Genomic markers of drug resistance in Mycobacterium tuberculosis populations with minority variants.

Minority variants of Mycobacterium tuberculosis harboring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug-resistant within-host subpopulations and the frequency of resistance-conferring mutations in minority variants remains limited. M. tuberculosis sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance-conferring minority variants were identified using LoFreq, and mixed populations of different M. tuberculosis strains (≥100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced M. tuberculosis strains were included in the analysis. Drug resistance-conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high-confidence drug resistance-conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed M. tuberculosis strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high-confidence drug resistance-conferring mutations were more frequently detected in mixed M. tuberculosis strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; P = 0.01). Drug-resistant minority variants require monitoring in settings that implement routine M. tuberculosis sequencing. The frequency with which drug-resistant minority variants are detected is likely influenced by pre-culture requirement. Culture-independent sequencing methods should provide a more accurate reflection of drug-resistant subpopulations.

SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes.

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 - Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

Understanding moral empathy: A verbatim-theatre supported phenomenological exploration of the empathy imperative.

OBJECTIVES: Several studies have measured a decline in empathy during medical training, speculating that factors within the formal, informal and hidden curricula are responsible for this phenomenon. Although the medical education literature describes the moral domain of empathy as most fundamental to the empathic response, most research into the decline has examined the cognitive, affective and behavioural domains. This study distinguishes itself by focusing on how moral empathy is affected through training. METHODS: Ten medical residents from core education specialties at McMaster University participated in lightly structured interviews concerning their training experiences. Interview transcripts were analysed by way of a descriptive phenomenological approach. Analyses afforded descriptions of the way medical training influences moral empathy. These descriptions were then used to generate a verbatim theatre play that was performed for an audience of residents, educators, learners, researchers and scholars. Following the play, audience participants completed a survey to member-check the descriptions and to glean other reflective experiences in resident training that impact moral empathy. The survey results informed revisions to the codebook that was subsequently used to re-analyse the interview transcripts. This resulted in a final, refined version of the influence of training on learner moral empathy. RESULTS: The findings suggest that a resident's sense of moral empathy relies upon the notion of an innate capacity for empathy, and is influenced by their clinical and classroom education, and specific experiences with patients during training. Importantly, these factors are rarely experienced as having a direct deleterious impact on residents' moral empathy but rather are experienced as challenges to their ability to act on their moral empathy. CONCLUSIONS: The study promotes reflection of what it means to experience empathy in the moral domain. The description offers a new perspective from which to view empathic declines that have been previously reported, while also highlighting a moral-behavioural tension that has implications for competency-based assessment and the way empathy is conceptualised in medical education.

Implementation of evidence on management of pleural diseases: insights from a territory-wide survey of clinicians in Hong Kong.

BACKGROUND: Major advances in management of common pleural diseases have taken place in the past decade. However, pleural diseases are often managed by physicians of diverse training background and research on implementation of new knowledge is scanty. We aim to evaluate the practice pattern in pleural medicine among physicians in Hong Kong, for identification of possible gaps for clinical service improvement. METHODS: The Hong Kong Thoracic Society undertook a cross-sectional questionnaire survey in 2019, targeting clinicians of various subspecialties in internal medicine and levels of experience (basic and higher trainees, specialists) from twelve regional hospitals of diverse service scopes throughout Hong Kong. Respondents were selected by non-probability quota sampling. The questionnaire tool consisted of 46 questions covering diagnostic and therapeutic aspects of common pleural diseases. The responses were anonymous, and analysed independently using SPSS statistics software. RESULTS: The survey collected 129 responses, 47(36%) were from clinicians specialized in respiratory medicine. Majority of the respondents (98%) managed pleural diseases, including performing pleural procedures in their practice. Fifty-five percent of all the respondents had not received any formal training in transthoracic ultrasonography. A significant proportion of clinicians were unaware of pleuroscopy for investigation of exudative pleural effusion, indwelling pleural catheter for recurrent malignant pleural effusion, and combined intra-pleural Alteplase plus DNase for treatment of pleural infection (30%, 15% and 70% of non-respiratory clinicians respectively). Significant heterogeneity was found in the management of pleural infection, malignant pleural effusion and pneumothorax among respiratory versus non-respiratory clinicians. Contributing factors to the observed heterogeneity included lack of awareness or training, limited accessibility of drugs, devices, or dedicated service support. CONCLUSION: Significant heterogeneity in management of pleural diseases was observed among medical clinicians in Hong Kong. Continuous medical education and training provision for both specialists and non-specialists has to be strengthened to enhance the implementation of advances, improve quality and equity of healthcare provision in pleural medicine.

Transition program: Initial implementation with adults with neuromuscular conditions.

PURPOSE: To identify current medical and psychosocial needs and to examine the effectiveness of healthcare transition program for adult-aged patients with neuromuscular conditions transitioning from pediatric to adult services. DESIGN AND METHODS: At Neuromuscular Transition Clinic visit, 46 patients were evaluated and referred to adult-based providers, if did not currently have one, from an acquired list of interested clinicians. At mean follow-up of 22 months, 42 were interviewed by phone regarding referrals for Core Services (primary care, physiatry, dental care and gynecology), Medical Specialties and Rehabilitation Services. Mean age was 30 years with 62% males. Majority (74%) had cerebral palsy. Sixty percent were non-ambulatory. RESULTS: As per protocol, all were indicated to need Core Services. Eighty-three percent already had adult primary care provider. Most referrals were given for physiatry (62%), vocational training (100%), and occupational therapy (88%). At follow-up, visits were completed most frequently with adult provider for primary care (100%), occupational therapy (78%), and neurology (75%). Referred provider was seen 100% for physiatry, neurology, physical therapy, occupational therapy and vocational training. Of the total 125 referrals given across all services, 73 (58%) participants had completed a visit with an adult provider. CONCLUSIONS: As only about 60% transitioned to adult-based services after referral, healthcare transition remains challenging and requires tailoring of services according to patients' needs, staff and willing-and-available adult-based providers. PRACTICE IMPLICATIONS: Transitioning healthcare of patients with neuromuscular conditions from pediatric- to adult-based providers remains challenging. This clinical specialty requires tailoring of services based on patient's needs, and availability of adult-based providers and resources.

Virtual Reality for Pediatric Needle Procedural Pain: Two Randomized Clinical Trials.

OBJECTIVE: To assess the efficacy and safety of a virtual reality distraction for needle pain in 2 common hospital settings: the emergency department (ED) and outpatient pathology (ie, outpatient laboratory). The control was standard of care (SOC) practice. STUDY DESIGN: In 2 clinical trials, we randomized children aged 4-11 years undergoing venous needle procedures to virtual reality or SOC at 2 tertiary Australian hospitals. In the first study, we enrolled children in the ED requiring intravenous cannulation or venipuncture. In the second, we enrolled children in outpatient pathology requiring venipuncture. In the ED, 64 children were assigned to virtual reality and 59 to SOC. In pathology, 63 children were assigned to virtual reality and 68 to SOC; 2 children withdrew assent in the SOC arm, leaving 66. The primary endpoint was change from baseline pain between virtual reality and SOC on child-rated Faces Pain Scale-Revised. RESULTS: In the ED, there was no change in pain from baseline with SOC, whereas virtual reality produced a significant reduction in pain (between-group difference, -1.78; 95% CI, -3.24 to -0.317; P = .018). In pathology, both groups experienced an increase in pain from baseline, but this was significantly less in the virtual reality group (between-group difference, -1.39; 95% CI, -2.68 to -0.11; P = .034). Across both studies, 10 participants experienced minor adverse events, equally distributed between virtual reality/SOC; none required pharmacotherapy. CONCLUSIONS: In children aged 4-11 years of age undergoing intravenous cannulation or venipuncture, virtual reality was efficacious in decreasing pain and was safe. TRIAL REGISTRATION: Australia and New Zealand Clinical Trial Registry: ACTRN12617000285358p.

Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia.

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008-2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (Prn). Multiple mechanisms of Prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of Prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing Prn arose independently multiple times since 2008, rather than by expansion of a single Prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.

Analysis of the accuracy and readability of herbal supplement information on Wikipedia.

OBJECTIVE: To determine the completeness and readability of information found in Wikipedia for leading dietary supplements and assess the accuracy of this information with regard to safety (including use during pregnancy/lactation), contraindications, drug interactions, therapeutic uses, and dosing. DESIGN: Cross-sectional analysis of Wikipedia articles. INTERVENTIONS: The contents of Wikipedia articles for the 19 top-selling herbal supplements were retrieved on July 24, 2012, and evaluated for organization, content, accuracy (as compared with information in two leading dietary supplement references) and readability. MAIN OUTCOME MEASURES: Accuracy of Wikipedia articles. RESULTS: No consistency was noted in how much information was included in each Wikipedia article, how the information was organized, what major categories were used, and where safety and therapeutic information was located in the article. All articles in Wikipedia contained information on therapeutic uses and adverse effects but several lacked information on drug interactions, pregnancy, and contraindications. Wikipedia articles had 26%-75% of therapeutic uses and 76%-100% of adverse effects listed in the Natural Medicines Comprehensive Database and/or Natural Standard. Overall, articles were written at a 13.5-grade level, and all were at a ninth-grade level or above. CONCLUSION: Articles in Wikipedia in mid-2012 for the 19 top-selling herbal supplements were frequently incomplete, of variable quality, and sometimes inconsistent with reputable sources of information on these products. Safety information was particularly inconsistent among the articles. Patients and health professionals should not rely solely on Wikipedia for information on these herbal supplements when treatment decisions are being made.

Pulmonary artery sarcoma diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration.

Pulmonary artery sarcoma is a rare disease with poor prognosis that has not been reported in Hong Kong. Its clinical and radiological presentation frequently mimics pulmonary embolism. Diagnosis is usually delayed until surgery, which is the treatment option that provides the best survival. Endobronchial ultrasound-guided transbronchial needle aspiration is an effective non-surgical technique for lymph node staging of lung cancer and diagnosis of mediastinal lesions via bronchoscopy. Here we discuss a case of pulmonary artery sarcoma diagnosed by this method, the second one in the literature, which serves to illustrate its potential use for early and minimally invasive diagnosis of the condition. Although such aspiration is a safe procedure, tissue sampling of extravascular extensions is advisable wherever possible.

Impact of Whole-Genome Sequencing of Mycobacterium tuberculosis on Treatment Outcomes for MDR-TB/XDR-TB: A Systematic Review.

The emergence and persistence of drug-resistant tuberculosis is a major threat to global public health. Our objective was to assess the applicability of whole-genome sequencing (WGS) to detect genomic markers of drug resistance and explore their association with treatment outcomes for multidrug-resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB). METHODS: Five electronic databases were searched for studies published in English from the year 2000 onward. Two reviewers independently conducted the article screening, relevant data extraction, and quality assessment. The data of the included studies were synthesized with a narrative method and are presented in a tabular format. RESULTS: The database search identified 949 published articles and 8 studies were included. An unfavorable treatment outcome was reported for 26.6% (488/1834) of TB cases, which ranged from 9.7 to 51.3%. Death was reported in 10.5% (194/1834) of total cases. High-level fluoroquinolone resistance (due to gyrA 94AAC and 94GGC mutations) was correlated as the cause of unfavorable treatment outcomes and reported in three studies. Other drug resistance mutations, like kanamycin high-level resistance mutations (rrs 1401G), rpoB Ile491Phe, and ethA mutations, conferring prothionamide resistance were also reported. The secondary findings from this systematic review involved laboratory aspects of WGS, including correlations with phenotypic DST, cost, and turnaround time, or the impact of WGS results on public health actions, such as determining transmission events within outbreaks. CONCLUSIONS: WGS has a significant capacity to provide accurate and comprehensive drug resistance data for MDR/XDR-TB, which can inform personalized drug therapy to optimize treatment outcomes.

Exploring programmatic indicators of tuberculosis control that incorporate routine Mycobacterium tuberculosis sequencing in low incidence settings: a comprehensive (2017-2021) patient cohort analysis.

Background: Routine whole genome sequencing of Mycobacterium tuberculosis has been implemented with increasing frequency. However, its value for tuberculosis (TB) control programs beyond individual case management and enhanced drug resistance detection has not yet been explored. Methods: We analysed routine sequencing data of culture-confirmed TB cases notified between 1st January 2017 and 31st December 2021 in New South Wales (NSW), Australia. Genomic surveillance included evidence of local TB transmission, defined by single nucleotide polymorphism (SNP) clustering over a variable (0-25) SNP threshold, and drug resistance conferring mutations. Findings: M. tuberculosis sequences from 1831 patients were examined, representing 64.8% of all notified TB cases and 96.2% of culture-confirmed cases. Applying a traditional 5-SNP cluster threshold identified 62 transmission clusters with 183 clustered cases; 101/183 (55.2%) had 0 SNP differences. Cluster assessment over a 5-year period, using a 5-SNP threshold, provided a comprehensive overview of likely recent transmission within NSW, Australia, as an indicator of local TB control. Genotypic drug susceptibility testing (DST) was highly concordant with phenotypic DST and provided a 6.8% increase in antimycobacterial resistance detection. Importantly, it detected mutations missed by routine molecular tests. Lineage 2 strains were more likely to be drug resistant (p < 0.0001) and locally transmitted if drug resistant (p < 0.0001). Interpretation: Performing routine prospective WGS in a low incidence country like Australia, provides genomically informed programmatic indicators of local TB control. A rolling 5-year cluster assessment reflects epidemic containment and progress towards 'zero TB transmission'. Genomic DST also provides valuable information for clinical care and drug resistance surveillance. Funding: NHMRC Centre for Research Excellence in Tuberculosis (www.tbcre.org.au) and NSW Health Prevention Research Support Program.

A laboratory framework for ongoing optimization of amplification-based genomic surveillance programs.

IMPORTANCE: This study provides a laboratory framework to ensure ongoing relevance and performance of amplification-based whole genome sequencing to strengthen public health surveillance during extended outbreaks or pandemics. The framework integrates regular reviews of the performance of a genomic surveillance system and highlights the importance of ongoing monitoring and the identification and implementation of improvements to whole genome sequencing methods to enhance public health responses to pathogen outbreaks.

Advancing pathogen genomics in resource-limited settings.

Genomic sequencing has emerged as a powerful tool to enhance early pathogen detection and characterization with implications for public health and clinical decision making. Although widely available in developed countries, the application of pathogen genomics among low-resource, high-disease burden settings remains at an early stage. In these contexts, tailored approaches for integrating pathogen genomics within infectious disease control programs will be essential to optimize cost efficiency and public health impact. We propose a framework for embedding pathogen genomics within national surveillance plans across a spectrum of surveillance and laboratory capacities. We adopt a public health approach to genomics and examine its application to high-priority diseases relevant in resource-limited settings. For each grouping, we assess the value proposition for genomics to inform public health and clinical decision-making, alongside its contribution toward research and development of novel diagnostics, therapeutics, and vaccines.

Catalysis-based inhibitors of the calcium signaling function of CD38.

CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.

Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose.

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca(2+) signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.

Functional interplay between LIS1, NDE1 and NDEL1 in dynein-dependent organelle positioning.

LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.

Multi-locus variable number tandem repeat analysis of 7th pandemic Vibrio cholerae.

BACKGROUND: Seven pandemics of cholera have been recorded since 1817, with the current and ongoing pandemic affecting almost every continent. Cholera remains endemic in developing countries and is still a significant public health issue. In this study we use multilocus variable number of tandem repeats (VNTRs) analysis (MLVA) to discriminate between isolates of the 7th pandemic clone of Vibrio cholerae. RESULTS: MLVA of six VNTRs selected from previously published data distinguished 66 V. cholerae isolates collected between 1961-1999 into 60 unique MLVA profiles. Only 4 MLVA profiles consisted of more than 2 isolates. The discriminatory power was 0.995. Phylogenetic analysis showed that, except for the closely related profiles, the relationships derived from MLVA profiles were in conflict with that inferred from Single Nucleotide Polymorphism (SNP) typing. The six SNP groups share consensus VNTR patterns and two SNP groups contained isolates which differed by only one VNTR locus. CONCLUSIONS: MLVA is highly discriminatory in differentiating 7th pandemic V. cholerae isolates and MLVA data was most useful in resolving the genetic relationships among isolates within groups previously defined by SNPs. Thus MLVA is best used in conjunction with SNP typing in order to best determine the evolutionary relationships among the 7th pandemic V. cholerae isolates and for longer term epidemiological typing.

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture.

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts. Discussion and Conclusion: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.