Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape.

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape.

Eighteen months into the COVID-19 pandemic: The prevalence of depression, anxiety, and stress symptoms in Southeast Asia and the associated demographic factors.

Mental health has become a growing concern in the wake of the COVID-19 pandemic. We sought to determine the prevalence of mental health symptoms 18 months after the pandemic's declaration. Our cross-sectional study conducted among 18- to 65-year-old adults (N = 33,454) in October 2021 using the Depression, Anxiety and Stress Scales (DASS-21) found a high prevalence of severe to extremely severe anxiety (49%), depression (47%) and stress (36%) symptoms in Malaysia, Indonesia, Thailand, and Singapore. Multiple logistic regression showed that female and non-binary genders were associated with increased odds of severe/extremely severe symptoms of anxiety (female: aOR 1.44 [95% CI 1.37-1.52]; non-binary aOR 1.46 [1.16-1.84]), depression (female: aOR 1.39 [1.32-1.47]; non-binary aOR 1.42 [1.13-1.79]), and stress (female: aOR 1.48 [CI 1.40-1.57]; non-binary aOR 1.42 [1.12-1.78]). In all three symptom domains, the odds of severe/extremely severe symptoms decreased across age groups. Middle- and high-income respondents had lower odds of reporting severe/extremely severe anxiety (middle-income: aOR 0.79 [0.75-0.84]; high-income aOR 0.77 [0.69-0.86]) and depression (middle-income: aOR 0.85 [0.80-0.90]; high-income aOR 0.84 [0.76-0.94]) symptoms compared to low-income respondents, while only middle-income respondents had lower odds of experiencing severe/extremely severe stress symptoms (aOR 0.89 [0.84-0.95]). Compared to residents of Malaysia, residents of Indonesia were more likely to experience severe/extremely severe anxiety symptoms (aOR 1.08 [1.03-1.15]) but less likely to experience depression (aOR 0.69 [0.65-0.73]) or stress symptoms (aOR 0.92 [0.87-0.97]). Respondents living in Singapore had increased odds of reporting severe/extremely severe depression symptoms (aOR 1.33 [1.16-1.52]), while respondents residing in Thailand were more likely to experience severe/extremely severe stress symptoms (aOR 1.46 [1.37-1.55]). This study provides insights into the impacts of the COVID-19 pandemic on the point prevalence of psychological distress in Southeast Asia one and a half years after the beginning of the pandemic.

Clinical and epidemiological characteristics of patients seeking COVID-19 testing in a private centre in Malaysia: Is there a role for private healthcare in battling the outbreak?

OBJECTIVE: This cross-sectional observational study summarized the baseline characteristics of subjects who underwent COVID-19 molecular testing in a private medical centre located in the state of Selangor in Malaysia between 1 Oct 2020 and 31 Jan 2021. We compared the baseline characteristics between subjects who were tested positive and negative of SARS-CoV-2 infection, and identified risk factors which may be predictive of SARS-CoV-2 positivity. METHODS AND FINDINGS: A total of 36603 subjects who were tested for COVID-19 infection via molecular assays at Sunway Medical Centre between Oct 1, 2020 and Jan 31, 2021, and consented to participate in this observation study were included for analysis. Descriptive statistics was used to summarize the study cohort, whereas logistic regression analysis was used to identify risk factors associated with SARS-CoV-2 positivity. Among the reasons listed for COVID-19 screening were those who needed clearance for travelling, clearance to return to work, or clearance prior to hospital admission. They accounted for 67.7% of tested subjects, followed by the self-referred group (27.3%). Most of the confirmed cases were asymptomatic (62.6%), had no travel history (99.6%), and had neither exposure to SARS-CoV-2 confirmed cases (61.9%) nor exposure to patients under investigation (82.7%) and disease clusters (89.2%). Those who presented with loss of smell or taste (OR: 26.91; 95% CI: 14.81-48.92, p<0.001), fever (OR:3.97; 95% CI: 2.54-6.20, p<0.001), running nose (OR: 1.75; 95% CI:1.10-2.79, p = 0.019) or other symptoms (OR: 5.63; 95% CI:1.68-18.91, p = 0.005) were significantly associated with SARS-CoV-2 positivity in the multivariate logistic regression analysis. CONCLUSION: Our study showed that majority of patients seeking COVID-19 testing in a private healthcare setting were mainly asymptomatic with low epidemiological risk. Consequently, the average positivity rate was 1.2% compared to the national cumulative positivity rate of 4.65%. Consistent with other studies, we found that loss of smell or taste, fever and running nose were associated with SARS-CoV-2 positivity. We believe that strengthening the capacity of private health institutions is important in the national battle against the COVID-19 pandemic, emphasizing the importance of public-private partnership to improve the quality of clinical care.

Soft Interactions with Model Crowders and Non-canonical Interactions with Cellular Proteins Stabilize RNA Folding.

Living cells contain diverse biopolymers, creating a heterogeneous crowding environment, the impact of which on RNA folding is poorly understood. Here, we have used single-molecule fluorescence resonance energy transfer to monitor tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene glycol and yeast cell extract as crowding agents. As expected, polyethylene glycol stabilizes the docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexpectedly, we found evidence that it additionally displays soft, non-specific interactions with the ribozyme. Yeast extract has a profound effect on folding at protein concentrations 1000-fold lower than found intracellularly, suggesting the dominance of specific interactions over volume exclusion. Gel shift assays and affinity pull-down followed by mass spectrometry identified numerous non-canonical RNA-binding proteins that stabilize ribozyme folding; the apparent chaperoning activity of these ubiquitous proteins significantly compensates for the low-counterion environment of the cell.

Plectosphaeroic acids A, B, and C, indoleamine 2,3-dioxygenase inhibitors produced in culture by a marine isolate of the fungus Plectosphaerella cucumerina.

Laboratory cultures of the fungus Plectosphaerella cucumerina obtained from marine sediments collected in Barkley Sound, British Columbia, yielded the novel alkaloids plectosphaeroic acids A (1) to C (3). The alkaloids 1-3 are inhibitors of indoleamine 2,3-dioxygenase (IDO).