An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Modification of proteins by the ubiquitin-like protein, UFM1, requires activation of UFM1 by the E1-activating enzyme, UBA5. In humans, UBA5 possesses two isoforms, each comprising an adenylation domain, but only one containing an N-terminal extension. Currently, the role of the N-terminal extension in UFM1 activation is not clear. Here we provide structural and biochemical data on UBA5 N-terminal extension to understand its contribution to UFM1 activation. The crystal structures of the UBA5 long isoform bound to ATP with and without UFM1 show that the N-terminus not only is directly involved in ATP binding but also affects how the adenylation domain interacts with ATP. Surprisingly, in the presence of the N-terminus, UBA5 no longer retains the 1:2 ratio of ATP to UBA5, but rather this becomes a 1:1 ratio. Accordingly, the N-terminus significantly increases the affinity of ATP to UBA5. Finally, the N-terminus, although not directly involved in the E2 binding, stimulates transfer of UFM1 from UBA5 to the E2, UFC1.Marie Curie Career Integration GrantIsrael Science FoundationIsraeli Cancer Associatio

Comparing computer-generated and pathologist-generated tumour segmentations for immunohistochemical scoring of breast tissue microarrays

BACKGROUND: Tissue microarrays (TMAs) have become a valuable resource for biomarker expression in translational research. Immunohistochemical (IHC) assessment of TMAs is the principal method for analysing large numbers of patient samples, but manual IHC assessment of TMAs remains a challenging and laborious task. With advances in image analysis, computer-generated analyses of TMAs have the potential to lessen the burden of expert pathologist review. METHODS: In current commercial software computerised oestrogen receptor (ER) scoring relies on tumour localisation in the form of hand-drawn annotations. In this study, tumour localisation for ER scoring was evaluated comparing computer-generated segmentation masks with those of two specialist breast pathologists. Automatically and manually obtained segmentation masks were used to obtain IHC scores for thirty-two ER-stained invasive breast cancer TMA samples using FDA-approved IHC scoring software. RESULTS: Although pixel-level comparisons showed lower agreement between automated and manual segmentation masks (κ=0.81) than between pathologists' masks (κ=0.91), this had little impact on computed IHC scores (Allred; [Image: see text]=0.91, Quickscore; [Image: see text]=0.92). CONCLUSIONS: The proposed automated system provides consistent measurements thus ensuring standardisation, and shows promise for increasing IHC analysis of nuclear staining in TMAs from large clinical trials

Skeletal trade-offs in coralline algae in response to ocean acidification

Ocean acidification is changing the marine environment, with potentially serious consequences for many organisms. Much of our understanding of ocean acidification effects comes from laboratory experiments, which demonstrate physiological responses over relatively short timescales. Observational studies and, more recently, experimental studies in natural systems suggest that ocean acidification will alter the structure of seaweed communities. Here, we provide a mechanistic understanding of altered competitive dynamics among a group of seaweeds, the crustose coralline algae (CCA). We compare CCA from historical experiments (1981-1997) with specimens from recent, identical experiments (2012) to describe morphological changes over this time period, which coincides with acidification of seawater in the Northeastern Pacific. Traditionally thick species decreased in thickness by a factor of 2.0-2.3, but did not experience a change in internal skeletal metrics. In contrast, traditionally thin species remained approximately the same thickness but reduced their total carbonate tissue by making thinner inter-filament cell walls. These changes represent alternative mechanisms for the reduction of calcium carbonate production in CCA and suggest energetic trade-offs related to the cost of building and maintaining a calcium carbonate skeleton as pH declines. Our classification of stress response by morphological type may be generalizable to CCA at other sites, as well as to other calcifying organisms with species-specific differences in morphological types

‘We have to wait in a queue for our turn quite a bit’ Examining children’s physical activity during primary physical education lessons

The overall purpose of this study was to examine children’s physical activity (PA) during primary physical education (PE). This was achieved through the following two research objectives: (1) to measure children’s PA, lesson context and teacher promotion of PA during PE lessons; and (2) to explore teachers’ and children’s perspectives on PA levels during PE lessons. Evidence suggests that children’s PA during PE is below recommended levels and further research is required to understand the reasons why. Through a mixed method design, 138 children were observed using the System for Observing Fitness and Instruction Time, 80 children participated in group interviews, and 13 teachers were interviewed, across three primary schools in England. Findings indicated that the mean percentage of lesson time allocated to moderate to vigorous PA (MVPA) was 42.4% and the average lesson length was 35.3 minutes. Qualitative themes identified were: ‘knowledge and beliefs’; ‘teacher pedagogy’; and ‘teacher development’. The findings indicate that a change in perspective is needed, which includes a focus on PA during primary PE lessons. Intervention work is required that targets teachers’ knowledge and beliefs towards PE along with the development of effective teaching strategies. However, this needs to be grounded in an ecological approach which will allow researchers and schools to target the various levels of influence. It is strongly recommended that interventions are grounded in behaviour change theory, as this study indicates that sharing knowledge about pedagogical strategies to increase children’s MVPA does not necessarily produce changes in teachers’ behaviours

Testing the theory of immune selection in cancers that break the rules of transplantation

Modification of cancer cells likely to reduce their immunogenicity, including loss or down-regulation of MHC molecules, is now well documented and has become the main support for the concept of immune surveillance. The evidence that these modifications, in fact, result from selection by the immune system is less clear, since the possibility that they may result from reorganized metabolism associated with proliferation or from cell de-differentiation remains. Here, we (a) survey old and new transplantation experiments that test the possibility of selection and (b) survey how transmissible tumours of dogs and Tasmanian devils provide naturally evolved tests of immune surveillance

Internal and external cooling methods and their effect on body temperature, thermal perception and dexterity

© 2018 The Authors. Published by PLOS. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0191416© 2018 Maley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objective The present study aimed to compare a range of cooling methods possibly utilised by occupational workers, focusing on their effect on body temperature, perception and manual dexterity. Methods Ten male participants completed eight trials involving 30 min of seated rest followed by 30 min of cooling or control of no cooling (CON) (34C, 58% relative humidity). The cooling methods utilised were: ice cooling vest (CV0), phase change cooling vest melting at 14C (CV14), evaporative cooling vest (CVEV), arm immersion in 10C water (AI), portable water-perfused suit (WPS), heliox inhalation (HE) and ice slushy ingestion (SL). Immediately before and after cooling, participants were assessed for fine (Purdue pegboard task) and gross (grip and pinch strength) manual dexterity. Rectal and skin temperature, as well as thermal sensation and comfort, were monitored throughout. Results Compared with CON, SL was the only method to reduce rectal temperature (P = 0.012). All externally applied cooling methods reduced skin temperature (P0.05). Conclusion The present study observed that ice ingestion or ice applied to the skin produced the greatest effect on rectal and skin temperature, respectively. AI should not be utilised if workers require subsequent fine manual dexterity. These results will help inform future studies investigating appropriate pre-cooling methods for the occupational worker.This project is financially supported by the US Government through the Technical Support Working Group within the Combating Terrorism Technical Support Office.Published versio

Randomized clinical trial to evaluate the effect of fecal microbiota transplant for initial Clostridium difficile infection in intestinal microbiome

Objective The aim of this study was to evaluate the impact of fecal donor-unrelated donor mix (FMT-FURM) transplantation as first-line therapy for C. difficile infection (CDI) in intestinal microbiome. Methods We designed an open, two-arm pilot study with oral vancomycin (250mg every 6 h for 10–14 days) or FMT-FURM as treatments for the first CDI episode in hospitalized adult patients in Hospital Universitario “Dr. Jose Eleuterio Gonzalez”. Patients were randomized by a closed envelope method in a 1: 1 ratio to either oral vancomycin or FMT-FURM. CDI resolution was considered when there was a reduction on the Bristol scale of at least 2 points, a reduction of at least 50% in the number of bowel movements, absence of fever, and resolution of abdominal pain (at least two criteria). From each patient, a fecal sample was obtained at days 0, 3, and 7 after treatment. Specimens were cultured to isolate C. difficile, and isolates were characterized by PCR. Susceptibility testing of isolates was performed using the agar dilution method. Fecal samples and FMT-FURM were analyzed by 16S rRNA sequencing. Results We included 19 patients; 10 in the vancomycin arm and 9 in the FMT-FURM arm. However, one of the patients in the vancomycin arm and two patients in the FMT-FURM arm were eliminated. Symptoms resolved in 8/9 patients (88.9%) in the vancomycin group, while symptoms resolved in 4/7 patients (57.1%) after the first FMT-FURM dose (P = 0.26) and in 5/7 patients (71.4%) after the second dose (P = 0.55). During the study, no adverse effects attributable to FMT-FURM were observed in patients. Twelve isolates were recovered, most isolates carried tcdB, tcdA, cdtA, and cdtB, with an 18-bp deletion in tcdC. All isolates were resistant to ciprofloxacin and moxifloxacin but susceptible to metronidazole, linezolid, fidaxomicin, and tetracycline. In the FMT-FURM group, the bacterial composition was dominated by Firmicutes, Bacteroidetes, and Proteobacteria at all-time points and the microbiota were remarkably stable over time. The vancomycin group showed a very different pattern of the microbial composition when comparing to the FMT-FURM group over time. Conclusion The results of this preliminary study showed that FMT-FURM for initial CDI is associated with specific bacterial communities that do not resemble the donors’ sample.Peer reviewedFinal Published versio

Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy.

BACKGROUND: We aimed to determine the mutation yield and clinical applicability of "molecular autopsy" following sudden arrhythmic death syndrome (SADS) by validating and utilizing low-cost high-throughput technologies: Fluidigm Access Array PCR-enrichment with Illumina HiSeq 2000 next generation sequencing (NGS). METHODS: We validated and optimized the NGS platform with a subset of 46 patients by comparison with Sanger sequencing of coding exons of major arrhythmia risk-genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, RYR2). A combined large multi-ethnic international SADS cohort was sequenced utilizing the NGS platform to determine overall molecular yield; rare variants identified by NGS were subsequently reconfirmed by Sanger sequencing. RESULTS: The NGS platform demonstrated 100% sensitivity for pathogenic variants as well as 87.20% sensitivity and 99.99% specificity for all substitutions (optimization subset, n = 46). The positive predictive value (PPV) for NGS for rare substitutions was 16.0% (27 confirmed rare variants of 169 positive NGS calls in 151 additional cases). The overall molecular yield in 197 multi-ethnic SADS cases (mean age 22.6 ± 14.4 years, 68% male) was 5.1% (95% confidence interval 2.0-8.1%), representing 10 cases carrying pathogenic or likely pathogenic risk-mutations. CONCLUSIONS: Molecular autopsy with Fluidigm Access Array and Illumina HiSeq NGS utilizing a selected panel of LQTS/BrS and CPVT risk-genes offers moderate diagnostic yield, albeit requiring confirmatory Sanger-sequencing of mutational variants

Geographic Coincidence of Increased Malaria Transmission Hazard and Vulnerability Occurring at the Periphery of two Tanzanian Villages.

The goal of malaria elimination necessitates an improved understanding of any fine-scale geographic variations in transmission risk so that complementary vector control tools can be integrated into current vector control programmes as supplementary measures that are spatially targeted to maximize impact upon residual transmission. This study examines the distribution of host-seeking malaria vectors at households within two villages in rural Tanzania. Host-seeking mosquitoes were sampled from 72 randomly selected households in two villages on a monthly basis throughout 2008 using CDC light-traps placed beside occupied nets. Spatial autocorrelation in the dataset was examined using the Moran's I statistic and the location of any clusters was identified using the Getis-Ord Gi* statistic. Statistical associations between the household characteristics and clusters of mosquitoes were assessed using a generalized linear model for each species. For both Anopheles gambiae sensu lato and Anopheles funestus, the density of host-seeking females was spatially autocorrelated, or clustered. For both species, houses with low densities were clustered in the semi-urban village centre while houses with high densities were clustered in the periphery of the villages. Clusters of houses with low or high densities of An. gambiae s.l. were influenced by the number of residents in nearby houses. The occurrence of high-density clusters of An. gambiae s.l. was associated with lower elevations while An. funestus was also associated with higher elevations. Distance from the village centre was also positively correlated with the number of household occupants and having houses constructed with open eaves. The results of the current study highlight that complementary vector control tools could be most effectively targeted to the periphery of villages where the households potentially have a higher hazard (mosquito densities) and vulnerability (open eaves and larger households) to malaria infection

Genome Wide Association Identifies PPFIA1 as a Candidate Gene for Acute Lung Injury Risk Following Major Trauma

Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research