RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo ß-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal ß-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.This work was supported by Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (H.H.-W.W.), Gates Cambridge (J.Q.L.), Fundac¸ a˜ o para a Cieˆ ncia e Tecnologia (C.M.R.), Wellcome Trust Senior Investigator Award (100329/Z/ 12/Z) (W.A.H.), EPSRC Grant (EP/H018301/1), MRC Grant (MR/K015850/1 and MR/K02292X/1), Wellcome Trust (089703/Z/09/Z) (C.F.K.), Wellcome Trust Programme Grant (085314/Z/08/Z), and ERC Advanced Grant (322817) (C.E.H.)

Studying Early Lethality of 45,XO (Turner's Syndrome) Embryos Using Human Embryonic Stem Cells

Turner's syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS 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. Findings Between 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. Interpretation Fluoxetine 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. Funding UK Stroke Association and NIHR Health Technology Assessment Programme

Are we paying too much attention to cardio-pulmonary nematodes and neglecting old-fashioned worms like <it>Trichuris vulpis</it>?

<p>Abstract</p> <p><it>Trichuris vulpis</it>, the dog whipworm, causes an intestinal parasitosis of relevance in current canine veterinary practice. Its occurrence is well-known in pets, kennelled dogs and stray animals, and its eggs contaminate the ground in urban areas all over the world. Moreover, <it>T. vulpis </it>has been occasionally incriminated, though not convincingly substantiated, as a cause of zoonosis. This nematode is erroneously considered an "old-fashioned" pathogen with a consequent lack of up- to- date knowledge on several aspects of the infection. These, in turn, are still controversial and need to be studied in greater depth. This article reviews current knowledge of <it>T. vulpis</it>, together with a discussion of critical points in epidemiology, zoonotic hazard, diagnosis and treatment of canine trichurosis.</p

Feasibility of reporting results of large randomised controlled trials to participants:experience from the Fluoxetine or Control under supervision (FOCUS) trial

Objectives Informing research participants of the results of studies in which they took part is viewed as an ethical imperative. However, there is little guidance in the literature about how to do this. The Fluoxetine Or Control Under Supervision trial randomised 3127 patients with a recent acute stroke to 6 months of fluoxetine or placebo and was published in the Lancet on 5 December 2018. The trial team decided to inform the participants of the results at exactly the same time as the Lancet publication, and also whether they had been allocated fluoxetine or placebo. In this report, we describe how we informed participants of the results.Design In the 6-month and 12-month follow-up questionnaires, we invited participants to provide an email address if they wished to be informed of the results of the trial. We re-opened our trial telephone helpline between 5 December 2018 and 31 March 2019.Setting UK stroke services.Participants 3127 participants were randomised. 2847 returned 6-month follow-up forms and 2703 returned 12-month follow-up forms; the remaining participants had died (380), withdrawn consent or did not respond.Results Of those returning follow-up questionnaires, a total of 1845 email addresses were provided and a further 50 people requested results to be sent by post. Results were sent to all email and postal addresses provided; 309 emails were returned unrecognised. Seventeen people replied, of whom three called the helpline and the rest responded by email.Conclusion It is feasible to disseminate results of large trials to research participants, though only around 60% of those randomised wanted to receive the results. The system we developed was efficient and required very little resource, and could be replicated by trialists in the future.Trial registration number ISRCTN83290762; Post-results