Targeted delivery of antisense oligonucleotides by molecular conjugates

Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly( l -lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 µM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 µM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45543/1/11188_2005_Article_BF01232652.pd

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Effects of sleep disturbance on dyspnoea and impaired lung function following hospital admission due to COVID-19 in the UK: a prospective multicentre cohort study

Background: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea. Methods: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2–7 months after hospital discharge and a later time point 10–14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107). Findings: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4–6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5–8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (–19%; 95% CI –20 to –16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18–39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27–41% of this effect. Interpretation: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition. Funding: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council

Effects of Heat Stress on the Lactation Performance of Ewes Accustomed to Tropical Conditions and the Total Fluid Intake of Their Lambs

The impact of heat stress on the feed intake, milk production, water intake and urine output of undernourished lactating ewes and on the growth rate and water intake of their lambs was measured both in a climate chamber and during exposure to natural high ambient temperature conditions. Similar liveweight increases occurred in both stressed and unstressed lambs. Freed intake was depressed in heat-stressed ewes in the climate chamber but not under natural heat-stress conditions. During the first and second weeks of lactation calculated milk yield (200--500 ml/day) and composition were unaffected by heat stress per se. However, undernutrition due to the poor quality roughage offered apparently depressed milk production of all ewes. Increased water intake (27%) and plasma prolactin concentrations (220%) were recorded in heat-stressed ewes, but daily urine output (27--36 ml/kg body wt) was unaffected. No relationship between prolactin concentrations, milk production and antidiuretic activity was obvious. At the age of 5--6 weeks water intake accounted for 67% (500 ml/day) and 80% (1000 ml/day) of total fluid intake (water plus milk) of unstressed and heat-stressed lambs respectively. These data indicate the importance of making water freely available to lambs subjected to extensive grazing systems of tropical regions

Social Stressors and Intoxicated Sex Among an Online Sample of Men who have Sex with Men (MSM) Drawn from Seven Countries

Background: Rates of drug and alcohol use are higher among men who have sex with men (MSM) than the general adult male population, and are often associated with increased sexual risk-taking. Objectives: We aim to examine the prevalence of drunk or high sex and their associations with socio-demographic characteristics, gay social network size, and social stress among an online sample of MSM drawn from seven countries. Methods: Sexually-active MSM aged over 18 residing in Australia, Brazil, Canada, South Africa, Thailand, the United Kingdom, or the United States were recruited through Facebook for a quantitative survey (n = 2,403) in 2012. Two outcomes were examined via logistic regression: reporting being buzzed/drunk at last sex, and reporting being high at last sex. Results: Results highlight the role of social stressors in shaping drug use among MSM. Results were context-specific, though commonalities were seen across countries. Being in a male–male sexual relationship was associated with lower odds of being buzzed/drunk at last sex in five countries. Higher scores on measures of external homonegative discrimination and internalized homonegativity were associated with greater odds of reporting being high at last sex in three countries. Conclusions/Importance: Social networks and minority stressors can have significant effects on drug use and sex while drunk or high. This points to the importance of focusing on structural issues when designing interventions for MSM aimed at reducing the transmission of HIV and other STIs. © 2018 Taylor & Francis Group, LLC