A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry (preprint)

Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence SARS-CoV-2 infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of COVID-19 patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 over-expression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Our study contributes to understanding the association between PLSCR1 variants and severe COVID-19 cases reported in a recent GWAS.

COVID-19: Using high-throughput flow cytometry to dissect clinical heterogeneity.

Here we consider how high-content flow cytometric methodology at appropriate scale and throughput rapidly provided meaningful biological data in our recent studies of COVID-19, which we discuss in the context of other similar investigations. In our work, high-throughput flow cytometry was instrumental to identify a consensus immune signature in COVID-19 patients, and to investigate the impact of SARS-CoV-2 exposure on patients with either solid or hematological cancers. We provide here some examples of our 'holistic' approach, in which flow cytometry data generated by lymphocyte and myelomonocyte panels were integrated with other analytical metrics, including SARS-CoV-2-specific serum antibody titers, plasma cytokine/chemokine levels, and in-depth clinical annotation. We report how selective differences between T cell subsets were revealed by a newly described flow cytometric TDS assay to distinguish actively cycling T cells in the peripheral blood. By such approaches, our and others' high-content flow cytometry studies collectively identified overt abnormalities and subtle but critical changes that discriminate the immuno-signature of COVID-19 patients from those of healthy donors and patients with non-COVID respiratory infections. Thereby, these studies offered several meaningful biomarkers of COVID-19 severity that have the potential to improve the management of patients and of hospital resources. In sum, flow cytometry provides an important means for rapidly obtaining data that can guide clinical decision-making without requiring highly expensive, sophisticated equipment, and/or "-omics" capabilities. We consider how this approach might be further developed.

Serum proteomics hint at an early T-cell response and modulation of SARS-CoV-2-related pathogenic pathways in COVID-19-ARDS treated with Ruxolitinib.

Background: Acute respiratory distress syndrome (ARDS) in corona virus disease 19 (COVID-19) is triggered by hyperinflammation, thus providing a rationale for immunosuppressive treatments. The Janus kinase inhibitor Ruxolitinib (Ruxo) has shown efficacy in severe and critical COVID-19. In this study, we hypothesized that Ruxo's mode of action in this condition is reflected by changes in the peripheral blood proteome. Methods: This study included 11 COVID-19 patients, who were treated at our center's Intensive Care Unit (ICU). All patients received standard-of-care treatment and n = 8 patients with ARDS received Ruxo in addition. Blood samples were collected before (day 0) and on days 1, 6, and 10 of Ruxo treatment or, respectively, ICU admission. Serum proteomes were analyzed by mass spectrometry (MS) and cytometric bead array. Results: Linear modeling of MS data yielded 27 significantly differentially regulated proteins on day 1, 69 on day 6 and 72 on day 10. Only five factors (IGLV10-54, PSMB1, PGLYRP1, APOA5, WARS1) were regulated both concordantly and significantly over time. Overrepresentation analysis revealed biological processes involving T-cells only on day 1, while a humoral immune response and complement activation were detected at day 6 and day 10. Pathway enrichment analysis identified the NRF2-pathway early under Ruxo treatment and Network map of SARS-CoV-2 signaling and Statin inhibition of cholesterol production at later time points. Conclusion: Our results indicate that the mechanism of action of Ruxo in COVID-19-ARDS can be related to both known effects of this drug as a modulator of T-cells and the SARS-CoV-2-infection.

Management of Post-Infectious Anosmia and Hyposmia: A Systematic Review.

BACKGROUND: Anosmia and hyposmia significantly affect patients' quality of life and have many etiologies, including trauma, inflammatory conditions including chronic rhinosinusitis, neoplasm, and viral infections, such as rhinovirus and SARS-CoV-2. OBJECTIVE: Our purpose was to establish whether a consensus exists regarding optimal management of olfactory dysfunction and to provide insight into the treatment of anosmia in the current climate of increased prevalence secondary to COVID-19. Thus, we aimed to systematically review the literature on the management of non-Chronic-rhinosinusitis- related anosmia/hyposmia. METHODS: PubMed, EMBASE, and Cochrane databases were searched for articles published since January 1990 using terms combined with Medical Subject Headings (MeSH). We included articles evaluating management of anosmia and hyposmia written in the English language, with original data, a minimum of 3 months of follow-up except for COVID-related studies, at least 2 patients, and well-defined and measurable outcomes. RESULTS: A total of 3013 unique titles were returned upon the initial search. Of these, 297 abstracts were examined, yielding 19 full texts meeting inclusion criteria (8 with level 1 evidence, 3 with level 2, 1 with level 3, and 7 with level 4). The studies included a total of 1522 subjects, with follow up ranging from 3 to 72 months, with an exception for COVID related studies. Endpoints were based on clinically significant improvements of olfactory functions as measured through validated smell tests. Treatments with the most robust data were intranasal corticosteroids and olfactory training. CONCLUSION: The literature on the treatment of anosmia and hyposmia includes randomized trials showing the efficacy of a few modalities. While further research is needed to expand therapeutic options for this debilitating condition, the current literature supports the use of olfactory training and topical corticosteroids.

Standardization of in-house anti-IgG and IgA ELISAs for the detection of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

A Comparison of Telehealth-Based Instruction with or without Instructive Feedback.

Language delays are commonly displayed by children on the autism spectrum. To help facilitate the development of verbal behavior, practitioners often implement intensive one-on-one, face-to-face instruction. However, the COVID-19 pandemic hindered typical face-to-face service delivery and caused practitioners to assess alternative approaches to facilitate clients' continued progress. Instructive feedback (IF) is one teaching strategy to enhance instruction or make it more efficient. During this teaching procedure, instructors provide formal teaching of target responses and embed demonstrations of secondary target responses within sequences of instruction. In the current study, we investigated the efficacy of IF provided within telehealth instruction. Four participants on the autism spectrum participated in the study. Participants received two forms of telehealth instruction that targeted speaker-responding. The first form consisted of discrete trial instruction (DTI), and the second form combined DTI with IF. These results indicate that both forms of instruction improved speaker-responding of primary targets for all participants. Additionally, a secondary analysis of secondary targets indicated that two of the four participants acquired some secondary targets. These results suggest that including IF within DTI might be beneficial for some participants receiving DTI via telehealth.

Covid-19 Vaccine-Induced Myositis

Background/Aims Through the COVID pandemic there have emerged reports of autoimmunity or new rheumatic diseases presenting in patients after they had COVID-19. This is thought to be caused by cross-reactivity of the COVID-19 spike protein to human antigens. Given the use of mRNA COVID-19 vaccinations which express the spike protein we might expect to see presentation of new rheumatic diseases following their use. We discuss a case where this appears to have occurred. Methods Our patient is a 24-year-old male with mixed phenotype acute leukaemia who had been treated with allogenic stem cell transplant and was currently in remission. He presented with fevers, palpitations, myalgia and bilateral arm and leg swelling. Symptoms began the day after receiving the first dose of an mRNA COVID-19 vaccination (Pfizer/BioNTech.) There were no other symptoms or recent change in medications. Physical examination revealed tender oedema in his forearms, biceps and thighs bilaterally with sparring of the hands. He had reduced power with shoulder (MRC 3/5), elbow (4), wrist (4+) and hip (4) movements. Observations revealed tachycardia and fevers up to 40C. Results Laboratory studies showed markedly elevated C-reactive protein (202), creatinine kinase (6697) and troponin (593) whilst investigations for infection were negative. An autoimmune panel was positive for anti- PM-SCL-75-Ab. An electrocardiogram showed sinus tachycardia. Echocardiogram was normal. Bilateral upper limb dopplers revealed no deep vein thrombus. An MRI of his thighs showed diffuse symmetrical oedema within the muscles, in keeping with an inflammatory myositis. A quadricep muscle biopsy showed evidence of MHC class 1 up-regulation, suggesting an inflammatory process. In addition, there were numerous macrophages evident in the endomysium. While this can be seen in graft-versus-host disease (GVHD), they would usually be found in the perimysium. After discussion between haematology, rheumatology and neurology, this was felt to be a case of vaccine induced myositis and myocarditis. Autoimmune myositis was thought to be less likely due to the relative sparing of the hands and the absence of Raynaud's phenomenon. 1 gram of intravenous methylprednisolone was then given for 3 days. The patient had a marked response with defervescence, improving laboratory markers, improved myalgia and decreased limb swelling. The patient was stepped down to a reducing regime of prednisolone and discharged. Due to relapse whilst weaning he has started on mycophenalate mofetil and rituximab and now continues to improve. Conclusion There are case reports of myositis following COVID-19 vaccination but our patient's case is complicated by the differential diagnosis of GVHD and concurrent myocarditis. Ongoing work is needed to clarify the exact link between vaccination and the presentation of a new inflammatory myositis, but it is important to recognise and start treatment early in order to preserve muscle bulk and ensure recovery.

A qualitative study into the barriers and facilitators to effective communication with people with age-related hearing loss in community pharmacy settings

Introduction: One in six adults are affected by hearing loss, and this number increases with age.(1) Inadequate communication is a barrier to the delivery of effective care for people with hearing loss.(2) Communication regarding medicines is particularly relevant to the pharmaceutical care of older people who are more likely to be living with hearing loss, have multiple comorbidities and experience polypharmacy, a risk factor for adverse drug events. This study sought to explore factors that facilitate and impede communication with people with age-related hearing loss (presbycusis) in the community pharmacy, to provide a base upon which to identify solutions/ interventions to improve these interactions. Aim(s): To explore the barriers and facilitators to effective communication with people with age-related hearing loss in the community pharmacy setting. Method(s): This qualitative study consisted of semistructured interviews with people with age-related hearing loss (presbycusis), older people (>50 years old) without hearing loss, and focus groups and interviews with community pharmacists, conducted between March - June 2022. Participants were recruited purposively via advert and snowballing. Interviews and focus groups were conducted using Microsoft Teams or Zoom;two interviews were held by telephone. Data was analysed using the framework method. Result(s): Sixteen people with age-related hearing loss and three older people without hearing loss took part. Eight community pharmacists took part across two focus groups and one interview. Four main themes were identified. The first was 'grappling with the environment'. This theme reflects the multitude of environmental barriers to communication and person-centred pharmaceutical care such as heavy workload, time constraints, lack of privacy, noise levels and Covid-19 safety measures on communication. The second was 'promoting acknowledgment and inclusion of hearing loss in the pharmacy'. This theme captures the perception among patients that their hearing loss is not relevant to the community pharmacy setting and that more could be done to signify that a pharmacy recognises the needs of those with hearing loss. Third, 'patient perceptions and use of community pharmacy services'. This theme highlights the awareness and perceptions of the role of the pharmacy that is, primarily or exclusively as a place to collect prescriptions and the limited interaction with pharmacy personnel. The final theme was 'differing perceptions of patients and pharmacists about communication'. This theme articulates the varying patient-provider perceptions about communication and levels of awareness among pharmacists about the key facilitators to communication. Conclusion(s): This study garnered rich insights about the experiences of accessing and providing pharmaceutical care for people with age-related hearing loss within the community pharmacy setting. However, due to the Covid- 19 pandemic, data collection was chiefly conducted online and the findings cannot be considered reflective of the experiences of older people facing digital exclusion. There is clearly a need to improve pharmacists' awareness of, access to and implementation of strategies, adaptations, and assistive technologies to facilitate communication with people with hearing loss. Future research should identify interventions to support the implementation of strategies/adaptations that are tailored to the needs of people with hearing loss and pharmacists in order to engender hearing-friendly community pharmacies.

Efficacy of the combination of monoclonal antibodies against the SARS-CoV-2 Beta and Delta variants.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the biggest healthcare issue worldwide. This study aimed to develop a monoclonal antibody against SARS-CoV-2 from B cells of recovered COVID-19 patients, which might have beneficial therapeutic purposes for COVID-19 patients. We successfully generated human monoclonal antibodies (hmAbs) against the receptor binding domain (RBD) protein of SARS-CoV-2 using developed hybridoma technology. The isolated hmAbs against the RBD protein (wild-type) showed high binding activity and neutralized the interaction between the RBD and the cellular receptor angiotensin-converting enzyme 2 (ACE2) protein. Epitope binning and crystallography results displayed target epitopes of these antibodies in distinct regions beneficial in the mix as a cocktail. The 3D2 binds to conserved epitopes among multi-variants. Pseudovirion-based neutralization results revealed that the antibody cocktail, 1D1 and 3D2, showed high potency in multiple variants of SARS-CoV-2 infection. In vivo studies showed the ability of the antibody cocktail treatment (intraperitoneal (i.p.) administration) to reduce viral load (Beta variant) in blood and various tissues. While the antibody cocktail treatment (intranasal (i.n.) administration) could not significantly reduce the viral load in nasal turbinate and lung tissue, it could reduce the viral load in blood, kidney, and brain tissue. These findings revealed that the efficacy of the antibody cocktail, 1D1 and 3D2, should be further studied in animal models in terms of timing of administration, optimal dose, and efficacy to mitigate inflammation in targeted tissue such as nasal turbinate and lung.

Nanotechnology and Medicine: The Interphase

In a lecture entitled ‘There is plenty of room in the bottom', American physicist and Nobel Prize laureate Richard Feynman introduced the concept of Nanotechnology to the world of science. K. Eric Drexler's 1986 publication of the first book on nanotechnology, titled ‘Engines of Creation: The Coming Era of Nanotechnology', set the way for a revolution in this field. This chapter discusses the properties of nanoparticles including their antimicrobial and antiviral properties. Being an interdisciplinary field, nanotechnology has three main extensively overlapping areas including nanoelectronics, nanomaterials, and nanobiotechnology all having applications in healthcare. Manufacturing approaches in nanotechnology comprises of two approaches;top-down approach and bottom-down approach. On the basis of their creation, composition, dimension, and structure, nanomaterials can be categorized. The concept of nanomedicine was proposed by Robert A. Freitas Jr. in the year 1993. Similar to this is the concept of nanodentistry. Nanotechnology has a vivid range of applications in the field of dentistry. Its application starts from diagnosis (nanodiagnostics) and is spread to different specialties of dentistry including endodontics, periodontics, and dental anaesthesia dental material science to name a few. In order to achieve and maintain the long-term stability of dental implants in bone tissue, surface qualities including chemistry and roughness are increasingly being modified using nanotechnology. Nanotechnology has made promising strides towards the management of the COVID pandemic. Nano-based antiviral and antimicrobial formulations, nanoparticle-incorporated facial masks, gloves, hand sanitizers, PPE kits, and nano-based vaccines have added new dimensions in managing viral infection. The drawbacks of the technology have been pondered in this chapter. Despite all the shortcomings, this technology is all set to revolutionize the field of dentistry and dental science. However, like any other technology, nanotechnology too carries the potential for misuse and overuse if not properly regulated. © 2023, The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

COVID-19: Findings in nuclear medicine from head to toe.

COVID-19 is a multisystemic disease, and hence its potential manifestations on nuclear medicine imaging can extend beyond the lung. Therefore, it is important for the nuclear medicine physician to recognize these manifestations in the clinic. While FDG-PET/CT is not indicated routinely in COVID-19 evaluation, its unique capability to provide a functional and anatomical assessment of the entire body means that it can be a powerful tool to monitor acute, subacute, and long-term effects of COVID-19. Single-photon scintigraphy is routinely used to assess conditions such as pulmonary embolism, cardiac ischemia, and thyroiditis, and COVID-19 may present in these studies. The most common nuclear imaging finding of COVID-19 vaccination to date is hypermetabolic axillary lymphadenopathy. This may pose important diagnostic and management dilemmas in oncologic patients, particularly those with malignancies where the axilla constitutes a lymphatic drainage area. This article aims to summarize the relevant literature published since the beginning of the pandemic on the intersection between COVID-19 and nuclear medicine.

Plain language summary of Pfizer-BioNTech BNT162b2 vaccine protection against COVID-19 and its safety in participants 12- to 15-years-old

What is this summary about? This is a summary of an article about part of a clinical study for the BNT162b2 COVID-19 vaccine, also called the Pfizer-BioNTech vaccine. The article was published in the New England Journal of Medicine in May 2021. This summary describes how the vaccine worked in participants 12- to 15-years old. The part of the study described in the article is ongoing and expected to finish March 2023. This means that the final results may be different from the results included in this summary. What happened in this study? The part of the study described in this summary included participants 12- to 15-years old who had no serious health issues. The BNT162b2 vaccine had already been studied in participants 16 years of age or older. In this part of the study, the researchers wanted to find out: * How effective and safe the vaccine was in participants 12- to 15-years old. * What the immune response to the vaccine and the vaccine safety were like in 12- to 15-year-olds compared with 16- to 25-year-olds. * How well the vaccine prevented SARS-CoV-2 infections in participants who received the vaccine compared to those who did not. This is also called efficacy of the BNT162b2 vaccine Half of the participants in this study received 2 injections of the BNT162b2 vaccine and half received 2 injections of a placebo in a muscle of the upper arm. The placebo looked like the BNT162b2 vaccine but did not have any active vaccine in it. What were the results? * BNT162b2 had a favorable safety profile. The most common reactions were pain at the injection site, fatigue, and headache. None of the participants had serious reactions to the vaccine. * The 12- to 15-year-old participants' immune system responses to the BNT162b2 vaccine were as good as or stronger than the 16- to 25-year-old participants' immune responses. * The participants who received the BNT162b2 vaccine were less likely to get COVID-19 compared with the participants who got the placebo.Copyright © 2023 The Authors.

"COVID-19 is an Opportunity to Rediscover Ourselves": Reflections of a Novice EFL Teacher in Central America

This article presents a case study that examined the principles and practices of one novice English as a foreign language (EFL) teacher at a prominent English language institution in Central America. This qualitative study sought to contribute to the discussion of the perceived interdependent influences of EFL teachers' thoughts, identities, and behaviors through five stages of self-reflection in Farrell's framework for reflective practice. The EFL teacher engaged in conscious reflection to subject their beliefs to critical analysis and interpretation expressed through their philosophy, principles, theory, practice, and beyond practice. Overall, the findings confirm that reflections in all five stages are connected to several common themes, but simultaneously reveal a complex relationship between the teacher's stated principles and actual practice. The discussion explores potential reasons for convergence and divergence in teachers' beliefs and classroom actions, concluding that the results correlate with previous research in the field of language education and teacher reflection. [ FROM AUTHOR] Copyright of RELC Journal is the property of Sage Publications, Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Virtual reality intervention alleviates dyspnea in patients recovering from COVID pneumonia

Immersive virtual reality (iVR)-based digital therapeutics (DTx) are gaining clinical attention in the field of pain management. Based on known analogies between pain and dyspnea, we investigated the effects of visualrespiratory feedback, on persistent dyspnea in patients recovering from COVID-19 pneumonia. We performed a controlled, randomized, single-blind, cross-over clinical study to evaluate an iVR-based intervention to alleviate dyspnea in patients recovering from COVID-19 pneumonia. Included patients reported persistent dyspnea and preserved cognitive function. Assignment was random and concealed. Patients received synchronous (intervention) or asynchronous (control) feedback of their breathing, embodied via a gender-matched virtual body. Outcomes were assessed using questionnaires and breathing recordings. Twenty-six patients were enrolled (27% women;age: median=55, interquartile range (IQR)=18). The median (IQR) rating on a 7-point Likert-scale of breathing comfort improved from 1(2) at baseline, to 2(1) for synchronous feedback, but remained unchanged at 1(1.5) for asynchronous feedback (p<0.05) between iVR conditions) Moreover, 91.2% of all patients were satisfied with the intervention (p<0.0001) and 66.7% perceived it as beneficial for their breathing (p<0.05). Based on these findings, our iVR-based DTx presents a feasible and safe respiratory rehabilitation tool that improves breathing comfort in patients recovering from COVID-19 infection presenting with persistent dyspnea. Future research should investigate the DTx's generalizability to persistent dyspnea with other etiologies and its potential for preventing chronification.

Duration of MERS - Coronavirus antibodies in a small closed dromedary camel herd in Dubai

This investigation shows that MERS-CoV ELISA antibodies remain in dromedary camels at least for 9 years, most probably lifelong. The viral infection was most likely acquired at a young age when maternal antibodies have receded between 4 to 8 month of age. Additionally, in the investigated herd, the 4 ELISA antibody negative dromedary camels remained negative also for 9 years, indicating, that no MERS-CoV was circulating in the herd. Until today, the specific source of MERS-CoV infection for young dromedaries remains unknown.