A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

Choice of Statistical Tools for Outlier Removal Causes Substantial Changes in Analyte Reference Intervals in Healthy Populations.

BACKGROUND: Reference intervals are an important aid in medical practice as they provide clinicians a guide as to whether a patient is healthy or diseased.Outlier results in population studies are removed by any of a variety of statistical measures. We have compared several methods of outlier removal and applied them to a large body of analytes from a large population of healthy persons. METHODS: We used the outlier exclusion criteria of Reed-Dixon and Tukey and calculated reference intervals using nonparametric and Harrell-Davis statistical methods and applied them to a total of 36 different analytes. RESULTS: Nine of 36 analytes had a greater than 20% difference in the upper reference limit, and for some the difference was 100% or more. CONCLUSIONS: For some analytes, great importance is attached to the reference interval. We have shown that different statistical methods for outlier removal can cause large changes to reported reference intervals. So that population studies can be readily compared, common statistical methods should be used for outlier removal.

Inflammatory responses to a pathogenic West Nile virus strain.

BACKGROUND: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. METHODS: To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). RESULTS: Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. CONCLUSION: NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

Using a thyroid disease-free population to define the reference interval for TSH and free T4 on the Abbott Architect analyser.

OBJECTIVE: Thyroid disease can be subtle in its presentation, and TSH reference intervals may be artefactually increased by including persons with subclinical thyroid disease. We have therefore used a thyroid disease-free population to determine TSH and fT4 reference intervals. DESIGN: Apparently healthy subjects were assessed by health questionnaire, drug history, clinical assessment and measurement of thyroid antibodies. PATIENTS: Healthy subjects in a community setting. MEASUREMENTS: TSH, free T4, antithyroglobulin and anti-TPO were measured on the Abbott Architect analyser. Subjects with clinical abnormalities, consumption of thyroid-active medications or with thyroid antibodies above the manufacturer-quoted reference intervals were excluded. TSH and fT4 data were log-transformed, and the central 95% was used to calculate reference intervals. We assessed whether these data were normally distributed. We compared samples spanning the reference intervals for both TSH and fT4 between different assays looking at biases. RESULTS: From a population of 1,606 subjects, 140 males (18%) and 284 females (34%) were excluded. The central population 95% for TSH was 0·43-3·28 mU/l and for fT4 10·8-16·8 pmol/l. There were no age- or sex-related differences. For both analytes, the distribution was not significantly different to a Gaussian distribution (P > 0·05). For 5 commonly used assays for TSH, the maximum difference in the upper limit of the TSH reference interval was 0·48 mU/l and for fT4 the maximum difference for the upper reference limit was 4·1 pmol/l. CONCLUSIONS: A substantial proportion of apparently healthy persons have subclinical thyroid disease. These subjects must be excluded for any thyroid hormone reference interval studies.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

A multifactorial evaluation of illness risk factors in athletes preparing for the Summer Olympic Games.

OBJECTIVES: Illness can disrupt training and competition performance of athletes. Few studies have quantified the relative contribution of the known medical, behavioural and lifestyle risk factors. DESIGN: Cross-sectional. METHODS: Olympic athletes from 11 sports (n=221) were invited to complete questionnaires administered nine months before the Rio 2016 Olympic Games. These included the Depression, Anxiety and Stress Questionnaire (DASS-21), Perceived Stress Scale (PSS), Dispositional Resilience Scale (DRS), Recovery-Stress Questionnaire (REST-Q-52 item), Low Energy in Females Questionnaire (LEAF-Q), a modified Personal and Household Hygiene questionnaire, Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index, and custom-made questionnaires on probiotic usage and travel. An illness (case) was defined as an event which limited training or competition for greater hours in the prior month. Odds ratios and attributable fractions in the population (AFP) were utilised for categorical variables with independent t-tests or Wilcoxon rank-sum for continuous variables. RESULTS: Eighty-one athletes responded (male, n=26; female, n=55). There were 16 illness cases and 65 controls. Female athletes were at higher odds of illness (OR=9.4, 95%CI 1.3-410, p=0.01, AFP=0.84). Low energy availability (LEAF-Q score ≥8: OR=7.4, 95%CI 0.78-352, p=0.04, AFP=0.76), depression symptoms (DASS-21: depression score >4, OR=8.4, 95%CI 1.1-59, p<0.01; AFP=0.39) and higher perceived stress (PSS: 10-item, p=0.04) were significantly associated with illness. CONCLUSIONS: Female sex, low energy availability, and mental health are associated with sports incapacity (time loss) due to illness. Low energy availability had high attributable fractions in the population and stands out as a primary association with illness.

Harmonising Reference Intervals for Three Calculated Parameters used in Clinical Chemistry.

For more than a decade there has been a global effort to harmonise all phases of the testing process, with particular emphasis on the most frequently utilised measurands. In addition, it is recognised that calculated parameters derived from these measurands should also be a target for harmonisation. Using data from the Aussie Normals study we report reference intervals for three calculated parameters: serum osmolality, serum anion gap and albumin-adjusted serum calcium. The Aussie Normals study was an a priori study that analysed samples from 1856 healthy volunteers. The nine analytes used for the calculations in this study were measured on Abbott Architect analysers. The data demonstrated normal (Gaussian) distributions for the albumin-adjusted serum calcium, the anion gap (using potassium in the calculation) and the calculated serum osmolality (using both the Bhagat et al. and Smithline and Gardner formulae). To assess the suitability of these reference intervals for use as harmonised reference intervals, we reviewed data from the Royal College of Pathologists of Australasia/Australasian Association of Clinical Biochemists (RCPA/AACB) bias survey. We conclude that the reference intervals for the calculated serum osmolality (using the Smithline and Gardner formulae) may be suitable for use as a common reference interval. Although a common reference interval for albumin-adjusted serum calcium may be possible, further investigations (including a greater range of albumin concentrations) are needed. This is due to the bias between the Bromocresol Green (BCG) and Bromocresol Purple (BCP) methods at lower serum albumin concentrations. Problems with the measurement of Total CO2 in the bias survey meant that we could not use the data for assessing the suitability of a common reference interval for the anion gap. Further study is required.