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Wastewater sequencing has become a powerful supplement to clinical testing in monitoring SARS-CoV-2 infections in the post-COVID-19 pandemic era. While its applications in measuring the viral burden and main circulating lineages in the community have proved their efficacy, the variations in sequencing quality and coverage across the different regions of the SARS-CoV-2 genome are not well understood. Furthermore, it is unclear how different sample origins, viral extraction and concentration methods and environmental factors impact the reads sequenced from wastewater. Using high-coverage, amplicon-based, paired-end read sequencing of viral RNA extracted from wastewater collected directly from aircraft, pooled from different aircraft and airport buildings or from regular wastewater plants, we assessed the genome coverage across the sample groups with a focus on the 5′-end region covering the leader sequence and investigated whether it was possible to detect subgenomic RNA from viral material recovered from wastewater. We identified distinct patterns in the persistence of the different genomic regions across the different types of wastewaters and the existence of chimeric reads mapping to non-amplified regions. Our findings suggest that preservation of the 5′-end of the genome and the ability to detect subgenomic RNA reads, though highly susceptible to environment and sample processing conditions, may be indicative of the quality and amount of the viral RNA present in wastewater.

Summary: Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide in the population since it was first detected in late 2019. The transcription and replication of coronaviruses, although not fully understood, is characterised by the production of genomic length RNA and shorter subgenomic RNAs to make viral proteins and ultimately progeny virions. Observed levels of subgenomic RNAs differ between sub-lineages and open reading frames but their biological significance is presently unclear. Methods: Using a large and diverse panel of virus sequencing data produced as part of the Danish COVID-19 routine surveillance together with information in electronic health registries, we assessed the association of subgenomic RNA levels with demographic and clinical variables of the infected individuals. Findings: Our findings suggest no significant statistical relationship between levels of subgenomic RNAs and host-related factors. Interpretation: Differences between lineages and subgenomic ORFs may be related to differences in target cell tropism, early virus replication/transcription kinetics or sequence features. Funding: The author(s) received no specific funding for this work.

Intro: We investigated two poultry mortality events in northwest Bangladesh, one in backyard poultry in Pabna in March 2017 and another in a commercial layer farm in Mymensingh in January 2018, to determine and molecularly characterize the etiological agent and to assess putative risk factors of disease outbreaks in both areas. Methods: We collected pooled swab samples from affected birds and environmental pooled swabs from affected and neighboring commercial poultry farms, backyard poultry, and live bird markets (LBM). We tested samples using specific real-time RT-PCR for influenza virus A (M gene), followed by HA and NA subtypes and sequenced and performed mutation analysis. Findings: We detected the H5N1 virus in the affected birds and all production and trading systems at both outbreak sites. Phylogenetic analysis of H5N1sequences revealed that the outbreaks were caused by reassortant of clade 2.3.2.1a H5N1 virus, which had also been detected in poultry at LBMs in Dhaka during an overlapping period. We identified several mutations likely to affect possible phenotypic changes, such as increased mammalian adaptation, reduced susceptibility to antiviral agents, and reduced host antiviral response. Investigation into poultry-keeping practices showed that most of the households, farms, and LBMs did not employ adequate biosecurity and hygiene, where (i) backyard poultry were typically reared in farmers’ dwellings, (ii) slaughter remnants were being discarded in the open where wild birds were scavenging waste, and (iii) farmers and vendors were generally not employing hand sanitizing after handling poultry. Conclusion: The investigation revealed that the relatedness of H5N1 strains across large spatial scales is high, suggesting a readily spreading and continuing evolution of the virus. Thus, the results contribute to sketching a bleak picture of the current AIV situation, the necessity for ongoing surveillance to track the evolution of H5N1 viruses in Bangladesh, and that adequate mitigation measures are urgently required.

Mind wandering (MW) is a mental phenomenon humans experience daily. Yet, we lack a complete understanding of the neural basis of this pervasive mental state. Over the past decade there has been an increase in publications using transcranial direct current stimulation (tDCS) to modulate the propensity to mind wander, but findings are diverse, and a satisfactory conclusion is missing. Recently, Boayue et al. (2020) reported successful reduction of mind wandering using high-definition tDCS (HD-tDCS) over the dorsolateral prefrontal cortex, providing preliminary evidence for the efficacy of HD-tDCS in interfering with mind wandering. The current study is a high-powered, pre-registered direct replication attempt of the effect found by Boayue et al. (2020). In addition, we investigated whether the effects of HD-tDCS on mind wandering would be prolonged and assessed the underlying processes of mind wandering using electroencephalography (EEG) and pupillometry during a finger-tapping random sequence generation task that requires the use of executive resources. We failed to find any evidence of the original effect of reduced MW during and after stimulation. When combining our data with the data from Boayue et al. (2020), the original effect of reduced MW caused by HD-tDCS disappeared. In addition, we observed increased occipital alpha power as task duration increased and increased midfrontal theta power preceding response patterns signaling high executive function use. Finally, tonic and phasic pupil size decreased as task duration increased yet, phasic responses were increased, while tonic responses were reduced preceding reports of MW. Additionally phasic pupil size also showed a tendency to be increased during periods of high executive function use. Importantly, none of the EEG or pupil measures were modulated by HD-tDCS. We conclude that HD-tDCS over the dorsolateral prefrontal cortex does not affect MW propensity and its neural signatures. Furthermore, we recommend that previously reported effects of tDCS on mind wandering and other cognitive functions should only be accepted after a successful pre-registered replication.

Purpose: Bangladesh has faced recurrent house crow (Corvus splendens) mortality incidents due to highly pathogenic avian influenza (HPAI) viruses since 2011. We investigated one such event in Dhaka in 2017 to detect and molecularly characterize the avian influenza virus (AIV) in crows and poultry from nearby live bird markets (LBMs). Methods & Materials: We collected cloacal and oropharyngeal swabs from moribund and dead crows (N=163) and environmental samples from 16 neighboring LBMs (N=145). We tested all samples using specific q-PCR for influenza A (M gene) H5/H7/H9/N1/N2/N6. All H5 positive samples were inoculated into embryonated eggs for virus culture. We performed conventional RT-PCR and Sanger sequencing of the isolated viruses and used MEGAX for phylogenetic analysis. Results: We identified 3 house crow roosts that had evidence of crow morbidity and mortality and crows feeding on poultry offal and wastage in neighboring LBMs. Ninety-one percent of crows (n=149; 95% Confidence Interval (CI):86-95) were positive for A/H5 including 89.3% for H5N1 (n =118), 2.0% for H5N6 (n = 3) and 8.7% H5Nx (n = 13). Among LBMs samples, 58.3% were positive with A/H5 (n=60; 95%CI:33-49), containing 88.3% H5N1 (n=53), 3.3% H5N6 (n=2), 8.3% H5NX (n=5); 8.7% for A/H9N2 (n=9), 6.8% for A/H5N1/H9N2 (n=7) and 26.2% for A/untypable (n=27). None of the crow and LBM samples were positive for H7. Phylogenetic analysis based on HA and NA genes of H5N1 and H5N6 suggests that the virus belonging to clade 2.3.2.1a and 2.3.4.4B respectively. Conclusion: Data findings suggest that multiple subtypes of H5 including H5Nx are circulating in both crows and poultry; surprisingly co-circulation of H5 subtypes in LBMs without mortality in chickens. The virus may thus have been transmitted to crows while they were feeding on poultry waste in the LBMs. Whether the virus is non-pathogenic in chickens but pathogenic in crows or it rapidly evolved in crows to become pathogenic and because mortality is unresolved. We suggest improved LBM biosecurity measures to reduce the risk of influenza virus spillover to wild birds, which would also benefit avoiding spill-over risks to humans and Bangladesh being at the epicenter of the next pandemic.

Chronic fibro-proliferative diseases are associated with nearly 45% of all deaths in the developed world. Matrix metalloproteinase (MMP) mediated remodeling of the extracellular matrix (ECM) plays an important role in disease development. Degradation of type I collagen is considered having a major role in this matter. C1M is a biomarker measuring type I collagen degradation fragments in blood. The aim of the current study was to investigate whether MMP mediated type I collagen degradation (C1M) was predictive of mortality in a large prospective cohort of Danish women aged 48–89 (n = 5855). Subjects with high serum C1M showed significant increased mortality. The adjusted three year HR was 2.02 [95% CI: 1.48–2.76] for all-cause mortality, 2.32 [95% CI: 1.51–3.56] for cancer and 1.77 [95% CI: 0.98–3.17] for cardiovascular diseases. The adjusted nine year HR was 1.50 [95% CI: 1.28–1.75] for all-cause mortality, 1.49 [95% CI: 1.16–1.90] for cancer and 1.69 [95% CI: 1.27–2.24] for cardiovascular diseases. High MMP-mediated type I collagen degradation was associated with increased mortality. Subjects with high C1M had a 2-fold increase in mortality compared to subjects with low levels of this collagen degradation product.

The significance of the age-related decline of androgens remains unclear in terms of cardiovascular risk, mood and cognition, and prostatic health. Although much research has been undertaken in this area and men's health has received still more attention in the latest years, there are no data based on randomized controlled clinical studies in aging men investigating the long-term effects of androgen replacement therapy on various aspects of the cardiovascular system, the immune system, body composition, and the brain. In men receiving long-term androgen replacement therapy, the safety aspects regarding the prostate are also an area of clinical importance. In this paper we present an up-dated review of the experimental and clinical evidence of androgen deficiency and androgen replacement therapy on carbohydrate metabolism, on coagulation and fibrinolysis, inflammatory effects, effects on lipoprotein metabolism, direct arterial effects, effects on body composition, effects on cognitive function and mood, and prostatic effects. The evidence clearly shows that data for the most part are conflicting, with only very few randomized studies available.

The present study investigated the effect of raloxifene, a selective estrogen receptor modulator (SERM), on aortic atherosclerosis in 80 ovariectomized, cholesterol-fed rabbits with pre-induced atherosclerosis. The animals were fed an atherogenic diet containing 240 mg cholesterol/day for 15 weeks, after this period a baseline control group was sacrificed. Thereafter, oral treatment was initiated with either estradiol 4 mg/day (n=20), raloxifene (210 mg/day) or placebo (n=20). In the treatment period of 39 weeks, the dietary cholesterol content was reduced to 80 mg cholesterol/day. Postmortem evaluation showed a significantly increased uterine weight induced by estradiol treatment (10.3+/-1.2 g), whereas raloxifene intervention caused a decreased uterus weight (1.21+/-0.1 g) when compared to placebo (2.48+/-0.47 g). Throughout the study, serum lipids increased in all groups to levels seen in very high risk humans. After 58 weeks the cholesterol content in the aorta was 3.18+/-0.54 micromol/cm(2) (38% reduction) in the estradiol group, 3.66+/-0.52 micromol/cm(2) (29% reduction) in the raloxifene group and 5.12+/-0.60 micromol/cm(2) in the placebo group. Analyses of the aortic cholesterol content corrected for time-averaged serum cholesterol revealed that both estradiol and raloxifene therapy significantly reduced the progression of atherosclerosis (P<0.01 for both) as compared to placebo.

Published studies dealing with the relationship between circulating levels of testosterone and dehydroepiandrosterone (sulfate) (DHEA(S)) and coronary heart disease (CHD) in males, as well as corresponding experimental animal studies are reviewed. One randomized intervention study, eight prospective and 30 cross-sectional studies have evaluated this relationship. In the intervention study, testosterone undecanoate given orally significantly improved angina pectoris in 62 patients with CHD as compared to placebo. No significant association between serum testosterone and CHD was reported in the prospective studies, whereas those studies concerning DHEAS found either no or an inverse association with CHD. Of 30 cross-sectional studies, 18 reported reduced concentrations of testosterone (primarily), and/or DHEA(S) in CHD patients as compared to normals, 11 found similar circulating levels of these androgens in controls and patients with CHD, and one study found elevated levels of DHEA(S) in patients. Animal studies (six male rabbits and one in male chicks) suggest an anti-atherogenic effect of testosterone and DHEA. In conclusion, one intervention, eight cohort and several cross-sectional studies suggest either a neutral or a favourable effect of testosterone and DHEA(S) on CHD in males.