Explore the vast range of titles available.

Gliomas are highly malignant brain tumours characterised by extensive areas of poor perfusion which subsequently leads to hypoxia and reduced survival. Therapies that address the hypoxic microenvironment are likely to significantly improve patient outcomes. Verteporfin, a benzoporphyrin-like drug, has been suggested to target the Yes-associated protein (YAP). Increased YAP expression and transcriptional activity has been proposed in other tumour types to promote malignant cell survival and thus YAP-inhibitor, verteporfin, may be predicted to impact glioma cell growth and viability. Due to the extensive hypoxic nature of gliomas, we investigated the effect of hypoxia on YAP expression and found that YAP transcription is increased under these conditions. Treatment of both primary and immortalised glioblastoma cell lines with verteporfin resulted in a significant decrease in viability but strikingly only under hypoxic conditions (1% O 2 ). We discovered that cell death occurs through a YAP-independent mechanism, predominately involving binding of free iron and likely through redox cycling, contributes to production of reactive oxygen species. This results in disruption of normal cellular processes and death in cells already under oxidative stress – such as those in hypoxia. We suggest that through repurposing verteporfin, it represents a novel means of treating highly therapy-resistant, hypoxic cells in glioma.

In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the U L / b ′ region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV.

Less than 20% of adolescents globally meet recommended levels of physical activity, and not meeting these recommended levels is associated with social disadvantage and rising disease risk. The determinants of physical activity in adolescents are multilevel and poorly understood, but the school's social environment likely plays an important role. We conducted a cluster randomised controlled trial to assess the effectiveness of a school-based programme (GoActive) to increase moderate-to-vigorous physical activity (MVPA) among adolescents. Non-fee-paying, co-educational schools including Year 9 students in the UK counties of Cambridgeshire and Essex were eligible for inclusion. Within participating schools (n = 16), all Year 9 students were eligible and invited to participate. Participants were 2,862 13- to 14-year-olds (84% of eligible students). After baseline assessment, schools were computer-randomised, stratified by school-level pupil premium funding (below/above county-specific median) and county (control: 8 schools, 1,319 participants, mean [SD] participants per school n = 165 [62]; intervention: 8 schools, 1,543 participants, n = 193 [43]). Measurement staff were blinded to allocation. The iteratively developed, feasibility-tested 12-week intervention, aligned with self-determination theory, trained older adolescent mentors and in-class peer-leaders to encourage classes to conduct 2 new weekly activities. Students and classes gained points and rewards for engaging in any activity in or out of school. The primary outcome was average daily minutes of accelerometer-assessed MVPA at 10-month follow-up; a mixed-methods process evaluation evaluated implementation. Of 2,862 recruited participants (52.1% male), 2,167 (76%) attended 10-month follow-up measurements; we analysed the primary outcome for 1,874 participants (65.5%). At 10 months, there was a mean (SD) decrease in MVPA of 8.3 (19.3) minutes in the control group and 10.4 (22.7) minutes in the intervention group (baseline-adjusted difference [95% confidence interval] -1.91 minutes [-5.53 to 1.70], p = 0.316). The programme cost £13 per student compared with control; it was not cost-effective. Overall, 62.9% of students and 87.3% of mentors reported that GoActive was fun. Teachers and mentors commented that their roles in programme delivery were unclear. Implementation fidelity was low. The main methodological limitation of this study was the relatively affluent and ethnically homogeneous sample. In this study, we observed that a rigorously developed school-based intervention was no more effective than standard school practice at preventing declines in adolescent physical activity. Interdisciplinary research is required to understand educational-setting-specific implementation challenges. School leaders and authorities should be realistic about expectations of the effect of school-based physical activity promotion strategies implemented at scale. ISRCTN Registry ISRCTN31583496.

Human cytomegalovirus (HCMV) extensively modulates host cells, downregulating >900 human proteins during viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a mass spectrometry-based interactome analysis of 169 tagged, stably-expressed canonical strain Merlin HCMV proteins, and two non-canonical HCMV proteins, in infected cells. This identified a network of >3400 virus-host and >150 virus-virus protein interactions, providing insights into functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to SH3 domains of NCK Adaptor Protein-1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection.

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SCF AFF1 revealed that this E3 ubiquitin ligase directly interacts with ARF19 and ARF7 to regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning. Mutants defective in AFF1 display attenuated auxin responsiveness, and developmental defects, suggesting that SCF AFF1 -mediated regulation of ARF protein drives aspects of auxin response and plant development. Auxin-driven transcriptional responses are mediated by ARF transcription factors. Here the authors characterize an F-box protein, AFF1, that regulates the accumulation, condensation, and nucleo-cytoplasmic partitioning of ARF19 and ARF7.

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks. It is unclear how gut-dwelling E. coli bacteria often emerge to cause systemic infection in chickens. Here, Mageiros et al. use population genomics and pangenome-wide association studies to identify genetic elements associated with pathogenicity in avian E. coli .

Cardiac pump function arises from a series of highly orchestrated events across multiple scales. Computational electromechanics can encode these events in physics-constrained models. However, the large number of parameters in these models has made the systematic study of the link between cellular, tissue, and organ scale parameters to whole heart physiology challenging. A patient-specific anatomical heart model, or digital twin, was created. Cellular ionic dynamics and contraction were simulated with the Courtemanche-Land and the ToR-ORd-Land models for the atria and the ventricles, respectively. Whole heart contraction was coupled with the circulatory system, simulated with CircAdapt, while accounting for the effect of the pericardium on cardiac motion. The four-chamber electromechanics framework resulted in 117 parameters of interest. The model was broken into five hierarchical sub-models: tissue electrophysiology, ToR-ORd-Land model, Courtemanche-Land model, passive mechanics and CircAdapt. For each sub-model, we trained Gaussian processes emulators (GPEs) that were then used to perform a global sensitivity analysis (GSA) to retain parameters explaining 90% of the total sensitivity for subsequent analysis. We identified 45 out of 117 parameters that were important for whole heart function. We performed a GSA over these 45 parameters and identified the systemic and pulmonary peripheral resistance as being critical parameters for a wide range of volumetric and hemodynamic cardiac indexes across all four chambers. We have shown that GPEs provide a robust method for mapping between cellular properties and clinical measurements. This could be applied to identify parameters that can be calibrated in patient-specific models or digital twins, and to link cellular function to clinical indexes.

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2′-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100–200 nucleotides. Note: In the version of this article initially published online, the value for the pH range in the second paragraph on page 2 was incorrect; it should have read 7.5–8.2. In addition, two sentences were included that should have been removed from page 1. These errors have been corrected in all versions of the article.

Protein N -myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N -myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N -myristoyltransferase (NMT) inhibition. Global quantification of N -myristoylation during normal growth or apoptosis allowed the identification of >100 N -myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N -myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N -myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. Protein N -myristoylation is a ubiquitous modification implicated in the regulation of multiple cellular processes. Here, Thinon et al. report the development of a general method to identify N -myristoylated proteins in human cells and identify over 100 endogenous post- and co-translational substrates of N -myristoyltransferase.

The immune reconstitution inflammatory syndrome (IRIS) has emerged as an important early complication of antiretroviral therapy (ART) in resource-limited settings, especially in patients with tuberculosis. However, there are no consensus case definitions for IRIS or tuberculosis-associated IRIS. Moreover, previously proposed case definitions are not readily applicable in settings where laboratory resources are limited. As a result, existing studies on tuberculosis-associated IRIS have used a variety of non-standardised general case definitions. To rectify this problem, around 100 researchers, including microbiologists, immunologists, clinicians, epidemiologists, clinical trialists, and public-health specialists from 16 countries met in Kampala, Uganda, in November, 2006. At this meeting, consensus case definitions for paradoxical tuberculosis-associated IRIS, ART-associated tuberculosis, and unmasking tuberculosis-associated IRIS were derived, which can be used in high-income and resource-limited settings. It is envisaged that these definitions could be used by clinicians and researchers in a variety of settings to promote standardisation and comparability of data.