pesticide without previously identified antibacterial activity, was among the most potent inhibitors. Tolfenpyrad appears to uniquely target Francisella, as related species were sensitive, whereas bacteria in other genera were resistant. To identify Francisella pathways conferring sensitivity to tolfenpyrad, whole-genome sequencing of tolfenpyrad-resistant isolates was performed. Point mutations in two genes conferred resistance to osrR, a transcriptional regulator, and nuoM, a subunit of the electron transport chain. OsrR point mutants displayed attenuated growth in interferon gamma (IFN-γ)-stimulated macrophages but not in untreated macrophages, indicating that OsrR plays a role in resistance to IFN-γ-stimulated clearance of intracellular bacteria. OsrR point mutants are hypersensitive to reactive oxygen species, suggesting a mechanism by which they are rapidly cleared from IFN-γ-treated macrophages. This work identifies a novel Francisella-specific antibiotic activity and the metabolic pathways targeted. Moreover, we provide amino acid-level details of a Francisella protein that governs sensitivity to free radicals. IMPORTANCE Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella. This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.

Background:. Multiple studies have compared different pharmacologic thromboprophylaxis agents after hip fracture surgery, including aspirin, unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), direct oral anticoagulants (DOAC), and warfarin, resulting in variability in clinical practice. To guide clinical management, a systematic review and network meta-analysis (NMA), which enables the simultaneous assessment of the effects of multiple interventions for the same patient population, was performed. This study aimed to determine the comparative effectiveness of thromboprophylaxis in reducing venous thromboembolism (VTE) in patients with surgically treated hip fractures. Methods:. The primary outcome was the effect of the treatment on the VTE rate, and the secondary outcome was the treatment effect on the bleeding rate. Relevant studies were identified by a systematic search of Embase, MEDLINE, and the Cochrane Central Register of Controlled Trials (CENTRAL) from January 2000 to February 2022. Title, abstract, and full-text screening; data extraction; and risk-of-bias assessment were performed. All studies examining thromboprophylaxis interventions (DOAC, LMWH, UFH, aspirin, and warfarin) in patients with a surgically treated hip fracture were included. Bayesian NMA was performed, and dichotomous outcome data were pooled using the odds ratio. Interventions were ranked using the surface under the cumulative ranking curve (SUCRA) for each outcome. Results:. A total of 19 studies were included after the screening of 466 citations and 77 full-text articles. Of the included studies, 15 studies had a high overall risk of bias. The NMA of the VTE outcome included 19 studies, 49,409 participants, and 6 thromboprophylaxis interventions. The NMA of the bleeding outcome included 3 studies, 18,163 participants, and 3 interventions. The mean age ranged from 43.5 to 86.2 years among the included studies. No thromboprophylaxis intervention was statistically different from any other intervention in its effect on the VTE or bleeding rate in hip fracture patients. Conclusions:. This NMA demonstrated that there was no difference between the thromboprophylaxis interventions in reducing VTE or bleeding rates in hip fracture patients. More robust randomized controlled trials are needed to determine the most effective thromboprophylaxis interventions for patients with hip fractures. Level of Evidence:. Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

For plants, distinguishing between mutualistic and pathogenic microbes is a matter of survival. All microbes contain microbe-associated molecular patterns (MAMPs) that are perceived by plant pattern recognition receptors (PRRs). Lysin motif receptor-like kinases (LysM-RLKs) are PRRs attuned for binding and triggering a response to specific MAMPs, including chitin oligomers (COs) in fungi, lipo-chitooligosaccharides (LCOs), which are produced by mycorrhizal fungi and nitrogen-fixing rhizobial bacteria, and peptidoglycan in bacteria. The identification and characterization of LysM-RLKs in candidate bioenergy crops including Populus are limited compared to other model plant species, thus inhibiting our ability to both understand and engineer microbe-mediated gains in plant productivity. As such, we performed a sequence analysis of LysM-RLKs in the Populus genome and predicted their function based on phylogenetic analysis with known LysM-RLKs. Then, using predictive models, molecular dynamics simulations, and comparative structural analysis with previously characterized CO and LCO plant receptors, we identified probable ligand-binding sites in Populus LysM-RLKs. Using several machine learning models, we predicted remarkably consistent binding affinity rankings of Populus proteins to CO. In addition, we used a modified Random Walk with Restart network-topology based approach to identify a subset of Populus LysM-RLKs that are functionally related and propose a corresponding signal transduction cascade. Our findings provide the first look into the role of LysM-RLKs in Populus-microbe interactions and establish a crucial jumping-off point for future research efforts to understand specificity and redundancy in microbial perception mechanisms.

Abstract Background Firearm injuries are the second leading cause of death in American youth aged 15 to 24, and over half of these deaths are suicides. Self-harm deaths in Wisconsin among adolescents have increased by 34% since 2006. Each year, our pediatric emergency department (ED) staff care for over 1100 children and adolescents who present with suicidal ideation or self-harming behaviors. We implemented an ED-based program aimed at improving the education given to families on reducing self-directed violence and providing firearm storage devices to families with the goal of reducing access to lethal means. Program description Our program takes place in the pediatric ED of an academic children’s hospital and seeks to assist families of all patients who present with suicidal ideation or suicide attempt (SI/SA). In collaboration with our social workers, we reviewed their processes for interviewing and counseling families of patients who present with SI/SA. Social workers previously used a hospital-wide patient and family education sheet for safety planning that included information about safely storing medications and community mental health services. We teamed with our hospital’s health literacy and education committees and revised the teaching sheet to include more in-depth information about safe firearm storage. For families who were interested, we developed a process to provide up to two firearm lockboxes equipped with a combination lock. Working with risk management, the parent injury prevention product liability form was updated to include firearm lockboxes. Conclusion We implemented a safe firearm storage program including development of a patient and family education sheet and provision of firearm lockboxes to families. Next steps under consideration include providing lockboxes for safe medication storage and establishing a follow-up system to assess proper use of firearm lockboxes and family and social worker satisfaction.

IntroductionDespite a recent global decrease in suicide rates, death by suicide has increased in the United States. It is therefore imperative to identify the risk factors associated with suicide attempts to combat this growing epidemic. In this study, we aim to identify potential risk factors of suicide attempt using geospatial features in an Artificial intelligence framework.MethodsWe use iterative Random Forest, an explainable artificial intelligence method, to predict suicide attempts using data from the Million Veteran Program. This cohort incorporated 405,540 patients with 391,409 controls and 14,131 attempts. Our predictive model incorporates multiple climatic features at ZIP-code-level geospatial resolution. We additionally consider demographic features from the American Community Survey as well as the number of firearms and alcohol vendors per 10,000 people to assess the contributions of proximal environment, access to means, and restraint decrease to suicide attempts. In total 1,784 features were included in the predictive model.ResultsOur results show that geographic areas with higher concentrations of married males living with spouses are predictive of lower rates of suicide attempts, whereas geographic areas where males are more likely to live alone and to rent housing are predictive of higher rates of suicide attempts. We also identified climatic features that were associated with suicide attempt risk by age group. Additionally, we observed that firearms and alcohol vendors were associated with increased risk for suicide attempts irrespective of the age group examined, but that their effects were small in comparison to the top features.DiscussionTaken together, our findings highlight the importance of social determinants and environmental factors in understanding suicide risk among veterans.

Gene-to-gene networks, such as Gene Regulatory Networks (GRN) and Predictive Expression Networks (PEN) capture relationships between genes and are beneficial for use in downstream biological analyses. There exists multiple network inference tools to produce these gene-to-gene networks from matrices of gene expression data. Random Forest-Leave One Out Prediction (RF-LOOP) is a method that has been shown to be efficient at producing these gene-to-gene networks, frequently known as GEne Network Inference with Ensemble of trees (GENIE3). Random Forest can be replaced in this process by iterative Random Forest (iRF), which performs variable selection and boosting. Here we validate that iterative Random Forest-Leave One Out Prediction (iRF-LOOP) produces higher quality networks than GENIE3 (RF-LOOP). We use both synthetic and empirical networks from the Dialogue for Reverse Engineering Assessment and Methods (DREAM) Challenges by Sage Bionetworks, as well as two additional empirical networks created from Arabidopsis thaliana and Populus trichocarpa expression data.

The COVID-19 pandemic has worsened mental health among health workers around the world. With a projected global shortage of 10.2 million health workers by 2030, further exacerbated by COVID-19, taking action to support health worker mental health needs to be an integral component of investments to overcome this gap and build resiliency of systems for the future. Health workers are functioning in highly stressful environments at great personal risk to provide services that improve quality of life and save lives. To reduce burnout and early exits from the workforce, health workers must be protected and equipped to work in supportive environments, manage stress, and access mental health services when needed. This article explores the impact of COVID-19 on health worker mental health and proposes actions for health systems and workplaces to support health workers which draw on available evidence and examples of USAID-supported partner activities.

Abstract Premise Reproducible seed propagation and production protocols were developed for Spiranthes and related taxa to facilitate ex situ conservation practices. Methods and Results Spiranthes seeds were scarified for 3‐ and 10‐min intervals in 10% sodium hypochlorite solution, then cultured on three seed germination media. After germination, seedlings were given one of the three photoperiod treatments, and then planted in one of four greenhouse substrates. Seed germination ranged from 0% to 90% and occurred on all three media only after the 3‐min scarification. Seedlings in the 24/0‐h light/dark and 16/8‐h light/dark photoperiods on P723 medium had significantly higher fresh weight than those in the dark treatment group. Ex vitro survival ranged from 55% to 95% across substrates. Conclusions Results show that Spiranthes seeds are damaged by extended chemical scarification, are adaptable to a variety of culture media, and require light for optimal development. Further experimentation showed that the propagation protocols described here can be applied broadly within the genus.

Abstract We studied mucosal immune responses in six HIV-1 vaccine trials investigating different envelope (Env)-containing immunogens. Regimens were classified into four categories: DNA/vector, DNA/vector plus protein, protein alone, and vector alone. We measured HIV-1-specific IgG and IgA in secretions from cervical (n = 111) and rectal swabs (n = 154), saliva (n = 141), and seminal plasma (n = 124) and compared to corresponding blood levels. Protein-containing regimens had up to 100% response rates and the highest Env-specific IgG response rates. DNA/vector groups elicited mucosal Env-specific IgG response rates of up to 67% that varied across specimen types. Little to no mucosal IgA responses were observed. Overall, gp41- and gp140-specific antibodies dominated gp120 mucosal responses. In one trial, prior vaccination with a protein-containing immunogen maintained durability of cervical and rectal IgG for up to 17 years. Mucosal IgG responses were boosted after revaccination. These findings highlight a role for protein immunization in eliciting HIV-1-specific mucosal antibodies and the ability of HIV-1 vaccines to elicit durable HIV-1-specific mucosal IgG.

Abstract Objective To examine, through photo-elicitation, the personal and professional impact of the COVID-19 pandemic on mental health professionals working with children and adolescents around the globe. Methods We invited the submission of images collected about the pandemic between May and August 2020. We encouraged participants to yoke personal reflections or voice memos to their images. Using snowball sampling, we began with two invitations, including one to the graduates of a mentorship program continuously hosted since 2004 by the International Association of Child and Adolescent Psychiatry and Allied Professions (IACAPAP). We analyzed de-identified images and anonymized transcripts through iterative coding using thematic analysis informed by rich picture analysis and aided by NVivo software. Results We collected submissions from child and adolescent mental health professionals (n = 134) working in 54 countries spread across the five continents. We identified four overarching domains with component themes that revealed both the commonality and the uniqueness of the pandemic experience around the globe: (1) Place (adjusting to emptiness and stillness; shifting timeframes; blending of spaces); (2) Person (disruption to life rhythms; emotional toll; positives of the pandemic); (3) Profession (changing practices; outreach efforts; guild pride—and guilt); and (4) Purpose (from pandemic to syndemic; from lamenting to embracing; planning toward a better tomorrow). Conclusions Photo-elicitation provided a disarming and efficient means to learn about individual, regional, and global similarities and differences regarding the professionals charged with addressing the mental health needs of children and adolescents around the globe. These findings may help inform practice changes in post-pandemic times.

Abstract Background A mechanistic understanding of the spread of SARS-CoV-2 and diligent tracking of ongoing mutagenesis are of key importance to plan robust strategies for confining its transmission. Large numbers of available sequences and their dates of transmission provide an unprecedented opportunity to analyze evolutionary adaptation in novel ways. Addition of high-resolution structural information can reveal the functional basis of these processes at the molecular level. Integrated systems biology-directed analyses of these data layers afford valuable insights to build a global understanding of the COVID-19 pandemic. Results Here we identify globally distributed haplotypes from 15,789 SARS-CoV-2 genomes and model their success based on their duration, dispersal, and frequency in the host population. Our models identify mutations that are likely compensatory adaptive changes that allowed for rapid expansion of the virus. Functional predictions from structural analyses indicate that, contrary to previous reports, the Asp614Gly mutation in the spike glycoprotein (S) likely reduced transmission and the subsequent Pro323Leu mutation in the RNA-dependent RNA polymerase led to the precipitous spread of the virus. Our model also suggests that two mutations in the nsp13 helicase allowed for the adaptation of the virus to the Pacific Northwest of the USA. Finally, our explainable artificial intelligence algorithm identified a mutational hotspot in the sequence of S that also displays a signature of positive selection and may have implications for tissue or cell-specific expression of the virus. Conclusions These results provide valuable insights for the development of drugs and surveillance strategies to combat the current and future pandemics.

This qualitative case study examines the college choice decisions of rural students enrolled in engineering majors to understand what conditions and experiences led rural students to pursue engineering at their institution. We found four themes that help illuminate rural engineering students’ college choice journeys (1) The Inextricable Nature of College, Major, and Career Choice (2) “The Smart Person Thing to Do:” The Power of Prestige, (3) “Are You Sure You Don’t Want to Change your Major?” Dissonance Between Aspirations and Expectations, and (4) School and Community as Crucial Resources in College and Major Exploration. These findings have implications for those working with rural high school students seeking engineering degrees and admissions processes at four-year colleges and universities.

Abstract Pancreatic cancer (PC) is characterized by racial/ethnic disparities and the debilitating muscle‐wasting condition, cancer cachexia. Florida ranks second in the number of PC deaths and has a large and understudied minority population. We examined the primary hypothesis that PC incidence and mortality rates may be highest among Black Floridians and the secondary hypothesis that biological correlates of cancer cachexia may underlie disparities. PC incidence and mortality rates were estimated by race/ethnicity, gender, and county using publicly available state‐wide cancer registry data that included approximately 2700 Black, 25 200 Non‐Hispanic White (NHW), and 3300 Hispanic/Latino (H/L) Floridians diagnosed between 2004 and 2014. Blacks within Florida experienced a significantly (P

Abstract Background Progressive cerebellar ataxia is a neurodegenerative disorder without effective treatment options that seriously hinders quality of life. Previously, transcranial direct current stimulation (tDCS) has been demonstrated to benefit cerebellar functions (including improved motor control, learning and emotional processing) in healthy individuals and patients with neurological disorders. While tDCS is an emerging therapy, multiple daily sessions are needed for optimal clinical benefit. This case study tests the symptomatic benefit of remotely supervised tDCS (RS-tDCS) for a patient with cerebellar ataxia. Methods We report a case of a 71-year-old female patient with progressive cerebellar ataxia, who presented with unsteady gait and balance impairment, treated with tDCS. tDCS was administered using our RS-tDCS protocol and was completed daily in the patient’s home (Monday – Friday) with the help of a trained study technician. tDCS was paired with 20 min of simultaneous cognitive training, followed by 20 min of physical exercises directed by a physical therapist. Stimulation consisted of 20 min of 2.5 mA direct current targeting the cerebellum via an anodal electrode and a cathodal electrode placed over the right shoulder. The patient completed baseline and treatment end visits with neurological, cognitive, and motor (Lafayette Grooved Pegboard Test, 25 ft walk test and Timed Up and Go Test) assessments. Results The patient successfully completed sixty tDCS sessions, 59 of which were administered remotely at the patient’s home with the use of real time supervision as enabled by video conferencing. Mild improvement was observed in the patient’s gait with a 7% improvement in walking speed, which she completed without a walking-aid at treatment end, which was in stark contrast to her baseline assessment. Improvements were also achieved in manual dexterity, with an increase in pegboard scores bilaterally compared to baseline. Conclusions Results from this case report suggest that consecutively administered tDCS treatments paired with cognitive and physical exercise hold promise for improving balance, gait, and manual dexterity in patients with progressive ataxia. Remotely supervised tDCS provides home access to enable the administration over an extended period. Further controlled study in a large group of those with cerebellar ataxia is needed to replicate these findings. Trial registration ClinicalTrials.gov Identifier: NCT03049969. Registered 10 February 2017- Retrospectively registered.

Background: α4β7 is a gut-homing integrin heterodimer that can act as a non-essential binding molecule for HIV. A previous study in heterosexual African women found that individuals with higher proportions of α4β7 expressing CD4+ T cells were more likely to become infected with HIV, as well as present with faster disease progression. It is unknown if this phenomenon is also observed in men who have sex with men (MSM) or people who inject drugs (PWID). Methods: MSM and transgender women who seroconverted as part of the HVTN 505 HIV vaccine trial and PWID who seroconverted during the ALIVE cohort study were selected as cases and matched to HIV-uninfected controls from the same studies (1:1 and 1:3, respectively). Pre-seroconversion PBMC samples from cases and controls in both studies were examined by flow cytometry to measure levels of α4β7 expression on CD4+ T cells. Multivariable conditional logistic regression was used to compare α4β7 expression levels between cases and controls. A Kaplan-Meier curve was used to examine the association of α4β7 expression pre-seroconversion with HIV disease progression. Findings: In MSM and transgender women (n = 103 cases, 103 controls), there was no statistically significant difference in the levels of α4β7 expression on CD4+ T cells between cases and controls (adjusted odds ratio [adjOR] =1.10, 95% confidence interval [CI]=0.94,1.29; p = 0.246). Interestingly, in PWID (n = 49 cases, 143 controls), cases had significantly lower levels of α4β7 expression compared to their matched controls (adjOR = 0.80, 95% CI = 0.68, 0.93; p = 0.004). Among HIV-positive PWID (n = 47), there was no significant association in HIV disease progression in individuals above or below the median level of α4β7 expression (log-rank p = 0.84). Interpretation: In contrast to findings in heterosexual women, higher α4β7 expression does not predict HIV acquisition or disease progression in PWID or MSM. Funding: This study was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. The study was also supported by extramural grants from NIAID T32AI102623 (E.U.P.), and UM1AI069470.

Background: The incidence of colorectal cancer (CRC) has decreased in recent years, due in large part to increased screening, particularly through colonoscopy. This study aimed to examine the level of readability of information on colonoscopy preparation written on 100 websites that were found via an internet search.Methods: In this cross-sectional study, the content of the first 100 websites in English found via an internet search were analyzed using established readability scales. Websites were compared based on whether they had a commercial or non-commercial URL extension.Results: The majority of websites were found to have information of a difficult reading level. Less than 10% of websites had an easy reading level. Readability did not differ significantly based on URL extension.Conclusion: The information currently posted on the internet regarding preparation for colonoscopy is written at a difficult reading level. If information presented was both accurate and easier to read, it could benefit a greater proportion of the general public and help inform decisions about preparing for a colonoscopy.

In this paper we take an in-depth look at the curation of a large longitudinal survey and activities and procedures involved in moving the data from its generation to the state that is needed to conduct scientific analysis. Using a case study approach, we describe how large surveys generate a range of data assets that require many decisions well before the data is considered for analysis and publication. We use the notion of active curation to describe activities and decisions about the data objects that are “live,” i.e., when they are still being collected and processed for the later stages of the data lifecycle. Our efforts illustrate a gap in the existing discussions on curation. On one hand, there is an acknowledged need for active or upstream curation as an engagement of curators close to the point of data creation. On the other hand, the recommendations on how to do that are scattered across multiple domain-oriented data efforts. In describing the complexities of active curation of survey data and providing general recommendations we aim to draw attention to the practices of active curation, stimulate the development of interoperable tools, standards, and techniques needed at the initial stages of research projects, and encourage collaborations between libraries and other academic units.

Background: Our study aimed to determine source of upload and content portrayed in the100 most-viewed videos on autism spectrum disorders (ASDs) on the video sharing public forum, YouTube. ASDs have become highly prevalent in the last decade, arousing a significant response from the media and psycho-educational health professions. Utilization of and reliance on social media for information on health matters has also proliferated. Some suggest that online videos could promote early detection (and intervention) of ASD by prompting caregivers to seek guidance. However, the usefulness of the available videos is unclear.Methods: The 100 most popular YouTube videos were examined for source of upload and information provided. Popularity was determined by number of views, using the filter tool.Results: The videos had more than 121 million views combined. Only one video had been uploaded by a professional (a clinical psychologist). The 99 (non-professional) videos provided minimal data and research into known ASD risk factors. Interestingly, discredited vaccine associated risks were promoted in 16% (95% CI = 09%–25%) of the 100 videos analyzed. Many videos featured a child with ASD exhibiting some characteristic patterns, such as engaging in a repetitive behavior (73%, 95% CI = 63%-81%); about as many videos referenced various therapies (75%, 95% CI = 65%-83%); and 54% (95% CI = 44%–64%) and 61% (95% CI =51%–71%) of the videos mentioned the economic and emotional toll of ASD on the family,respectively. Additional variables are discussed.Conclusion: The most popular online videos were primarily uploaded by non-professionals and provided limited content regarding ASD. Given the wide reach of social media and its potential for providing valuable information and guidance to the public on matters pertaining to ASD, we wish to underscore the necessity for a professional presence in this medium.

This paper focuses on recent work by the TEI Archiving, Preservation, and Access Service (TAPAS) on pedagogy with TEI, and specifically on a recent initiative called “TAPAS Classroom” focused on exploring pedagogical uses of TEI. We provide some background on TAPAS, describe several case studies involving pedagogical partners who used TAPAS in teaching, and finally describe the TAPAS Classroom initiative and its outcomes.

Summary: The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1−/− mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with β-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy. : Surfactant deficiency due to lung immaturity is a major cause of respiratory distress in premature newborns. Coulombe et al. show that endothelial SAM and SH3 domain containing protein 1 (Sash1) drives perinatal lung maturation via nitric oxide signaling to alveolar cells. Sash1 interacts with β-arrestin1 to activate Akt-eNOS and induce alveolar epithelial cell maturation and surfactant synthesis. Keywords: TLR4, Sash1, surfactant, endothelium, alveolar type 2 cells, respiratory distress, lung development, β-arrestin, nitric oxide

An amendment to this paper has been published and can be accessed via the original article.

Dramatic changes in the health system due to national health reform are raising important questions regarding the educational preparation of social workers for the new health arena. While dual-degree programs in public health and social work can be an important response to what is needed educationally, little is known about them. The National MSW/MPH Programs Study surveyed MSW/MPH program administrators to better understand the prevalence, models, structure, and challenges of these dual-degree programs. Forty-two programs were identified, and 97.6% of those contacted participated (n=41). Findings indicate that MSW/MPH programs are popular, increasing, geographically dispersed, and drawing talented students interested in trans-disciplinary public health social work practice. Challenges for these programs include the need for greater institutional support, particularly funding, and a general lack of best practices for MSW/MPH education. While findings from this study suggest graduates appear especially well-prepared for leadership and practice in the new health environment, additional research is needed to assess their particular contributions and career trajectories.

This article reviews recent research on perception of tangible pictures in sighted and blind people. Haptic picture naming accuracy is dependent upon familiarity and access to semantic memory, just as in visual recognition. Performance is high when haptic picture recognition tasks do not depend upon semantic memory. Viewpoint matters for the ease or difficulty of interpreting haptic pictures of solid objects. Top views were easiest for sighted and blind persons when geometrical solids had constant crosssections in the vertical axis. The presence or absence of viewpoint effects depends upon the nature of the solids that are represented. Congenitally blind people do not spontaneously produce perspective drawings, but recent data suggests that depictions including linear perspective can be understood after minimal experience. The results suggest that two-dimensional configurations are not necessarily problematic for touch.

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.

Abstract Background To ensure the suitability of an infant formula as the sole source of nutrition or provide benefits similar to outcomes in breastfed infants, advancements in formula composition are warranted as more research detailing the nutrient composition of human milk becomes available. This study was designed to evaluate growth and tolerance in healthy infants who received one of two investigational cow’s milk-based formulas with adjustments in carbohydrate, fat, and calcium content and supplemented with a prebiotic blend of polydextrose (PDX) and galactooligosaccharides (GOS) or GOS alone. Methods In this multi-center, double-blind, parallel-designed, gender-stratified prospective study 419 infants were randomized and consumed either a marketed routine cow’s milk-based infant formula (Control; Enfamil® LIPIL®, Mead Johnson Nutrition, Evansville, IN) (n = 142) or one of two investigational formulas from 14 to 120 days of age. Investigational formulas were supplemented with 4 g/L (1:1 ratio) of a prebiotic blend of PDX and GOS (PDX/GOS; n = 139) or 4 g/L of GOS alone (GOS; n = 138). Anthropometric measurements were taken at 14, 30, 60, 90, and 120 days of age. Daily recall of formula intake, tolerance, and stool characteristics was collected during study weeks 1 and 2 and 24-h recall was collected at 60, 90, and 120 days of age. Medically-confirmed adverse events were recorded throughout the study. Results There were no group differences in growth rate from 14 to 120 days of age. Discontinuation rates were not significantly different among study groups. No differences in formula intake or infant fussiness or gassiness were observed. During study weeks 1 and 2 and at 60 days of age stool consistency ratings were higher (i.e. softer stools) for infants in the PDX/GOS and GOS groups versus Control and remained higher at 120 days for the PDX/GOS group (all P Conclusions Investigational routine infant formulas supplemented with 4 g/L of either a prebiotic blend of PDX and GOS or GOS alone were well-tolerated and supported normal growth. Compared to infants who received the unsupplemented control formula, infants who received prebiotic supplementation experienced a softer stooling pattern similar to that reported in breastfed infants. Trial registration ClinicalTrials.gov Identifier: NCT00712608

A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of EC 50 titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay robustness, with consistent EC 50 titres for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Despite concerted efforts to tackle the COVID-19 pandemic, the persistent transmission of SARS-CoV-2 demands continued research into novel vaccination strategies to combat the virus. In light of this, intranasally administered peptide vaccines, particularly those conjugated to an immune adjuvant to afford so-called "self-adjuvanted vaccines", remain underexplored. Here, we describe the synthesis and immunological evaluation of self-adjuvanting peptide vaccines derived from epitopes of the spike glycoprotein of SARS-CoV-2 covalently fused to the potent adjuvant, Pam 2 Cys, that targets toll-like receptor 2 (TLR2). When administered intranasally, these vaccines elicited a strong antigen-specific CD4 + and CD8 + T-cell response in the lungs as well as high titers of IgG and IgA specific to the native spike protein of SARS-CoV-2. Unfortunately, serum and lung fluid from mice immunized with these vaccines failed to inhibit viral entry in spike-expressing pseudovirus assays. Following this, we designed and synthesized fusion vaccines composed of the T-cell epitope discovered in this work, covalently fused to epitopes of the receptor-binding domain of the spike protein reported to be neutralizing. While antibodies elicited against these fusion vaccines were not neutralizing, the T-cell epitope retained its ability to stimulate strong antigen-specific CD4 + lymphocyte responses within the lungs. Given the Spike (883-909) region is still completely conserved in SARS-CoV-2 variants of concern and variants of interest, we envision the self-adjuvanting vaccine platform reported here may inform future vaccine efforts.

Background: Glioblastoma (GB) is a lethal and aggressive brain tumour. While molecular characteristics of GB is studied extensively, the aetiology of GB remains uncertain. The interest in exploring viruses as a potential contributor to the development of GB stems from the notion that viruses are known to play a key role in pathogenesis of other human cancers such as cervical cancer. Nevertheless, the role of viruses in GB remains controversial., Methods: This review delves into the current body of knowledge surrounding the presence of viruses in GB as well as provide updates on clinical trials examining the potential inclusion of antiviral therapies as part of the standard of care protocol., Conclusions: The review summarises current evidences and important gaps in our knowledge related to the presence of viruses in GB., (© 2024. The Author(s).)

Objectives: SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T-cell response to disease resolution or progression is still unclear. As newly emerging variants of concern have the capacity to partially escape NAb responses, defining the contribution of individual T-cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines., Methods: Immunophenotyping of T-cell responses in unvaccinated individuals was performed, representing the full spectrum of COVID-19 clinical presentation. Computational and manual analyses were used to identify T-cell populations associated with distinct disease states., Results: Critical SARS-CoV-2 infection was characterised by an increase in activated and cytotoxic CD4 + lymphocytes (CTL). These CD4 + CTLs were largely absent in asymptomatic to severe disease states. In contrast, non-critical COVID-19 was associated with high frequencies of naïve T cells and lack of activation marker expression., Conclusion: Highly activated and cytotoxic CD4 + T-cell responses may contribute to cell-mediated host tissue damage and progression of COVID-19. Induction of these potentially detrimental T-cell responses should be considered when developing and implementing effective COVID-19 control strategies., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

The fall of 2021 was the first school semester to begin with widespread in-person learning since the COVID-19 pandemic began. Understanding dietary and physical activity behaviors of adolescents during this time can provide insight into potential health equity gaps and programmatic needs in schools and communities. This report uses data from the 2021 national Youth Risk Behavior Survey conducted among a nationally representative sample of U.S. public and private school students in grades 9-12 to update estimates of dietary and physical activity behaviors among U.S. high school students overall and by sex and race and ethnicity. In addition, 2-year comparisons (2019 versus 2021) of these behaviors were examined. In 2021, daily consumption of fruits, vegetables, and breakfast during the past 7 days remained low and decreased overall with specific disparities by sex and race and ethnicity from 2019 to 2021. The overall prevalence of students attending physical education classes daily, exercising to strengthen muscles on ≥3 days/week (i.e., met the guideline for muscle-strengthening activity), and playing on at least one sports team decreased from 2019 to 2021; whereas being physically active for ≥60 minutes/day on all 7 days (i.e., met the guideline for aerobic activity) and meeting both aerobic and muscle-strengthening guidelines remained low but did not change. These findings underscore the need for strategies to increase healthy dietary and physical activity behaviors both in the recovery phase of COVID-19 and longer term., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Commercial tobacco use is the leading cause of preventable disease and death in the United States. Despite declines in overall tobacco product use among youths, disparities persist. This report uses biennial data from the 2015-2021 cycles of the nationally representative Youth Risk Behavior Survey to assess prevalence and trends in electronic vapor product (EVP) use among high school students, including ever use, current use (past 30 days), and daily use. Data from 2021 also included usual source of EVPs among students who currently used EVPs. Overall, in 2021, 36.2% had ever used EVPs, 18.0% currently used EVPs, and 5.0% used EVPs daily, with variation in prevalence by demographic characteristics. Prevalence of ever use and current use of EVPs was higher among female students than male students. Prevalence of ever use, current use, and daily use of EVPs was lower among Asian students than Black or African American (Black), Hispanic, Native Hawaiian or other Pacific Islander, White, and multiracial students. Prevalence of ever use, current use, and daily use of EVPs was higher among bisexual students than among students who were not bisexual. During 2015-2021, although ever use of EVPs decreased overall (from 44.9% to 36.2%) and current use of EVPs was stable overall, daily EVP use increased overall (from 2.0 to 5.0%) and among female (from 1.1% to 5.6%), male (from 2.8% to 4.5%), Black (from 1.1% to 3.1%), Hispanic (from 2.6% to 3.4%), multiracial (from 2.8% to 5.3%) and White (from 1.9% to 6.5%) students. Among students who currently use EVPs, 54.1% usually got or bought EVPs from a friend, family member, or someone else. Continued surveillance of EVP and other tobacco product use is necessary to document and understand youth tobacco product usage. These findings can be used to inform youth-focused tobacco prevention and control strategies at the local, state, tribal, and national levels., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Community violence, including homicides involving firearms, is a significant public health concern. From 2019 to 2020, firearm-related homicides increased by 39% for youths and young adults aged 10-24 years, and rates of suicide by firearm increased by approximately 15% among the same age group. Findings from the nationally representative 2021 Youth Risk Behavior Survey were used to analyze disparities and correlates of witnessing community violence and gun carrying among a nationally representative sample of high school students. Chi-square tests and logistic regression accounting for the complex sampling of the survey were used to assess demographic differences by student sex, race and ethnicity, age, and sexual identity in ever witnessing community violence, gun carrying in the past 12 months, and their associations with substance use and suicide risk. Measures of substance use included current binge drinking and marijuana use and lifetime prescription opioid misuse and illicit drug use. Suicide risk included seriously considered attempting suicide and attempted suicide in the past 12 months. Overall, approximately 20% of students witnessed community violence and 3.5% of students carried a gun. American Indian or Alaska Native, Black, and Hispanic students were more likely to witness community violence and to report carrying a gun than their White peers. Males were more likely to witness community violence and carry a gun than females. Lesbian, gay, or bisexual students were more likely to witness community violence than their heterosexual peers. Also, witnessing community violence consistently was associated with increased odds of gun carrying, substance use, and suicide risk for both males and females and when comparing Black, White, and Hispanic students. These findings highlight the importance of comprehensive violence prevention strategies that incorporate health equity to mitigate the effects of violence exposure on substance use and suicide risk among youths., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells)., Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout., Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells., Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ashley, McSharry, McWilliam, Stanton, Fielding, Mathias, Fairlie, McCluskey, Villadangos, Rossjohn, Abendroth and Slobedman.)

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam 2 Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam 2 Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses., (© 2022. The Author(s).)

Long non-coding RNAs (lncRNAs) are RNA transcripts that are over 200 nucleotides and rarely encode proteins or peptides. They regulate gene expression and protein activities and are heavily involved in many cellular processes such as cytokine secretion in respond to viral infection. In severe COVID-19 cases, hyperactivation of the immune system may cause an abnormally sharp increase in pro-inflammatory cytokines, known as cytokine release syndrome (CRS), which leads to severe tissue damage or even organ failure, raising COVID-19 mortality rate. In this review, we assessed the correlation between lncRNAs expression and cytokine release syndrome by comparing lncRNA profiles between COVID-19 patients and health controls, as well as between severe and non-severe cases. We also discussed the role of lncRNAs in CRS contributors and showed that the lncRNA profiles display consistency with patients' clinic symptoms, thus suggesting the potential of lncRNAs as drug targets or biomarkers in COVID-19 treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhong, Ashley, Steain and Ataide.)

This study adopts a socio-ecological framework and examines school- and district-level influences on sexual behaviors among high school students from 16 school districts that were federally funded to conduct a school-based, multilevel sexual health program. We drew cross-sectional data from the 2015 and 2017 Youth Risk Behavior Survey from funded school districts containing 648 schools and 101,728 students. We used multilevel modeling to determine the percentage of variance in sexual health outcomes explained at school and district levels, overall and by race/ethnicity and biological sex. We found protective behaviors such as using hormonal birth control had considerable district-level variance (10.1%) while sexual risk behaviors such as having multiple sex partners showed considerable school-level variance (12.7%). We also found significant subgroup heterogeneity in the variance. Findings indicate school-based interventions should address all levels of influences of the educational system to effectively improve a myriad of student sexual health outcomes.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality, and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein, and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants ( K D ) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect the spike protein in solution by ELISA, and the cocrystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a β-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD, and the ligands discovered in this work may find future use as reagents for diagnostic applications., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The antigen-presenting molecule MR1 presents microbial metabolites related to vitamin B2 biosynthesis to mucosal-associated invariant T cells (MAIT cells). Although bacteria and fungi drive the MR1 biosynthesis pathway, viruses have not previously been implicated in MR1 expression or its antigen presentation. We demonstrate that several herpesviruses inhibit MR1 cell surface upregulation, including a potent inhibition by herpes simplex virus type 1 (HSV-1). This virus profoundly suppresses MR1 cell surface expression and targets the molecule for proteasomal degradation, whereas ligand-induced cell surface expression of MR1 prior to infection enables MR1 to escape HSV-1-dependent targeting. HSV-1 downregulation of MR1 is dependent on de novo viral gene expression, and we identify the Us3 viral gene product as functioning to target MR1. Furthermore, HSV-1 downregulation of MR1 disrupts MAIT T cell receptor (TCR) activation. Accordingly, virus-mediated targeting of MR1 defines an immunomodulatory strategy that functionally disrupts the MR1-MAIT TCR axis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The critical role of interferons (IFNs) in mediating the innate immune response to cytomegalovirus (CMV) infection is well established. However, in recent years the functional importance of the IFN-independent antiviral response has become clearer. IFN-independent, IFN regulatory factor 3 (IRF3)-dependent interferon-stimulated gene (ISG) regulation in the context of CMV infection was first documented 20 years ago. Since then several IFN-independent, IRF3-dependent ISGs have been characterized and found to be among the most influential in the innate response to CMV. These include virus inhibitory protein, endoplasmic reticulum-associated IFN-inducible (viperin), ISG15, members of the interferon inducible protein with tetratricopeptide repeats (IFIT) family, interferon-inducible transmembrane (IFITM) proteins and myxovirus resistance proteins A and B (MxA, MxB). IRF3-independent, IFN-independent activation of canonically IFN-dependent signaling pathways has also been documented, such as IFN-independent biphasic activation of signal transducer and activator of transcription 1 (STAT1) during infection of monocytes, differential roles of mitochondrial and peroxisomal mitochondrial antiviral-signaling protein (MAVS), and the ability of human CMV (HCMV) immediate early protein 1 (IE1) protein to reroute IL-6 signaling and activation of STAT1 and its associated ISGs. This review examines the role of identified IFN-independent ISGs in the antiviral response to CMV and describes pathways of IFN-independent innate immune response induction by CMV., (Copyright © 2019 Ashley, Abendroth, McSharry and Slobedman.)

The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-β neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-β pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-β in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-β.