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Aim: There are few age‐ and fitness‐specific, evidence‐based guidelines for colorectal cancer surgery. The uptake of different assessment and optimization strategies is variable. The aim of this study was to explore healthcare professional opinion about these issues using a mixed methods design. Methods: Semi‐structured qualitative interviews were undertaken with healthcare professionals from a single UK region involved in the treatment, assessment and optimization of colorectal surgery patients. Interviews were analysed using the framework approach. An online questionnaire survey was subsequently designed and disseminated to UK surgeons to quantitatively assess the importance of interview themes. Descriptive statistics were used to analyse questionnaire data. Results: Thirty‐seven healthcare professionals out of 42 approached (response rate 88%) were interviewed across five hospitals in the south Yorkshire region. Three broad themes were developed: attitudes towards treatment of the older patient, methods of assessment of suitability and optimization strategies. The questionnaire was completed by 103 out of an estimated 256 surgeons (estimated response rate 40.2%). There was a difference in opinion regarding the role of major surgery in older patients, particularly when there is coexisting dementia. Assessment was not standardized. Access to optimization strategies was limited, particularly in the emergency setting. Conclusion: There is wide variation in the process of assessment and provision of optimization strategies in UK practice. Lack of evidence‐based guidelines, cost and time constraints restrict the development of services and pathways. Differences in opinion between surgeons towards patients with frailty or dementia may account for some of the variation in colorectal cancer outcomes.

Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

The endovascular aneurysm repair-2 (EVAR-2) trial suggested that EVAR in patients unfit for open surgical repair (OSR) failed to provide a significant overall survival advantage compared with conservative management. The aim is to compare survival and cost-effectiveness in patients with poor cardiopulmonary exercise test (CPET) metrics who underwent EVAR or were managed conservatively.A prospective database of all CPETs (1435 patients) performed to assess preoperative fitness for abdominal aortic aneurysm repair was maintained. A total of 350 patients deemed unfit for OSR underwent EVAR or were managed conservatively. A 1:1 propensity-matched analysis incorporating age, gender, anaerobic threshold, and aneurysm size was used to compare survival. Cost-effectiveness analysis was based on the economic model for the National Institute for Health and Care Excellence clinical guideline on abdominal aortic aneurysm treatment.Propensity matching produced 122 pairs of patients in the EVAR and conservative management groups. The median overall survival for the EVAR group was significantly longer than that for the conservative management group (84 vs 30 months, P .001). One-, three-, and five-year mortality in the EVAR group was 7%, 40%, and 68%, respectively, compared with 25%, 68%, and 82% in the conservative management group, all P .001. The increment cost-effectiveness ratio for EVAR was £8023 (US$11,644) per quality-adjusted life year gained compared with £430,602 (US$624,967) in the National Institute for Health and Care Excellence guideline, which is based on EVAR-2 results.EVAR offers a survival advantage and is cost-effective in selected patients deemed unfit for OSR based on CPET compared with conservative management.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science., To help meet the ensuing demand for COVID-19 reagents, this article presents an openly available ‘coronavirus toolkit’ (https://mrcppu-covid.bio) and describes the generation and validation of these tools, including a simple SARS-CoV-2 reverse genetics system and a near-comprehensive panel of SARS-CoV-2 antibodies.

Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides that are not translated into functional proteins. Cellular lncRNAs have been shown to act as regulators by interacting with target nucleic acids or proteins and modulating their activities. We investigated the role of RNA1.2, which is one of four major lncRNAs expressed by human cytomegalovirus (HCMV), by comparing the properties of parental virus in vitro with those of deletion mutants lacking either most of the RNA1.2 gene or only the TATA element of the promoter. In comparison with parental virus, these mutants exhibited no growth defects and minimal differences in viral gene expression in human fibroblasts. In contrast, 76 cellular genes were consistently up- or down-regulated by the mutants at both the RNA and protein levels at 72 hours after infection. Differential expression of the gene most highly upregulated by the mutants (Tumor protein p63-regulated gene 1-like protein; TPRG1L) was confirmed at both levels by RT-PCR and immunoblotting. Consistent with the known ability of TPRG1L to upregulate IL-6 expression via NF-kappaB stimulation, RNA1.2 mutant-infected fibroblasts were observed to upregulate IL-6 in addition to TPRG1L. Comparable surface expression of TNF receptors and responsiveness to TNF-α in cells infected by the parental and mutant viruses indicated that activation of signalling by TNF-α is not involved in upregulation of IL-6 by the mutants. In contrast, inhibition of NF-kappaB activity and knockdown of TPRG1L expression reduced the extracellular release of IL-6 by RNA1.2 mutant-infected cells, thus demonstrating that upregulation of TPRG1L activates NF-kappaB. The levels of MCP-1 and CXCL1 transcripts were also increased in RNA1.2 mutant-infected cells, further demonstrating the presence of active NF-kappaB signalling. These results suggest that RNA1.2 plays a role in manipulating intrinsic NF-kappaB-dependent cytokine and chemokine release during HCMV infection, thereby impacting downstream immune responses.

peer-reviewed Equid herpesvirus 8 (EHV-8), formerly known as asinine herpesvirus 3, is an alpha herpesvirus that is closely related to equid herpesviruses 1 and 9 (EHV-1 and EHV-9). The pathogenesis of EHV-8 is relatively little studied and to date has only been associated with respiratory disease in donkeys in Australia and horses in China. A single EHV-8 genome sequence has been generated for strain Wh in China, but is apparently incomplete and contains frame shifts in two genes. In this study, the complete genome sequences of four EHV-8 strains isolated in Ireland between 2003 and 2015 were determined by Illumina sequencing. Two of these strains were isolated from cases of abortion in horses, and were misdiagnosed initially as EHV-1, and two were isolated from donkeys, one with neurological disease. The four genome sequences are very similar to each other, exhibiting greater than 98.4% nucleotide identity, and their phylogenetic clustering together demonstrated that genomic diversity is not dependent on the host. Comparative genomic analysis revealed 24 of the 76 predicted protein sequences are completely conserved among the Irish EHV-8 strains. Evolutionary comparisons indicate that EHV-8 is phylogenetically closer to EHV-9 than it is to EHV-1. In summary, the first complete genome sequences of EHV-8 isolates from two host species over a twelve year period are reported. The current study suggests that EHV-8 can cause abortion in horses. The potential threat of EHV-8 to the horse industry and the possibility that donkeys may act as reservoirs of infection warrant further investigation.

Background This study assessed the feasibility of a preoperative high-intensity interval training (HIT) programme in patients awaiting elective abdominal aortic aneurysm repair. Methods In this feasibility trial, participants were allocated by minimization to preoperative HIT or usual care. Patients in the HIT group were offered three exercise sessions per week for 4 weeks, and weekly maintenance sessions if surgery was delayed. Feasibility and acceptability outcomes were: rates of screening, eligibility, recruitment, retention, outcome completion, adverse events and adherence to exercise. Data on exercise enjoyment (Physical Activity Enjoyment Scale, PACES), cardiorespiratory fitness (anaerobic threshold and peak oxygen uptake), quality of life, postoperative morbidity and mortality, duration of hospital stay and healthcare utilization were also collected. Results Twenty-seven patients were allocated to HIT and 26 to usual care (controls). Screening, eligibility, recruitment, retention and outcome completion rates were 100 per cent (556 of 556), 43·2 per cent (240 of 556), 22·1 per cent (53 of 240), 91 per cent (48 of 53) and 79–92 per cent respectively. The overall exercise session attendance rate was 75·8 per cent (276 of 364), and the mean(s.d.) PACES score after the programme was 98(19) (‘enjoyable’); however, the intensity of exercise was generally lower than intended. The mean anaerobic threshold after exercise training (adjusted for baseline score and minimization variables) was 11·7 ml per kg per min in the exercise group and 11·4 ml per kg per min in controls (difference 0·3 (95 per cent c.i. –0·4 to 1·1) ml per kg per min). There were trivial-to-small differences in postoperative clinical and patient-reported outcomes between the exercise and control groups. Conclusion Despite the intensity of exercise being generally lower than intended, the findings support the feasibility and acceptability of both preoperative HIT and the trial procedures. A definitive trial is warranted. Registration number: ISRCTN09433624 (https://www.isrctn.com/).

A randomized controlled trial was used to evaluate the effects of a pro-social behavior after-school program called Mate-Tricks for nine and ten year old children and their parents living in an area of significant socioeconomic disadvantage. The children were randomly assigned to an intervention (n=220) or a control group (n=198). Children were compared on measures of pro-social behavior, anti-social behavior and related outcome measures. The trial found adverse effects on four outcomes among the intervention group compared to the control group: anti-social behavior increased on two different measures (d=+0.20) and (d=+0.18); child reported liberal parenting increased (d=+0.16); and child reported authoritarian parenting also increased (d=+0.20). In addition, parental participation was significantly associated with several program outcomes. It was concluded, that group based after-school behavior programs may have the potential to cause iatrogenic effects and must be designed, piloted, evaluated and implemented with a high degree of care.

A herpesvirus genome was sequenced directly from a biopsy specimen of a rectal lesion from a female common bottlenose dolphin. This genome sequence comprises a unique region (161,235 bp) flanked by multiple copies of a terminal repeat (4,431 bp) and contains 72 putative genes. The virus was named common bottlenose dolphin gammaherpesvirus 1.

Purpose: Intervention fidelity refers to the extent an experimental manipulation has been implemented as intended. Our aim was to evaluate the fidelity of high-intensity interval training (HIT) in patients awaiting repair of large abdominal aortic aneurysms.\ud \ud Methods: Following a baseline cardiopulmonary exercise test, 27 participants performed a hospital-based, supervised HIT intervention in the 4 weeks preceding surgery. The intervention was performed thrice weekly on a cycle ergometer and involved either 8 × 2-min intervals, each interspersed by 2-min recovery periods, or 4 × 4-min intervals interspersed with 4-min recovery periods. When surgery was delayed, participants undertook one maintenance HIT session per week until surgery. Session one power output was set to baseline anaerobic threshold power output and then increased on subsequent sessions until ratings of perceived exertion (RPE; Borg CR-10) for the legs (RPE-L) and sense of breathlessness/ chest (RPE-C) were hard (5) to very hard (7) at the end of each interval. For safety, power output was maintained or reduced if systolic blood pressure exceeded 180 mm Hg or heart rate exceeded 95% of maximum.\ud \ud Results: Overall session attendance across the 4-week HIT intervention was 74%. Seventeen participants met our compliance criteria of ≥75% of intervention sessions and all maintenance sessions. When compared to non-compliance, compliant participants had higher fitness, performed more HIT sessions and were able to exercise at higher exercise intensities with a lower proportion of exercise safety breaches. In the 17 compliant participants, the proportion of repetitions meeting the HIT criterion was 30% (RPE-L) and 16% (RPE-C). Mean repetition intensity was 4.1 ± 2.0 Arbitrary Units [AU] (RPE-L) and 3.5 ± 1.9 AU (RPE-C) with a within-subject variability of ±1.4 AU and ±1.6 AU, respectively. We observed higher RPE scores (~0.5 AU) following 2-min intervals when compared to 4-min intervals and exercise power output increased 23% across the 4-week HIT intervention. One participant experienced an adverse event but were still able to complete their remaining exercise sessions.\ud \ud Conclusions: Despite an inconsistent and lower than prescribed intensity, it is possible to exercise this high-risk patient population at moderate to hard intensities with a low risk of adverse events.\ud \ud Clinical Trial Registration: http://www.isrctn.com/, registration number ISRCTN09433624.

The World Health Organization recommends that all countries develop a cancer control program. Qatar is the first country in the Gulf Cooperation Council to develop such a plan, with its National Cancer Strategy 2011-2016. Three years into implementation, meaningful progress has been made, particularly in reducing patient waiting times, creating a multidisciplinary approach to cancer treatment, and fostering international research collaboration. Challenges include attracting sufficient numbers of trained health care workers, reaching a diverse population with messages tailored to their needs, and emphasizing cancer prevention and early detection in addition to research and treatment. Qatar's example shows that best practices developed in North America, Western Europe, and Australasia can be assimilated in a very different demographic and cultural context when such approaches are tailored to local characteristics and circumstances.

Johne's disease (JD), or paratuberculosis is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). There may be a long subclinical phase with no signs of clinical disease. Diagnosis of JD is problematic and no test can reliably detect sub-clinical disease. Th1 responses to Map are believed to be activated first with a later switch to Th2 responses and progression to clinical disease. Detection of a cell-mediated response, indicated by interferon gamma (IFN-γ) produced in response to mycobacterial antigens, may give an early indication of infection. Crude extracts of Map (PPDj) have been used to detect the cell-mediated response, but more specific, quantifiable antigens would improve the test. Thirty Map-specific proteins were screened for their ability to raise a cell-mediated response in subclinically infected sheep. Four proteins were selected and tested using blood from subclinical animals and controls from a JD-free flock. Three proteins elicited IFN-γ levels which were higher in the subclinical group than in the control group, two were statistically significant. Thus these proteins have the ability to discriminate groups of infected and uninfected animals and may have use in diagnosis of JD.

The Hamlyn Symposium is an annual international forum for clinicians, engineers and researchers to exchange ideas and explore new challenges and opportunities in medical robotics and allied technologies. The 5th Symposium was another success, due to the range of papers presented from leading international teams, and the growing workshop series, which is organised by teams of senior academics and young research fellows to further stimulate academic networking and collaboration. For this special issue, we have selected five representative manuscripts from the 52 papers from 11 countries presented at the Symposium, based on the initial review scores and the quality of the presentation during the meeting. They represent the unique multidisciplinary blend of the Symposium and our increasing focus on imaging and navigation, and usability by the surgeon to improve patient outcomes through technological innovation. The selected papers focus on novel developments in visualisation and navigation, and explore usability and skills assessment of new techniques. Visualisation and imaging are both important factors to consider in robotic surgery. The development and use of video augmentation for intraoperative image guidance for transoral robotic surgery is described by Liu et al. Using preoperative planning data, the team have demonstrated that the data can be registered to the intraoperative endoscopic view to augment and define targets, which will improve the operation through improved navigation, orientation and ultimately tumour resection. This has been piloted in situ to show improvements over and above simulated standard practice. Lee et al. explore improvements in navigation in vascular surgery, proposing a novel motionadaptive catheter navigation scheme to compensate for physiological motion and tortuous vessels. A simulated aortic branch cannulation procedure was used to demonstrate improved visualisation through dynamic shape modelling and real-time instantiation in conjunction with a reconstructed view of the catheter tip to enhance the usability for the surgeon. Improvements in efficiency and usability of robotic systems are increasingly being explored. In the paper by Simaan et al., a robotic system for single port access undergoes preliminary evaluation, demonstrating its design and functionality for basic tasks in minimally invasive surgery, manipulation, knot tying and visualisation/tracking. Ruiter et al. describe a novel robotic system that can be appended to a traditional flexible endoscope for advanced procedures, in particular those that need a team for complex and enhanced manoeuvrability. Initial results indicate that the robotic system reduces the time required to complete the investigation and that usability is enhanced from the physician’s perspective. Given the increased use of robotic systems, it is important to monitor and assess the surgeon’s skills as well as the technical developments. This is highlighted in the paper by Jun et al., which explores the use of a quantitative computer-based surgical performance evaluation scheme for assessing surgeons of varying levels. The Symposium would not be possible without the commitment and hard work of the programme and organising committees. We are also very grateful to all our distinguished invited speakers, particularly Professor Shigeo Hirose from Tokyo Institute of Technology, who provided an overview of ‘‘Biologically Inspired Robots: Snake-like, Spider-like Robots and Others’’, and Professor Richard Satava, who gave the inaugural Karl Storz–Harold Hopkins lecture on ‘‘Developing Next Generation Robots’’ that stimulated the panel debate.

We resequenced the genome of equid herpesvirus 2 (EHV2) strain 86/67 and sequenced the genomes of EHV2 strain G9/92 and equid herpesvirus 5 (EHV5) strain 2-141/67. The most prominent genetic differences are the dissimilar locations of the interleukin-10 (IL-10)-like genes and the presence of an OX-2-like gene in EHV5 only.

A latex agglutination test (LAT) has been developed for the diagnosis of contagious bovine pleuropneumonia (CBPP). The latex microspheres were coated with MmmSC polyclonal immunoglobulin G antiserum and detected MmmSC antigen in the serum of cattle infected with CBPP and in growth medium containing MmmSC. The specific antigen recognizsed by this test appeared to be the capsular polysaccharide (CPS). The LAT recognized all 23 strains of MmmSC examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 5 × 10 3 CFU, in a reaction volume of 0.03 ml. Therefore, rapid identification of MmmSC cultures should be possible. Agglutination was also observed with the related goat pathogens and “ Mycoplasma mycoides ” cluster members Mycoplasma mycoides subsp. mycoides large colony biotype (four of six strains positive) and Mycoplasma mycoides subsp. capri (three of six strains positive), in agreement with the suggestion that these latter two mycoplasmas may in fact represent a single species (although collectively exhibiting two capsular serotypes). Comparisons in diagnosis with the complement fixation test (CFT) were made by using African field sera from CBPP-infected cattle. After 2 (or 3) min of incubation, the test detected 55% (or 61%) of CFT-positive sera and 29% (or 40%) of CFT-negative sera, with an overall correlation in diagnosis of 62% (or 61%). The rates for false-positive diagnoses made by using “known” CBPP-negative sera from the United Kingdom were 3 or 13% after 2 or 3 min of incubation, respectively. The data agree with previous findings that some CBPP CFT-negative misdiagnoses may occur due to “antibody eclipsing” by excess circulating antigen. The LAT combines low cost and high specificity with ease of application in the field, without the need for any specialist training or equipment.

Infections of Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus (EEHV) can cause a rapid, highly lethal, hemorrhagic disease, which primarily affects juvenile animals up to the age of four years. So far, the majority of deaths have been attributed to infections with genotype EEHV1 or, more rarely, EEHV3 and EEHV4. Here, we report the pathological characteristics of the first fatality linked to EEHV5 infection, and describe the complete viral DNA sequence. Gross post-mortem and histological findings were indistinguishable from lethal cases previously attributed to other EEHV genotypes, and the presence of characteristic herpesviral inclusions in capillary endothelial cells at several sites was consistent with the diagnosis of acute EEHV infection. Molecular analysis confirmed the presence of EEHV5 DNA and was followed by sequencing of the viral genome directly from post-mortem material. The genome is 180,800 bp in size and contains 120 predicted protein-coding genes, five of which are fragmented and presumably nonfunctional. The seven families of paralogous genes recognized in EEHV1 are also represented in EEHV5. The overall degree of divergence (37%) between the EEHV5 and EEHV1 genomes, and phylogenetic analysis of eight conserved genes, support the proposed classification of EEHV5 into a new species (Elephantid herpesvirus 5).

The global epidemic of obesity continues to escalate. Obesity accounts for an increasing proportion of the international socioeconomic burden of noncommunicable disease. Online social networking services provide an effective medium through which information may be exchanged between obese and overweight patients and their health care providers, potentially contributing to superior weight-loss outcomes. We performed a systematic review and meta-analysis to assess the role of these services in modifying body mass index (BMI). Our analysis of twelve studies found that interventions using social networking services produced a modest but significant 0.64 percent reduction in BMI from baseline for the 941 people who participated in the studies' interventions. We recommend that social networking services that target obesity should be the subject of further clinical trials. Additionally, we recommend that policy makers adopt reforms that promote the use of anti-obesity social networking services, facilitate multistakeholder partnerships in such services, and create a supportive environment to confront obesity and its associated noncommunicable diseases.

Introduction In patients with large abdominal aortic aneurysm (AAA), open surgical or endovascular aneurysm repair procedures are often used to minimise the risk of aneurysm-related rupture and death; however, aneurysm repair itself carries a high risk. Low cardiopulmonary fitness is associated with an increased risk of early post-operative complications and death following elective AAA repair. Therefore, fitness should be enhanced before aneurysm repair. High-intensity interval exercise training (HIT) is a potent, time-efficient strategy for enhancing cardiopulmonary fitness. Here, we describe a feasibility study for a definitive trial of a pre-operative HIT intervention to improve post-operative outcomes in patients undergoing elective AAA repair. Methods and analysis A minimum of 50 patients awaiting elective repair of a 5.5–7.0 cm infrarenal AAA will be allocated by minimisation to HIT or usual care control in a 1:1 ratio. The patients allocated to HIT will complete three hospital-based exercise sessions per week, for 4 weeks. Each session will include 2 or 4 min of high-intensity stationary cycling followed by the same duration of easy cycling or passive recovery, repeated until a total of 16 min of high-intensity exercise is accumulated. Outcomes to be assessed before randomisation and 24–48 h before aneurysm repair include cardiopulmonary fitness, maximum AAA diameter and health-related quality of life. In the post-operative period, we will record destination (ward or critical care unit), organ-specific morbidity, mortality and the durations of critical care and hospital stay. Twelve weeks after the discharge, participants will be interviewed to reassess quality of life and determine post-discharge healthcare utilisation. The costs associated with the exercise intervention and healthcare utilisation will be calculated. Ethics and dissemination Ethics approval was secured through Sunderland Research Ethics Committee. The findings of the trial will be disseminated through peer-reviewed journals, and national and international presentations. Trial registration Current Controlled Trials ISRCTN09433624.

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.

A highly lethal hemorrhagic disease associated with infection by elephant endotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephant husbandry. We have used high-throughput methods to sequence the genomes of the two genotypes that are involved in most fatalities, namely, EEHV1A and EEHV1B (species Elephantid herpesvirus 1 , genus Proboscivirus , subfamily Betaherpesvirinae , family Herpesviridae ). The sequences were determined from postmortem tissue samples, despite the data containing tiny proportions of viral reads among reads from a host for which the genome sequence was not available. The EEHV1A genome is 180,421 bp in size and consists of a unique sequence (174,601 bp) flanked by a terminal direct repeat (2,910 bp). The genome contains 116 predicted protein-coding genes, of which six are fragmented, and seven paralogous gene families are present. The EEHV1B genome is very similar to that of EEHV1A in structure, size, and gene layout. Half of the EEHV1A genes lack orthologs in other members of subfamily Betaherpesvirinae , such as human cytomegalovirus (genus Cytomegalovirus ) and human herpesvirus 6A (genus Roseolovirus ). Notable among these are 23 genes encoding type 3 membrane proteins containing seven transmembrane domains (the 7TM family) and seven genes encoding related type 2 membrane proteins (the EE50 family). The EE50 family appears to be under intense evolutionary selection, as it is highly diverged between the two genotypes, exhibits evidence of sequence duplications or deletions, and contains several fragmented genes. The availability of the genome sequences will facilitate future research on the epidemiology, pathogenesis, diagnosis, and treatment of EEHV-associated disease.

Localization of mRNAs, a crucial step in the early development of some animals, has been shown to be directed by cis-acting elements that presumably interact with localization factors. Here we identify a protein, exl, that binds to BLE1, an RNA localization element from the Drosophila bicoid mRNA. Using mutations in BLE1, we demonstrate a correlation between in vitro exl binding and one phase of in vivo localization directed by BLE1, implicating exl in that localization event. Furthermore, the same phase of localization is disrupted in exuperantia mutants, suggesting that exl and exuperantia proteins interact. Identification of a protein that binds specifically to an mRNA localization element and acts in mRNA localization opens the way for a biochemical analysis of this process.

Oskar (osk) protein directs the deployment of nanos (nos), the posterior body-patterning morphogen in Drosophila. To avoid inappropriate activation of nos, osk activity must appear only at the posterior pole of the oocyte, where the osk mRNA becomes localized during oogenesis. Here, we show that translation of osk mRNA is, and must be, repressed prior to its localization; absence of repression allows osk protein to accumulate throughout the oocyte, specifying posterior body patterning throughout the embryo. Translational repression is mediated by an ovarian protein, bruno, that binds specifically to bruno response elements (BREs), present in multiple copies in the osk mRNA 3′UTR. Addition of BREs to a heterologous mRNA renders it sensitive to translational repression in the ovary.

Spermatogenesis is a complex process that cannot be modelledin vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-downin vivo. We describe here a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC functionin vivoand future work will therefore focus on the use of lentiviral delivery systems.

ERBB2 is a receptor tyrosine kinase present on the basolateral membrane of polarized epithelia and has important functions in organ development and tumorigenesis. Using mutagenic analyses and Madin-Darby canine kidney (MDCK) cells, we have investigated the signals that regulate basolateral targeting of ERBB2. We show that basolateral delivery of ERBB2 is dependent on a novel bipartite juxtamembrane sorting signal residing between Gln-692 and Thr-701. The signal shows only limited sequence homology to known basolateral targeting signals and is both necessary and sufficient for correct sorting of ERBB2. In addition we demonstrate that this motif can function as a dominant basolateral targeting signal by its ability to redirect the apically localized P75 neurotrophin receptor to the basolateral membrane domain of polarized epithelial cells. Interestingly, LLC-PK1 cells, which are deficient for the micro 1B subunit of the AP1B adaptor complex, missort a large proportion of ERBB2 to the apical membrane domain. This missorting can be partially corrected by the introduction of micro 1B, suggesting a possible role for AP1B in ERBB2 endosomal trafficking. Furthermore, we find that the C-terminal ERBIN binding domain of ERBB2 is not necessary for its basolateral targeting in MDCK cells.

Localization signals are RNA regulatory elements that direct the localization of mRNAs to subcellular sites. Localization signals presumably function by mediating RNA recognition events through which the mRNA becomes associated with the localization machinery. At present little is known about individual RNA recognition events, which in turn has limited progress in identifying the trans-acting binding factors involved in these events. Here we describe a detailed characterization of the RNA elements required for the RNA recognition event, event A, that initiates localization of bicoid mRNA in the Drosophila ovary. One element is a helix in which nucleotide identities are not important, suggesting that it plays a primarily structural role. Immediately adjacent to the helix is a recognition domain in which the identities of some, but not all, nucleotides are important for function. Comparison of two related but different RNAs that both support recognition event A further defines the important features of the recognition domain.

nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.

Deployment of the bicoid morphogen gradient in early Drosophila embryos requires the prelocalization of bicoid mRNA to the anterior pole of the egg. This anterior localization is mediated by a cis-acting localization signal contained within the 3 ′ untranslated region of the bicoid mRNA. Here we use a series of bicoid transgenes carrying small deletions in the 3 ′ untranslated region to survey for functional elements that constitute the localization signal. We identify and characterize one essential element, BLE1, which specifically directs the early steps of localization. In addition, we find that many deletions within the bicoid mRNA 3 ′ untranslated region impair but do not prevent localization. One such deletion specifically interferes with a later step in localization. Thus the bicoid mRNA localization signal appears to consist of multiple different elements, each responsible for different steps in the localization process.

While there is evidence for effective in-school programmes for struggling beginning readers, the evidence in relation to after-schools interventions focusing on academic outcomes is particularly weak. This study seeks to contribute to this body of evidence through a randomized trial (n=464) of an after-school literacy programme (DoodleDen) for struggling beginning readers in a deprived area of Dublin, Ireland. The programme based on a balanced literacy framework, was delivered in group settings, and had a strong focus on staff development. Doodle Den was found to improve the children’s overall literacy (d=+0.17), teacher assessments of their literacy ability (d=+0.28) and to positively impact on problem behaviours in regular school class (d=-0.18). The implications for the development of after-school programmes are discussed.

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb isoform. FgfR2-IIIb−/−lacZ mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth.

Background Currently, there is no standardised tool used to capture morbidity following abdominal aortic aneurysm (AAA) repair. The aim of this prospective observational study was to validate the Postoperative Morbidity Survey (POMS) according to its two guiding principles: to only capture morbidity substantial enough to delay discharge from hospital and to be a rapid, simple screening tool. Methods A total of 64 adult patients undergoing elective infrarenal AAA repair participated in the study. Following surgery, the POMS was recorded daily, by trained research staff with the clinical teams blinded, until hospital discharge or death. We modelled the data using Cox regression, accounting for the competing risk of death, with POMS as a binary time-dependent (repeated measures) internal covariate. For each day for each patient, ‘discharged’ (yes/no) was the event, with the elapsed number of days post-surgery as the time variable. We derived the hazard ratio for any POMS morbidity (score 1–9) vs. no morbidity (zero), adjusted for type of repair (endovascular versus open), age and aneurysm size. Results The hazard ratio for alive discharge with any POMS-recorded morbidity versus no morbidity was 0.130 (95 % confidence interval 0.070 to 0.243). The median time-to-discharge was 13 days after recording any POMS morbidity vs. 2 days after scoring zero for POMS morbidity. Compliance with POMS completion was 99.5 %. Conclusions The POMS is a valid tool for capturing short-term postoperative morbidity following elective infrarenal AAA repair that is substantial enough to delay discharge from hospital. Daily POMS measurement is recommended to fully capture morbidity and allow robust analysis. The survey could be a valuable outcome measure for use in quality improvement programmes and future research.