The splicing of U12-type introns is a rate-limiting step in gene expression

Metazoan genes have recently been found to contain a novel class of introns that display non-canonical consensus sequences and are excised by a distinct splicing machinery. These introns occur very rarely, and have been called U12-type introns because recognition of their branch point sequences requires the U12 snRNP, which performs an analogous function to the U2 snRNP in splicing major-class introns. The persistence of two spliceosomes throughout virtually all of metazoan evolution suggests that the two spliceosomes play distinct and probably indispensable cellular roles. One enticing possibility is that the U12-type spliceosome functions in post-transcriptional regulation, serving as the rate-determining step in the splicing pathway of genes containing U12-type introns. To test this idea I have investigated the timing of U12-type intron removal relative to the removal of major-class introns from pre-mRNAs that contain both intron types. I have addressed this question using two different experimental approaches.One way to evaluate the order of intron removal from a transcript is to document the relative amounts of unspliced intron sequences within a steady-state population of partially-processed transcripts: if the splicing of U12-type introns is rate limiting and occurs last, then their sequences should be more abundant than those of their major-class intron neighbors. I have developed an accurate assay based on the technique of quantitative RT-PCR to measure the relative abundance of unspliced introns within several genes. Here, I present results from the analysis of three human genes showing that in all three cases splicing of the U12-type intron proceeds more slowly than splicing of the U2-type introns from the same transcript.The second experimental approach aimed to address the question of whether replacement of a naturally occurring U12-type intron with U2-type intron consensus sequences can affect the rate of production of mature mRNA and protein. Constructs were created which expressed either cyan or yellow fluorescent proteins only when completely spliced. The constructs contained either a U12-type or a U2-type intron in an arrangement which permitted correlation of color with type of splicing. By observing the relative intensities of the two fluorescent colors, it was possible to infer the relative efficiencies of the two splicing pathways within transfected Drosophila melanogaster tissue culture cells. Results of these experiments showed that replacement of a U12-type intron with canonical consensus sequences did indeed dramatically increase expression of the corresponding mRNA and protein.These results provide direct evidence that in vivo gene expression can be altered by the presence of a U12-type intron and implicate the U12-type spliceosome as a potential target in the post-transcriptional regulation of gene expression

The six-year outcome of alcohol use disorders in men: A population based study from India.

BACKGROUND: Despite the large and growing public health problem of alcohol use disorders (AUD) in India there is a dearth of evidence about the longitudinal outcomes in AUD. The aim of this study is to describe the course and outcomes of AUD in a population based sample of men in India. METHODS: A community cohort of 1899 adult (18-49 years at baseline) men who participated in a cross-sectional survey in Goa, India between 2006 and 08, were re-interviewed face to face 6 years later (2012-14). A range of outcomes including social problems (e.g., workplace problems, domestic violence), morbidity (e.g., range of physical and mental health problems), biological parameters (e.g., mean corpuscular volume [MCV], gamma-glutamyl transpeptidase [GGT]) and mortality were measured at follow up. For the association of AUD at baseline with outcomes at follow-up, multivariable logistic regression was used to estimate odds ratios (OR). Analyses were weighted to account for baseline sampling design, age distribution, rural and urban sample sizes, number of adults aged 18-49 years in the household (at baseline), and non-response (at baseline). RESULTS: 1514 (79.7%) were seen at follow-up; a loss to follow up of 20.3%. At follow up, 3.7% of baseline non-drinkers and 15.0% of baseline casual drinkers had AUD. 46.9% of baseline hazardous drinkers and 55.4% baseline harmful drinkers continued to have AUD at follow up. Of those with AUD at baseline, 21.8% had stopped drinking at follow-up. Compared to being abstinent, harmful drinking at baseline was associated with several outcomes at follow-up: workplace/social problems, hypertension, death, tobacco use, suicidality, anxiety disorders, and raised GGT (p<0.002). Hazardous drinking at baseline was associated with tobacco use and raised GGT and MCV (p<0.002) at follow-up. CONCLUSION: Our findings of high persistent and new AUD in the community and the association with a range of long term adverse events are an important addition to the limited evidence about the course and outcomes of AUD in India, which have the potential for informing health policy

Safety and Efficacy of Paracetamol + Lignocaine Injection in Patients with High-grade Fever:A Prospective Analysis

Paracetamol is frequently used as an analgesic and antipyretic across the world. However, there is no data on Indian patients regarding the safety and efficacy of paracetamol + lignocaine injection in patients with high-grade fever. Hence, a prospective analysis was conducted to assess the safety and efficacy of paracetamol and lignocaine injection in patients with high-grade fever and mild-to-moderate body pain. The study is a real-world prospective study. The results showed that following intervention with paracetamol and lignocaine injection, 98% patients showed resolution of fever and 58% patients showed improvement in pain symptoms. The authors suggest that clinicians should consider paracetamol and lignocaine injection in patients with high-grade fever and associated pain and discomfort

A lay-counsellor delivered brief psychological treatment for men with comorbid Alcohol Use Disorder and depression in primary care: Secondary analysis of data from a randomized controlled trial.

BACKGROUND: We investigated the feasibility, acceptability, safety, and preliminary effectiveness of the Counselling for Alcohol Problems (CAP) psychological intervention delivered by non-specialist health workers (NSHW) to participants with alcohol use disorder (AUD) and comorbid depression in primary care. METHODS: We used data from a single blind randomised controlled trial conducted in ten primary health care centres in Goa, India. Adult male harmful or dependent drinkers with or without depression were randomized (1:1) to receive either CAP & enhanced usual care (EUC) or EUC only. Process indicators such as the number of completed counselling sessions were assessed and compared between comorbid and non-comorbid participants. Remission from AUD and depression along with abstinence were measured at 3 and 12 months post randomisation. Analyses were on an intention-to-treat basis, employing multivariable regression analyses. RESULTS: 271 participants had symptoms of comorbid depression; 241 did not. Both groups completed a similar number of counselling sessions (adjusted Mean Difference 0.05, 95 %CI -0.24-0.34;p = 0.72). Among comorbid participants, CAP did not lead to more frequent adverse events compared to EUC only (adjusted Odds Ratio [aOR] 0.84, 0.43-1.64;p = 0.62), and there was no evidence for an effect of CAP on remission from AUD or depression at 3 months (aOR 1.51, 0.84-2.74;p = 0.17 and aOR 0.74, 0.43-1.27;p = 0.28) and 12 months follow-up, respectively (aOR 1.69, 0.96-3.01;p = 0.08 and aOR 1.08, 0.62-1.87;p = 0.79). CONCLUSIONS: Brief therapies like CAP can be safely delivered by NSHWs to patients with comorbid AUD and depression, but their effectiveness may be limited and requires further investigation

Counselling for Alcohol Problems (CAP), a lay counsellor-delivered brief psychological treatment for harmful drinking in men, in primary care in India : a randomised controlled trial

Background Although structured psychological treatments are recommended as first-line interventions for harmful drinking, only a small fraction of people globally receive these treatments because of poor access in routine primary care. We assessed the effectiveness and cost-effectiveness of Counselling for Alcohol Problems (CAP), a brief psychological treatment delivered by lay counsellors to patients with harmful drinking attending routine primary health-care settings. Methods In this randomised controlled trial, we recruited male harmful drinkers defined by an Alcohol Use Disorders Identification Test (AUDIT) score of 12–19 who were aged 18–65 years from ten primary health centres in Goa, India. We excluded patients who needed emergency medical treatment or inpatient admission, who were unable to communicate clearly, and who were intoxicated at the time of screening. Participants were randomly allocated (1:1) by trained health assistants based at the primary health centres to enhanced usual care (EUC) alone or EUC combined with CAP, in randomly sized blocks of four to six, stratified by primary health centre, and allocation was concealed with use of sequential numbered opaque envelopes. Physicians providing EUC and those assessing outcomes were masked. Primary outcomes were remission (AUDIT score of <8) and mean daily alcohol consumed in the past 14 days, at 3 months. Secondary outcomes were the effect of drinking, disability score, days unable to work, suicide attempts, intimate partner violence, and resource use and costs of illness. Analyses were on an intention-to-treat basis. We used logistic regression analysis for remission and zero-inflated negative binomial regression analysis for alcohol consumption. We assessed serious adverse events in the per-protocol population. This trial is registered with the ISCRTN registry, number ISRCTN76465238. Findings Between Oct 28, 2013, and July 29, 2015, we enrolled and randomly allocated 377 participants (188 [50%] to the EUC plus CAP group and 190 [50%] to the EUC alone group [one of whom was subsequently excluded because of a protocol violation]), of whom 336 (89%) completed the 3 month primary outcome assessment (164 [87%] in the EUC plus CAP group and 172 [91%] in the EUC alone group). The proportion with remission (59 [36%] of 164 in the EUC plus CAP group vs 44 [26%] of 172 in the EUC alone group; adjusted prevalence ratio 1·50 [95% CI 1·09–2·07]; p=0·01) and the proportion abstinent in the past 14 days (68 [42%] vs 31 [18%]; adjusted odds ratio 3·00 [1·76–5·13]; p<0·0001) were significantly higher in the EUC plus CAP group than in the EUC alone group, but we noted no effect on mean daily alcohol consumed in the past 14 days among those who reported drinking in this period (37·0 g [SD 44·2] vs 31·0 g [27·8]; count ratio 1·08 [0·79–1·49]; p=0·62). We noted an effect on the percentage of days abstinent in the past 14 days (adjusted mean difference [AMD] 16·0% [8·1–24·1]; p<0·0001), but no effect on the percentage of days of heavy drinking (AMD −0·4% [–5·7 to 4·9]; p=0·88), the effect of drinking (Short Inventory of Problems score AMD–0·03 [–1·93 to 1·86]; p=0.97), disability score (WHO Disability Assessment Schedule score AMD 0·62 [–0·62 to 1·87]; p=0·32), days unable to work (no days unable to work adjusted odds ratio 1·02 [0·61–1·69]; p=0.95), suicide attempts (adjusted prevalence ratio 1·8 [–2·4 to 6·0]; p=0·25), and intimate partner violence (adjusted prevalence ratio 3·0 [–10·4 to 4·4]; p=0·57). The incremental cost per additional remission was $217 (95% CI 50–1073), with an 85% chance of being cost-effective in the study setting. We noted no significant difference in the number of serious adverse events between the two groups (six [4%] in the EUC plus CAP group vs 13 [8%] in the EUC alone group; p=0·11). Interpretation CAP delivered by lay counsellors plus EUC was better than EUC alone was for harmful drinkers in routine primary health-care settings, and might be cost-effective. CAP could be a key strategy to reduce the treatment gap for alcohol use disorders, one of the leading causes of the global burden among men worldwide. Funding Wellcome Trust

Feasibility, acceptability, and cost-effectiveness of a brief, lay counsellor delivered psychological treatment for men with alcohol dependence in primary care: an exploratory randomized controlled trial

Aims: To examine the feasibility, acceptability and preliminary cost-effectiveness of a lay counsellor delivered psychological treatment for men with alcohol dependence in primary care. Design: Single-blind individually randomized trial comparing counselling for alcohol problems (CAP) plus enhanced usual care (EUC) versus EUC only. Setting: Ten primary health centres in Goa, India. Participants: Men (n = 135) scoring ≥ 20 on the Alcohol Use Disorder Identification Test (AUDIT). Sixty-six participants were randomized to EUC and 69 to CAP + EUC. Interventions: CAP, a lay counsellor-delivered psychological treatment for harmful drinking, with referral to de-addiction centre for medically assisted detoxification. EUC comprised consultation with physician, providing screening results and referral to a de-addiction centre. Measurements: Baseline socio-demographic data, readiness to change and perceived usefulness of counselling. Acceptability and feasibility process indicators such as data on screening and therapy. Outcomes were measured at 3 and 12 months post-randomization and included remission, mean daily alcohol consumed, percentage of days abstinent (PDA), percentage of days of heavy drinking (PDHD), recovery, uptake of detoxification services, impacts of alcohol dependence, resource use and costs. Findings: Participants in the CAP + EUC arm had more numerically but not statistically significantly favourable outcomes compared with those in the EUC arm for (a) remission at 3 months [adjusted odds ratio (aOR) = 1.95, 95% confidence interval (CI) = 0.74–5.15] and 12 months (aOR = 1.90, 95% CI = 0.72–5.00), (b) proportion of non-drinkers at 3 months (aOR = 1.26; 95% CI = 0.58–2.75) and 12 months (aOR = 1.25; 95% CI = 0.58–2.64) and (c) ethanol consumption among drinkers at 3 months (count ratio = 0.91; 95% CI = 0.58–1.45) and 12 months (count ratio = 1.06; 95% CI = 0.73–1.54). There was no statistically significant evidence of a difference in the occurrence of serious adverse events between the two arms. From a societal perspective, there was a 53% chance of CAP + EUC being cost-effective in achieving remission at 12 months at the willingness-to-pay threshold of $415. Conclusions: Lay counsellor-delivered psychological treatment for men with alcohol dependence (AD) in primary care may be effective in managing AD in low- and middle-income countries. A definitive trial of the intervention is warranted

The effectiveness and cost-effectiveness of lay counsellor-delivered psychological treatments for harmful and dependent drinking and moderate to severe depression in primary care in India: PREMIUM study protocol for randomized controlled trials.

BACKGROUND: The leading mental health causes of the global burden of disease are depression in women and alcohol use disorders in men. A major hurdle to the implementation of evidence-based psychological treatments in primary care in developing countries is the non-availability of skilled human resources. The aim of these trials is to evaluate the effectiveness and cost-effectiveness of two psychological treatments developed for the treatment of depression and alcohol use disorders in primary care in India. METHODS/DESIGN: This study protocol is for parallel group, randomized controlled trials (Healthy Activity Program for moderate to severe depression, Counselling for Alcohol Problems for harmful and dependent drinking) in eight primary health centres in Goa, India. Adult primary care attendees will be screened with the Patient Health Questionnaire for depression and, in men only, the Alcohol Use Disorders Identification Test for drinking problems. Screen-positive attendees will be invited to participate; men who screen positive for both disorders will be invited to participate in the Counselling for Alcohol Problems trial. Those who consent will be allocated in a 1:1 ratio to receive either the respective psychological treatment plus enhanced usual care or enhanced usual care only using a computer generated allocation sequence, stratified by primary health centre and, for depression, by sex. The enhanced usual care comprises providing primary health centre doctors with contextualized World Health Organization guidelines and screening results. Psychological treatments will be delivered by lay counsellors, over a maximum period of three months. Primary outcomes are severity of disorder and remission rates at three months post-enrolment and, for the Counselling for Alcohol Problems trial, drinking and the impact of drinking on daily lives. Secondary outcomes include severity of disorder and remission rates at 12 months, disability scores, suicidal behaviour and economic impact, and cost-effectiveness at three and 12 months. 500 participants with depression and 400 participants with harmful drinking will be recruited. Primary analyses will be intention-to-treat. DISCUSSION: These trials may offer a new approach for the treatment of moderate-severe depression and drinking problems in primary care that is potentially scalable as it relies on delivery by a single pool of lay counsellors. TRIAL REGISTRATION: Both trials are registered with the International Society for the Registration of Clinical Trials (Healthy Activity Programme registration number ISRCTN95149997; Counselling for Alcohol Problems registration number ISRCTN76465238)

Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2

Activation of the angiotensin 1-7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1-7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1-7 action. Wild type male C57BL/6 mice (10-12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1-7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1-7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1-7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1-7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1-7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1-7, and increased Ang 1-7 action represents a potential therapeutic strategy for cardiovascular diseases. KEY MESSAGES: Activation of the renin-angiotensin system (RAS) plays a key pathogenic role in cardiovascular disease. ACE2, a monocarboxypeptidase, negatively regulates pathological effects of Ang II. Antagonizing Ang 1-7 prevents the therapeutic effects of recombinant human ACE2. Our results highlight a key protective role of Ang 1-7 in cardiovascular disease

Sustained effectiveness and cost-effectiveness of the Healthy Activity Programme, a brief psychological treatment for depression delivered by lay counsellors in primary care: 12-month follow-up of a randomised controlled trial

BACKGROUND:The Healthy Activity Programme (HAP), a brief behavioural intervention delivered by lay counsellors, enhanced remission over 3 months among primary care attendees with depression in peri-urban and rural settings in India. We evaluated the sustainability of the effects after treatment termination, the cost-effectiveness of HAP over 12 months, and the effects of the hypothesized mediator of activation on clinical outcomes. METHODS AND FINDINGS:Primary care attendees aged 18-65 years screened with moderately severe to severe depression on the Patient Health Questionnaire 9 (PHQ-9) were randomised to either HAP plus enhanced usual care (EUC) (n = 247) or EUC alone (n = 248), of whom 95% completed assessments at 3 months, and 91% at 12 months. Primary outcomes were severity on the Beck Depression Inventory-II (BDI-II) and remission on the PHQ-9. HAP participants maintained the gains they showed at the end of treatment through the 12-month follow-up (difference in mean BDI-II score between 3 and 12 months = -0.34; 95% CI -2.37, 1.69; p = 0.74), with lower symptom severity scores than participants who received EUC alone (adjusted mean difference in BDI-II score = -4.45; 95% CI -7.26, -1.63; p = 0.002) and higher rates of remission (adjusted prevalence ratio [aPR] = 1.36; 95% CI 1.15, 1.61; p < 0.009). They also fared better on most secondary outcomes, including recovery (aPR = 1.98; 95% CI 1.29, 3.03; p = 0.002), any response over time (aPR = 1.45; 95% CI 1.27, 1.66; p < 0.001), higher likelihood of reporting a minimal clinically important difference (aPR = 1.42; 95% CI 1.17, 1.71; p < 0.001), and lower likelihood of reporting suicidal behaviour (aPR = 0.71; 95% CI 0.51, 1.01; p = 0.06). HAP plus EUC also had a marginal effect on WHO Disability Assessment Schedule score at 12 months (aPR = -1.58; 95% CI -3.33, 0.17; p = 0.08); other outcomes (days unable to work, intimate partner violence toward females) did not statistically significantly differ between the two arms. Economic analyses indicated that HAP plus EUC was dominant over EUC alone, with lower costs and better outcomes; uncertainty analysis showed that from this health system perspective there was a 95% chance of HAP being cost-effective, given a willingness to pay threshold of Intl$16,060-equivalent to GDP per capita in Goa-per quality-adjusted life year gained. Patient-reported behavioural activation level at 3 months mediated the effect of the HAP intervention on the 12-month depression score (β = -2.62; 95% CI -3.28, -1.97; p < 0.001). Serious adverse events were infrequent, and prevalence was similar by arm. We were unable to assess possible episodes of remission and relapse that may have occurred between our outcome assessment time points of 3 and 12 months after randomisation. We did not account for or evaluate the effect of mediators other than behavioural activation. CONCLUSIONS:HAP's superiority over EUC at the end of treatment was largely stable over time and was mediated by patient activation. HAP provides better outcomes at lower costs from a perspective covering publicly funded healthcare services and productivity impacts on patients and their families. TRIAL REGISTRATION:ISRCTN registry ISRCTN95149997

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy