In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl ␥-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals.Molecules found in bacteria that consist of a lipid moiety conjugated to an amino acid have been termed lipoamino acids (1-3). Burstein et al. (4) found that the lipoamino acid Narachidonylglycine (NAGly) 1 causes hot plate analgesia in mice, indicating its possible biological relevance in mammals. NAGly was first synthesized (5) as a structural analog of the endogenous cannabinoid anandamide (6), and it was found to lack affinity for the cannabinoid CB1 receptor. We hypothesized that NAGly may be produced by mammalian tissues because it is composed of the naturally occurring compounds glycine and arachidonic acid. Herein we show that at least three arachidonyl amino acids are natural constituents in mammalian brain: NAGly, N-arachidonyl ␥-aminobutyric acid (NAGABA), and N-arachidonylalanine (NAAla). One member of this group, NAGly, is characterized in detail here. It is synthesized in situ in rat brain tissue from the precursors arachidonic acid and glycine, and it is hydrolyzed by the enzyme fatty acid amide hydrolase (FAAH). NAGly is widely distributed among mammalian tissues, implying multiple functions. One possible physiological function of NAGly is pain suppression, indicated by its marked suppression of formalininduced pain behavior in rats, confirming a previous report of analgesic activity in mice (4). EXPERIMENTAL PROCEDURESTissue Extraction and Purification-The procedure comprised a liquidliquid extraction modified from that described by Folch et al. (7) followed by a series of solid-phase separations. Fresh bovine brain and rat organs were homogenized in the methanol fraction of 20 volumes of 2:1 chloroform:methanol and centrifuged for 15 min at 31,000 ϫ g at 4°C. Chloroform was then added to the supernatant. NaCl (0.2 volume, 0.73%) was mixed with the crude homogenate, and the solution was allowed to separate overnight at 4°C or centrifuged at 1,000 ϫ g for 15 min. The upper phase was discarded and the interphase washed twice. The lower phase was then applied to diethylaminopropyl silica-based...
SummaryBackgroundResults of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects.MethodsFOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762.FindingsBetween Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months.InterpretationFluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function.FundingUK Stroke Association and NIHR Health Technology Assessment Programme.
Background Earlier serosurveys in India revealed SARS-CoV-2 seroprevalence of 0.73% during May-June and 7.1% during August-September 2020. We conducted the third serosurvey during Dec 2020 and Jan 2021, to estimate the seroprevalence of SARS-CoV-2 infection among general population and healthcare workers (HCWs) in India. Methods We conducted the serosurvey in the same 70 districts selected for the first and second serosurveys. From each district, we enrolled at least 400 individuals aged ≥ 10 years from general population and 100 HCWs from sub-district level health facilities. Sera from general population were tested for presence of IgG antibodies against nucleocapsid (N) and spike protein (S1-RBD) of SARS-CoV-2, whereas sera from HCWs were tested for anti-S1-RBD. We estimated weighted seroprevalence adjusted for assay characteristics. Results Of the 28,598 sera from general population, 4585 (16%) had IgG antibodies against N, 6647 (23.2%) against S1-RBD and 7436 (26%) against either. The weighted and assay characteristic adjusted seroprevalence against either of the antibodies was 24.1 (95%CI: 23.0%-25.3%). Among 7385 HCWs, the seroprevalence of anti-S1-RBD IgG antibodies was 25.6% (95% CI: 23.5%-27.8%). Conclusions Nearly one in four individuals aged > = 10 years from general population as well as HCWs in India were exposed to SARS-CoV-2 by December 2020.
SummaryBackgroundIntensive antiplatelet therapy with three agents might be more effective than guideline treatment for preventing recurrent events in patients with acute cerebral ischaemia. We aimed to compare the safety and efficacy of intensive antiplatelet therapy (combined aspirin, clopidogrel, and dipyridamole) with that of guideline-based antiplatelet therapy.MethodsWe did an international, prospective, randomised, open-label, blinded-endpoint trial in adult participants with ischaemic stroke or transient ischaemic attack (TIA) within 48 h of onset. Participants were assigned in a 1:1 ratio using computer randomisation to receive loading doses and then 30 days of intensive antiplatelet therapy (combined aspirin 75 mg, clopidogrel 75 mg, and dipyridamole 200 mg twice daily) or guideline-based therapy (comprising either clopidogrel alone or combined aspirin and dipyridamole). Randomisation was stratified by country and index event, and minimised with prognostic baseline factors, medication use, time to randomisation, stroke-related factors, and thrombolysis. The ordinal primary outcome was the combined incidence and severity of any recurrent stroke (ischaemic or haemorrhagic; assessed using the modified Rankin Scale) or TIA within 90 days, as assessed by central telephone follow-up with masking to treatment assignment, and analysed by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN47823388.Findings3096 participants (1556 in the intensive antiplatelet therapy group, 1540 in the guideline antiplatelet therapy group) were recruited from 106 hospitals in four countries between April 7, 2009, and March 18, 2016. The trial was stopped early on the recommendation of the data monitoring committee. The incidence and severity of recurrent stroke or TIA did not differ between intensive and guideline therapy (93 [6%] participants vs 105 [7%]; adjusted common odds ratio [cOR] 0·90, 95% CI 0·67–1·20, p=0·47). By contrast, intensive antiplatelet therapy was associated with more, and more severe, bleeding (adjusted cOR 2·54, 95% CI 2·05–3·16, p<0·0001).InterpretationAmong patients with recent cerebral ischaemia, intensive antiplatelet therapy did not reduce the incidence and severity of recurrent stroke or TIA, but did significantly increase the risk of major bleeding. Triple antiplatelet therapy should not be used in routine clinical practice.FundingNational Institutes of Health Research Health Technology Assessment Programme, British Heart Foundation.
The internal cyclopropanation of several diversely substituted dienic diazo esters is described. Thermolysis of the resulting vinylcyclopropanes yielded cyclopentene-annulated lactones in good yields. Depending on the choice of the dienyl unit, either guaiane or pseudoguaiane substitution patterns of the cyclopentene portion were obtained. Stereochemical assignments based on 13C NMR data are provided for all of these lactones. Subsequent transformations of the bicyclic lactones to differentially functionalized cyclopentenes are described. The potential of these synthons in the synthesis of perhydroazulene sesquiterpenes and several monoterpene cyclopentanoid natural products is addressed.
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