Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples

‘It was like coming back from the clouds’:a qualitative analysis of the lived experience of overdose consequent to drug use among a cohort of people who use drugs in Scotland

Background: Globally, non-fatal overdose (NFOD) rates consequent to drug use, typically opioids, continue increasing at a startling rate. Existing quantitative research has revealed myriad factors and characteristics linked to experiencing NFOD, but it is critically important to explore the lived context underlying these associations. In this qualitative study, we sought to understand the experiences of NFOD among people who use drugs in a Scottish region in order to: enhance public policy responses; inform potential intervention development to mitigate risk; and contribute to the literature documenting the lived experience of NFOD.Methods: From June to July 2021, two peer researchers conducted face-to-face semi-structured interviews with people who use drugs who had experienced recent NFOD attending harm reduction services in Tayside, Scotland. These were transcribed verbatim and evaluated using thematic analysis with an inductive approach which had an experiential and essentialist orientation.Results: Twenty people were interviewed across two sites. Of those, 15 (75%) were male and mean age was 38.2 (7.7) years. All had experienced at least one NFOD in the prior six months, and all reported polydrug use. Five themes were identified, within which 12 subthemes were situated. The themes were: social context; personal risk-taking triggers; planned and impulsive consumption; risk perception; and overdose reversal. The results spoke to the environmental, behavioural, cognitive, economic, and marketplace, factors which influence the context of NFOD in the region.Conclusions: A complex interplay of behavioural, psychological, and situational factors were found to impact the likelihood of experiencing NFOD. Structural inequities which policy professionals and civic leaders should seek to remedy were identified, while service providers may seek to reconfigure healthcare provision for people who use drugs to account for the interpersonal, psychological, and social factors identified, which appear to precipitate NFOD.Trial registration: Not applicable

“Back to square one”: The experience of straddling adolescence and early adulthood in unemployed UK university graduates with common mental health issues

We investigated the experiences of unemployed university graduates with common mental health issues. After conducting semistructured interviews with 12 unemployed bachelor’s degree graduates with common mental health issues, we used interpretative phenomenological analysis to generate three superordinate themes: “fall from grace,” “vulnerability,” and “life on hold.” Our focus in this article is life on hold and its constituent themes: “stagnation,” “moving backward,” and “feeling left behind.” Graduates struggled to complete the broader structural life transition from university student to the adult world of work, experiencing a nebulous state of straddling adolescence and early adulthood. This undermined their sense of adult maturity, leaving them vulnerable to becoming entrenched in their mental health-related difficulties. We discuss these findings in relation to the developmental perspectives of life-course theory, status passages, and separation–individuation in early adulthood, which raise important issues for the applicability of life-course frameworks for these graduates, who are a disadvantaged minority group

Psychosocial factors associated with overdose subsequent to Illicit Drug use:a systematic review and narrative synthesis

Background and aims: Psychological and social status, and environmental context, may mediate the likelihood of experiencing overdose subsequent to illicit drug use. The aim of this systematic review was to identify and synthesise psychosocial factors associated with overdose among people who use drugs.Methods: This review was registered on Prospero (CRD42021242495). Systematic record searches were undertaken in databases of peer-reviewed literature (Medline, Embase, PsycINFO, and Cinahl) and grey literature sources (Google Scholar) for work published up to and including 14 February 2023. Reference lists of selected full-text papers were searched for additional records. Studies were eligible if they included people who use drugs with a focus on relationships between psychosocial factors and overdose subsequent to illicit drug use. Results were tabulated and narratively synthesised.Results: Twenty-six studies were included in the review, with 150,625 participants: of those 3,383–4072 (3%) experienced overdose. Twenty-one (81%) studies were conducted in North America and 23 (89%) reported polydrug use. Psychosocial factors associated with risk of overdose (n = 103) were identified and thematically organised into ten groups. These were: income; housing instability; incarceration; traumatic experiences; overdose risk perception and past experience; healthcare experiences; perception of own drug use and injecting skills; injecting setting; conditions with physical environment; and social network traits.Conclusions: Global rates of overdose continue to increase, and many guidelines recommend psychosocial interventions for dependent drug use. The factors identified here provide useful targets for practitioners to focus on at the individual level, but many identified will require wider policy changes to affect positive change. Future research should seek to develop and trial interventions targeting factors identified, whilst advocacy for key policy reforms to reduce harm must continue

Psychosocial factors associated with overdose subsequent to Illicit Drug use:a systematic review and narrative synthesis

Background and aims: Psychological and social status, and environmental context, may mediate the likelihood of experiencing overdose subsequent to illicit drug use. The aim of this systematic review was to identify and synthesise psychosocial factors associated with overdose among people who use drugs.Methods: This review was registered on Prospero (CRD42021242495). Systematic record searches were undertaken in databases of peer-reviewed literature (Medline, Embase, PsycINFO, and Cinahl) and grey literature sources (Google Scholar) for work published up to and including 14 February 2023. Reference lists of selected full-text papers were searched for additional records. Studies were eligible if they included people who use drugs with a focus on relationships between psychosocial factors and overdose subsequent to illicit drug use. Results were tabulated and narratively synthesised.Results: Twenty-six studies were included in the review, with 150,625 participants: of those 3,383–4072 (3%) experienced overdose. Twenty-one (81%) studies were conducted in North America and 23 (89%) reported polydrug use. Psychosocial factors associated with risk of overdose (n = 103) were identified and thematically organised into ten groups. These were: income; housing instability; incarceration; traumatic experiences; overdose risk perception and past experience; healthcare experiences; perception of own drug use and injecting skills; injecting setting; conditions with physical environment; and social network traits.Conclusions: Global rates of overdose continue to increase, and many guidelines recommend psychosocial interventions for dependent drug use. The factors identified here provide useful targets for practitioners to focus on at the individual level, but many identified will require wider policy changes to affect positive change. Future research should seek to develop and trial interventions targeting factors identified, whilst advocacy for key policy reforms to reduce harm must continue

The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments

AbstractFörster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ∼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research

Long-term risk factors for developing Barrett's oesophagus in patients with gastro-oesophageal reflux disease:a longitudinal cohort study

Background and aims: Several characteristics are known to affect the risk of Barrett's oesophagus (BO) in the general population, with symptomatic gastro-oesophageal reflux disease (GORD) being a critical risk factor. In this study, we examined factors that influence BO development in people living with GORD.Design: People living with GORD were recruited from an endoscopy unit with lifestyle, medical and prescribing history collected. Logistic regression analysis was undertaken to assess the effects of multiple parameters on the likelihood of developing BO.Results: 1197 participants were recruited. Most were Caucasian (n=1188, 99%), had no formal educational qualifications (n=714; 59.6%) and lived with overweight (mean body mass index &gt;25 kg/m2). Many lived in areas of least socioeconomic resource (n=568; 47.4%). 139 (11.6%) had BO at baseline. In adjusted baseline analysis (n=1197), male sex (adjusted OR, aOR 2.04 (95% CI 1.92 to 4.12), p≤0.001), increasing age (aOR 1.03 (95% CI 1.01 to 1.04), p≤0.0001) and proton pump inhibitor use (aOR 3.03 (95% CI 1.80 to 5.13), p≤0.0001) were associated with higher odds of BO. At follow-up (n=363), 22 (6.1%) participants developed BO; male sex (aOR 3.18 (95% CI 1.28 to 7.86), p=0.012), pack-years cigarettes smoked (aOR 1.04 (95% CI 1.00 to 1.08), p=0.046) and increased alcohol intake (aOR 1.02 (95% CI 1.00 to 1.04), p=0.013), were associated with increased odds of BO.Conclusion: Male sex, pack-years cigarettes smoked, and increasing alcohol intake, were independently associated with increased odds of developing BO over 20-year follow-up. These results align with research linking male sex and smoking with BO and extend this by implicating the potential role of alcohol in developing BO, which may require communication through public health messaging.</p

Alcohol consumption and metabolic syndrome : Clinical and epidemiological impact on liver disease

Alcohol use and metabolic syndrome are highly prevalent in the population and frequently co-exist. Both are implicated in a large range of health problems, including chronic liver disease, hepatocellular carcinoma, and liver-related outcomes (i.e. decompensation or liver transplantation). Studies have yielded mixed results regarding the effects of mild-moderate alcohol consumption on the risk of metabolic syndrome and fatty liver disease, possibly due to methodological differences. The few available prospective studies have indicated that mild-moderate alcohol use is associated with an increase in liver-related outcomes. This conclusion was substantiated by systems biology analyses suggesting that alcohol and metabolic syndrome may play a similar role in fatty liver disease, potentiating an already existing dysregulation of common vital homeostatic pathways. Alcohol and metabolic factors are independently and jointly associated with liver-related outcomes. Indeed, metabolic syndrome increases the risk of liver-related outcomes, regardless of alcohol intake. Moreover, the components of metabolic syndrome appear to have additive effects when it comes to the risk of liver-related outcomes. A number of population studies have implied that measures of central/abdominal obesity, such as the waist-to-hip ratio, can predict liver-related outcomes more accurately than BMI, including in individuals who consume harmful quantities of alcohol. Many studies even point to synergistic interactions between harmful alcohol use and many metabolic components. This accumulating evidence showing independent, combined, and modifying effects of alcohol and metabolic factors on the onset and progression of chronic liver disease highlights the multifactorial background of liver disease in the population. The available evidence suggests that more holistic approaches could be useful for risk prediction, diagnostics and treatment planning. (c) 2022 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Long-term risk factors for developing Barrett's oesophagus in patients with gastro-oesophageal reflux disease:a longitudinal cohort study

Background and aims: Several characteristics are known to affect the risk of Barrett's oesophagus (BO) in the general population, with symptomatic gastro-oesophageal reflux disease (GORD) being a critical risk factor. In this study, we examined factors that influence BO development in people living with GORD.Design: People living with GORD were recruited from an endoscopy unit with lifestyle, medical and prescribing history collected. Logistic regression analysis was undertaken to assess the effects of multiple parameters on the likelihood of developing BO.Results: 1197 participants were recruited. Most were Caucasian (n=1188, 99%), had no formal educational qualifications (n=714; 59.6%) and lived with overweight (mean body mass index &gt;25 kg/m2). Many lived in areas of least socioeconomic resource (n=568; 47.4%). 139 (11.6%) had BO at baseline. In adjusted baseline analysis (n=1197), male sex (adjusted OR, aOR 2.04 (95% CI 1.92 to 4.12), p≤0.001), increasing age (aOR 1.03 (95% CI 1.01 to 1.04), p≤0.0001) and proton pump inhibitor use (aOR 3.03 (95% CI 1.80 to 5.13), p≤0.0001) were associated with higher odds of BO. At follow-up (n=363), 22 (6.1%) participants developed BO; male sex (aOR 3.18 (95% CI 1.28 to 7.86), p=0.012), pack-years cigarettes smoked (aOR 1.04 (95% CI 1.00 to 1.08), p=0.046) and increased alcohol intake (aOR 1.02 (95% CI 1.00 to 1.04), p=0.013), were associated with increased odds of BO.Conclusion: Male sex, pack-years cigarettes smoked, and increasing alcohol intake, were independently associated with increased odds of developing BO over 20-year follow-up. These results align with research linking male sex and smoking with BO and extend this by implicating the potential role of alcohol in developing BO, which may require communication through public health messaging.</p