Factors associated with anxiety during the first two years of the COVID-19 pandemic in the United States: An analysis of the COVID-19 Citizen Science study.

COVID-19 increased the prevalence of clinically significant anxiety in the United States. To investigate contributing factors we analyzed anxiety, reported online via monthly Generalized Anxiety Disorders-7 (GAD-7) surveys between April 2020 and May 2022, in association with self-reported worry about the health effects of COVID-19, economic difficulty, personal COVID-19 experience, and subjective social status. 333,292 anxiety surveys from 50,172 participants (82% non-Hispanic white; 73% female; median age 55, IQR 42-66) showed high levels of anxiety, especially early in the pandemic. Anxiety scores showed strong independent associations with worry about the health effects of COVID-19 for oneself or family members (GAD-7 score +3.28 for highest vs. lowest category; 95% confidence interval: 3.24, 3.33; p<0.0001 for trend) and with difficulty paying for basic living expenses (+2.06; 1.97, 2.15, p<0.0001) in multivariable regression models after adjusting for demographic characteristics, COVID-19 case rates and death rates, and personal COVID-19 experience. High levels of COVID-19 health worry and economic stress were each more common among participants reporting lower subjective social status, and median anxiety scores for those experiencing both were in the range considered indicative of moderate to severe clinical anxiety disorders. In summary, health worry and economic difficulty both contributed to high rates of anxiety during the first two years of the COVID-19 pandemic in the US, especially in disadvantaged socioeconomic groups. Programs to address both health concerns and economic insecurity in vulnerable populations could help mitigate pandemic impacts on anxiety and mental health.

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq).

N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Small RNA Modifications: Integral to Function and Disease.

Small RNAs have the potential to store a secondary layer of labile biological information in the form of modified nucleotides. Emerging evidence has shown that small RNAs including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and tRNA-derived small RNAs (tsRNAs) harbor a diversity of RNA modifications. These findings highlight the importance of RNA modifications in the modulation of basic properties such as RNA stability and other complex physiological processes involved in stress responses, metabolism, immunity, and epigenetic inheritance of environmentally acquired traits, among others. High-resolution, high-throughput methods for detecting, mapping and screening these small RNA modifications now provide opportunities to uncover their diagnostic potential as sensitive disease markers.

ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.

High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules.

Archaeal and eukaryotic homologs of Hfq: A structural and evolutionary perspective on Sm function.

Hfq and other Sm proteins are central in RNA metabolism, forming an evolutionarily conserved family that plays key roles in RNA processing in organisms ranging from archaea to bacteria to human. Sm-based cellular pathways vary in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in each of these pathways being mediated by an RNA-associated molecular assembly built upon Sm proteins. Though the first structures of Sm assemblies were from archaeal systems, the functions of Sm-like archaeal proteins (SmAPs) remain murky. Our ignorance about SmAP biology, particularly vis-à-vis the eukaryotic and bacterial Sm homologs, can be partly reduced by leveraging the homology between these lineages to make phylogenetic inferences about Sm functions in archaea. Nevertheless, whether SmAPs are more eukaryotic (RNP scaffold) or bacterial (RNA chaperone) in character remains unclear. Thus, the archaeal domain of life is a missing link, and an opportunity, in Sm-based RNA biology.

Reclassification of Thermoproteus neutrophilus Stetter and Zillig 1989 as Pyrobaculum neutrophilum comb. nov. based on phylogenetic analysis.

The hyperthermophilic crenarchaeon Thermoproteus neutrophilus V24Sta(T) was originally classified before sequence-based phylogenetic analysis became standard for bacterial taxonomy. Subsequent phylogenetic analyses by various groups have shown that strain V24Sta(T) groups more closely with strains of the genus Pyrobaculum than with those in the genus Thermoproteus. Based on phylogenetic comparison of rRNA gene sequences and ribosomal proteins, we propose that strain V24Sta(T) be reclassified as Pyrobaculum neutrophilum comb. nov., with the type strain V24Sta(T) ( = DSM 2338(T) = JCM 9278(T)). An emended description of the genus Pyrobaculum is also presented.

Complete genome sequence of Pyrobaculum oguniense.

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative analyses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, reveals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illuminated some of the genome dynamics potentially shared with other species in this genus.

Discovery of permuted and recently split transfer RNAs in Archaea.

BACKGROUND: As in eukaryotes, precursor transfer RNAs in Archaea often contain introns that are removed in tRNA maturation. Two unrelated archaeal species display unique pre-tRNA processing complexity in the form of split tRNA genes, in which two to three segments of tRNAs are transcribed from different loci, then trans-spliced to form a mature tRNA. Another rare type of pre-tRNA, found only in eukaryotic algae, is permuted, where the 3' half is encoded upstream of the 5' half, and must be processed to be functional. RESULTS: Using an improved version of the gene-finding program tRNAscan-SE, comparative analyses and experimental verifications, we have now identified four novel trans-spliced tRNA genes, each in a different species of the Desulfurococcales branch of the Archaea: tRNA(Asp(GUC)) in Aeropyrum pernix and Thermosphaera aggregans, and tRNA(Lys(CUU)) in Staphylothermus hellenicus and Staphylothermus marinus. Each of these includes features surprisingly similar to previously studied split tRNAs, yet comparative genomic context analysis and phylogenetic distribution suggest several independent, relatively recent splitting events. Additionally, we identified the first examples of permuted tRNA genes in Archaea: tRNA(iMet(CAU)) and tRNA(Tyr(GUA)) in Thermofilum pendens, which appear to be permuted in the same arrangement seen previously in red alga. CONCLUSIONS: Our findings illustrate that split tRNAs are sporadically spread across a major branch of the Archaea, and that permuted tRNAs are a new shared characteristic between archaeal and eukaryotic species. The split tRNA discoveries also provide new clues to their evolutionary history, supporting hypotheses for recent acquisition via viral or other mobile elements.

Discovery of a minimal form of RNase P in Pyrobaculum.

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5' leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5' tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5' pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5' tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

Transcriptional map of respiratory versatility in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.

Hyperthermophilic crenarchaea in the genus Pyrobaculum are notable for respiratory versatility, but relatively little is known about the genetics or regulation of crenarchaeal respiratory pathways. We measured global gene expression in Pyrobaculum aerophilum cultured with oxygen, nitrate, arsenate and ferric iron as terminal electron acceptors to identify transcriptional patterns that differentiate these pathways. We also compared genome sequences for four closely related species with diverse respiratory characteristics (Pyrobaculum arsenaticum, Pyrobaculum calidifontis, Pyrobaculum islandicum, and Thermoproteus neutrophilus) to identify genes associated with different respiratory capabilities. Specific patterns of gene expression in P. aerophilum were associated with aerobic respiration, nitrate respiration, arsenate respiration, and anoxia. Functional predictions based on these patterns include separate cytochrome oxidases for aerobic growth and oxygen scavenging, a nitric oxide-responsive transcriptional regulator, a multicopper oxidase involved in denitrification, and an archaeal arsenate respiratory reductase. We were unable to identify specific genes for iron respiration, but P. aerophilum exhibited repressive transcriptional responses to iron remarkably similar to those controlled by the ferric uptake regulator in bacteria. Together, these analyses present a genome-scale view of crenarchaeal respiratory flexibility and support a large number of functional and regulatory predictions for further investigation. The complete gene expression data set can be viewed in genomic context with the Archaeal Genome Browser at archaea.ucsc.edu.

Growth and population dynamics of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a continuous-flow bioreactor.

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (mu(ANME-2) = 0.167 . week(-1)), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (mu(ANME-1) = 0.218 . week(-1)). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 . week(-1), and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.

Overexpression of urokinase by macrophages or deficiency of plasminogen activator inhibitor type 1 causes cardiac fibrosis in mice.

Several studies implicate elevated matrix metalloproteinase activity as a cause of cardiac fibrosis. However, it is unknown whether other proteases can also initiate cardiac fibrosis. Because absence of urokinase plasminogen activator (uPA) prevents development of cardiac fibrosis after experimental myocardial infarction in mice, we hypothesized that elevated activity of uPA or deficiency of the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1) might cause cardiac fibrosis. We used mice with scavenger-receptor (SR)-directed, macrophage-targeted uPA overexpression (SR-uPA+/0 mice) and PAI-1 null mice to test these hypotheses. Our studies revealed that SR-uPA+/0 mice developed cardiac fibrosis beginning between 5 and 10 weeks of age. Fibrosis was preceded by cardiac macrophage accumulation, implicating uPA-secreting macrophages as important contributors to development of fibrosis. A key role for uPA-secreting macrophages in development of cardiac fibrosis was supported by experiments in which recipients of bone marrow transplants from SR-uPA+/0 donors but not nontransgenic donors developed cardiac macrophage accumulation and fibrosis. SR-uPA+/0 mice and recipients of SR-uPA+/0 bone marrow had neither macrophage accumulation nor fibrosis in other major organs despite the presence of higher levels of uPA in these organs than in hearts. PAI-1 null mice but not congenic, age-matched controls also developed macrophage accumulation and fibrosis in hearts but not in other organs. We conclude: (1) either elevated macrophage uPA expression or PAI-1 deficiency is sufficient to cause cardiac macrophage accumulation and fibrosis; (2) macrophages are important contributors to the development of cardiac fibrosis; and (3) the heart is particularly sensitive to the effects of excess uPA activity.

Macrophage-targeted overexpression of urokinase causes accelerated atherosclerosis, coronary artery occlusions, and premature death.

BACKGROUND: Human atherosclerotic lesions contain elevated levels of urokinase plasminogen activator (uPA), expressed predominantly by macrophages. METHODS AND RESULTS: To test the hypothesis that macrophage-expressed uPA contributes to the progression and complications of atherosclerosis, we generated transgenic mice with macrophage-targeted overexpression of uPA. The uPA transgene was bred into the apolipoprotein E-null background, and transgenic mice and nontransgenic littermate controls were fed an atherogenic diet. uPA-transgenic mice had significantly elevated uPA activity in the atherosclerotic artery wall, of a magnitude similar to elevations reported in atherosclerotic human arteries. Compared with littermate controls, uPA-transgenic mice had accelerated atherosclerosis, dilated aortic roots, occlusive proximal coronary artery disease, myocardial infarcts, and early mortality. CONCLUSIONS: These data support the hypothesis that overexpression of uPA by artery wall macrophages is atherogenic and suggest that uPA inhibitors might be therapeutically useful.