A bead-based method for high-throughput mapping of the sequence- and force-dependence of T cell activation.

Adaptive immunity relies on T lymphocytes that use αß T cell receptors (TCRs) to discriminate among peptides presented by major histocompatibility complex molecules (pMHCs). Identifying pMHCs capable of inducing robust T cell responses will not only enable a deeper understanding of the mechanisms governing immune responses but could also have broad applications in diagnosis and treatment. T cell recognition of sparse antigenic pMHCs in vivo relies on biomechanical forces. However, in vitro screening methods test potential pMHCs without force and often at high (nonphysiological) pMHC densities and thus fail to predict potent agonists in vivo. Here, we present a technology termed BATTLES (biomechanically assisted T cell triggering for large-scale exogenous-pMHC screening) that uses biomechanical force to initiate T cell triggering for peptides and cells in parallel. BATTLES displays candidate pMHCs on spectrally encoded beads composed of a thermo-responsive polymer capable of applying shear loads to T cells, facilitating exploration of the force- and sequence-dependent landscape of T cell responses. BATTLES can be used to explore basic T cell mechanobiology and T cell-based immunotherapies.

Constipation prevalence and risk from prescribed medications in people with intellectual disability: Findings from an English mortality programme.

Constipation is common in people with intellectual disability, with case reports of associated deaths. Risk factors include lifestyle factors, health conditions, and certain medications. We aimed to explore constipation in a sample of people with intellectual disability who died in 2021. We described prevalence of constipation, causes of death and the risk of secondary constipation from prescribed medications. Medications were scored based on the risk of constipation indicated in the drug profile. Forty-eight percent of the sample had constipation. Half of the sample were prescribed at least two medications that are commonly associated with side effects of constipation. There were high rates of antipsychotic (30%) and laxative (40%) drug prescription. Five people with a history of constipation died of causes of death associated with constipation. Our findings highlight the risk of secondary constipation due to prescribed medication and the seriousness of the condition in people with intellectual disability.

Global mpox lineage discovery and rapid outbreak tracking with nanopore sequencing.

Insufficient tracking of virus introduction, spread, and new lineage emergence for the human monkeypox (mpox) virus 1 (hMPXV1) outbreak of 2022 hindered epidemiological studies and public health response. hMPXV1 mutations accumulated unexpectedly faster than predicted. Thus, new variants with altered pathogenicity could emerge and spread without early detection. Whole genome sequencing addresses this gap when implemented but requires widely accessible and standardized methodologies to be effective both regionally and globally. Here we developed a rapid nanopore whole genome sequencing method complete with working protocols, from DNA extraction to phylogenetic analysis tools. Using this method, we sequenced 84 complete hMPXV1 genomes from Illinois, a Midwestern region of the United States, spanning the first few months of the outbreak. The resulting five-fold increase in hMPXV1 genomes from this region established two previously unnamed global lineages, several mutational profiles not seen elsewhere, multiple separate introductions of the virus into the region, and the likely emergence and spread of new lineages from within this region. These results demonstrate that a dearth of genomic sequencing of hMPXV1 slowed our understanding and response to the mpox outbreak. This accessible nanopore sequencing approach makes near real-time mpox tracking and rapid lineage discovery straightforward and creates a blueprint for how to deploy nanopore sequencing for genomic surveillance of diverse viruses and future outbreaks.

Compulsory collisions and corporate interests in school rugby: challenging distortions in the framing of childhood injury.

This article examines how 'framing' is used to resist a proposal to remove rugby tackling from UK schools. It focuses on rugby tackling for UK school children, which is often a compulsory part of many schools' curricula. Specifically, we explore the importance of framing in how the problem is described in various academic publications, how ideas about risk are articulated and how advocates themselves are represented. We show how the corporate interests of rugby governing bodies can become entangled with distortions about injury prevention. These distortions (or framing practices) include omitting arguments, conflating arguments, changing the argument, misrepresenting advocacy positions and skewing advocate identities. Next, the article demonstrates how a combination of recent advocacy, political interventions, research and cultural shifts appears to be changing perceptions about the risks associated with rugby tackling for children in school settings. In conclusion, we argue that while framing can be a useful strategy for policy advocates, there is value in paying attention to how framing is used by different stakeholder groups.

An exploratory study into the relationship between playing at home or away and concussion.

PRIMARY OBJECTIVE: To investigate the effect of home and away game travel on risk of concussion across different levels of rugby union. RESEARCH DESIGN: Exploration study across school, university, and professional rugby teams. METHODS AND PROCEDURES: Retrospective analysis of concussion incidence and symptomology of surveillance data and prospective data collection for potential concussions via surveys. Data was collected from school rugby teams (n = 344 matches, over 2 years), a university rugby (n = 6 matches), and a professional rugby team (n = 64 matches, over two seasons). MAIN OUTCOMES AND RESULTS: School level rugby had an increased prevalence of concussions in away matches (p = 0.02). Likewise, there was a significant increase (p < 0.05) in concussions at away matches in university rugby. In addition, the professional rug by team had significant differences in recovery times and symptoms with away fixtures, including longer recovery times (p < 0.01), more initial symptoms (p < 0.01), as well as greater and more severe symptoms at 48 hours (p < 0.05). CONCLUSIONS: This research highlights an increased prevalence of concussion in school and university-aged rugby players away from home, as well as increased symptoms, symptom severity, and recovery times in professional rugby players.

From eye-blinks to state construction: Diagnostic benchmarks for online representation learning.

We present three new diagnostic prediction problems inspired by classical-conditioning experiments to facilitate research in online prediction learning. Experiments in classical conditioning show that animals such as rabbits, pigeons, and dogs can make long temporal associations that enable multi-step prediction. To replicate this remarkable ability, an agent must construct an internal state representation that summarizes its interaction history. Recurrent neural networks can automatically construct state and learn temporal associations. However, the current training methods are prohibitively expensive for online prediction-continual learning on every time step-which is the focus of this paper. Our proposed problems test the learning capabilities that animals readily exhibit and highlight the limitations of the current recurrent learning methods. While the proposed problems are nontrivial, they are still amenable to extensive testing and analysis in the small-compute regime, thereby enabling researchers to study issues in isolation, ultimately accelerating progress towards scalable online representation learning methods.

Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

Evidence in support of the call to ban the tackle and harmful contact in school rugby: a response to World Rugby.

In a paper published in BJSM (June 2016), World Rugby employees Ross Tucker and Martin Raftery and a third coauthor Evert Verhagen took issue with the recent call to ban tackling in school rugby in the UK and Ireland. That call (to ban tackling) was supported by a systematic review published in BJSM Tucker et al claim that: (1) the mechanisms and risk factors for injury along with the incidence and severity of injury in youth rugby union have not been thoroughly identified or understood; (2) rugby players are at no greater risk of injury than other sports people, (3) this is particularly the case for children under 15 years and (4) removing the opportunity to learn the tackle from school pupils might increase rates of injuries. They conclude that a ban 'may be unnecessary and may also lead to unintended consequences such as an increase in the risk of injury later in participation.' Here we aim to rebut the case by Tucker et al We share new research that extends the findings of our original systematic review and meta-analysis. A cautionary approach requires the removal of the tackle from school rugby as the quickest and most effective method of reducing high injury rates in youth rugby, a public health priority.

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

High-throughput microfluidic single-cell RT-qPCR.

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

Predicting recovery following stroke: Deep learning, multimodal data and feature selection using explainable AI.

Machine learning offers great potential for automated prediction of post-stroke symptoms and their response to rehabilitation. Major challenges for this endeavour include the very high dimensionality of neuroimaging data, the relatively small size of the datasets available for learning and interpreting the predictive features, as well as, how to effectively combine neuroimaging and tabular data (e.g. demographic information and clinical characteristics). This paper evaluates several solutions based on two strategies. The first is to use 2D images that summarise MRI scans. The second is to select key features that improve classification accuracy. Additionally, we introduce the novel approach of training a convolutional neural network (CNN) on images that combine regions-of-interests (ROIs) extracted from MRIs, with symbolic representations of tabular data. We evaluate a series of CNN architectures (both 2D and a 3D) that are trained on different representations of MRI and tabular data, to predict whether a composite measure of post-stroke spoken picture description ability is in the aphasic or non-aphasic range. MRI and tabular data were acquired from 758 English speaking stroke survivors who participated in the PLORAS study. Each participant was assigned to one of five different groups that were matched for initial severity of symptoms, recovery time, left lesion size and the months or years post-stroke that spoken description scores were collected. Training and validation were carried out on the first four groups. The fifth (lock-box/test set) group was used to test how well model accuracy generalises to new (unseen) data. The classification accuracy for a baseline logistic regression was 0.678 based on lesion size alone, rising to 0.757 and 0.813 when initial symptom severity and recovery time were successively added. The highest classification accuracy (0.854), area under the curve (0.899) and F1 score (0.901) were observed when 8 regions of interest were extracted from each MRI scan and combined with lesion size, initial severity and recovery time in a 2D Residual Neural Network (ResNet). This was also the best model when data were limited to the 286 participants with moderate or severe initial aphasia (with area under curve = 0.865), a group that would be considered more difficult to classify. Our findings demonstrate how imaging and tabular data can be combined to achieve high post-stroke classification accuracy, even when the dataset is small in machine learning terms. We conclude by proposing how the current models could be improved to achieve even higher levels of accuracy using images from hospital scanners.

Sequencing-guided design of genetically encoded small RNAs targeting CAG repeats for selective inhibition of mutant huntingtin.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal, but reduction of wild-type HTT (wtHTT) is undesirable, as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing (RNA-seq) to monitor the processing precision of a limited set of CAG repeat-targeted small RNAs expressed from multiple scaffold contexts. Small RNA-seq identified expression constructs with high-guide strand 5' processing precision and promising allele-selective inhibition of mutHTT. Transcriptome-wide mRNA-seq also identified an allele-selective small RNA with a favorable off-target profile. These results support continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.

A Qualitative Exploration of Parents' Perceptions of Risk in Youth Contact Rugby.

The purpose of this study was to explore the understandings and perceptions of risk related to brain trauma amongst parents of children that play contact rugby. A qualitative approach was taken, using semi-structured interviews with 7 mothers and 27 fathers of children that participate in contact rugby. A thematic analysis of data suggests that parents used two primary cognitive strategies to process the risk they consented to with their children's participation in rugby; (1) minimalizing rugby risk to be equivalent to less injurious sports; and (2) elevating physical and social advantages above what they think other sports are capable of providing. From the findings it is suggested that parents who permit their children to play contact rugby are both aware of the high risks of injury in the sport, but simultaneously utilize two cognitive distortion techniques to rectify the dissonance caused between their choice to have their children play, and the salient number of concussions they observe. These results suggest that it will take properly informed consent, inclusive of concussion rates compared to other sports, in order to reduce cognitive distortion and effectively communicate risks associated with participation in contact rugby.

Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs.

Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.

An assessment of current concussion identification and diagnosis methods in sports settings: a systematic review.

BACKGROUND: Concussion in sport is an ongoing global concern. The head injury assessment (HIA) by the field of play is acknowledged as the first step in recognising and identifying concussion. While previous systematic literature reviews have evaluated the sensitivity of side-line screening tools and assessment protocols, no systematic review has evaluated the research designs and assessments used in a field setting. This systematic review investigated existing screening and diagnostic tools used in research as part of the HIA protocol to identify concussion that are currently used in professional, semi-professional and amateur (club) sports settings. METHODS: A systematic searching of relevant databases was undertaken for peer-reviewed literature between 2015 and 2020. RESULTS: Twenty-six studies met the inclusion criteria. Studies were of moderate to good quality, reporting a variety of designs. The majority of studies were undertaken in professional/elite environments with medical doctors and allied health practitioners (e.g., physical therapists) involved in 88% of concussion assessments. While gender was reported in 24 of the 26 studies, the majority of participants were male (77%). There was also a variety of concussion assessments (n = 20) with the sports concussion assessment tool (SCAT) used in less than half of the included studies. CONCLUSION: The majority of studies investigating concussion HIAs are focused on professional/elite sport. With concussion an issue at all levels of sport, future research should be directed at non-elite sport. Further, for research purposes, the SCAT assessment should also be used more widely to allow for consistency across studies.

Metasurface optofluidics for dynamic control of light fields.

The ability to manipulate light and liquids on integrated optofluidics chips has spurred a myriad of important developments in biology, medicine, chemistry and display technologies. Here we show how the convergence of optofluidics and metasurface optics can lead to conceptually new platforms for the dynamic control of light fields. We first demonstrate metasurface building blocks that display an extreme sensitivity in their scattering properties to their dielectric environment. These blocks are then used to create metasurface-based flat optics inside microfluidic channels where liquids with different refractive indices can be directed to manipulate their optical behaviour. We demonstrate the intensity and spectral tuning of metasurface colour pixels as well as on-demand optical elements. We finally demonstrate automated control in an integrated meta-optofluidic platform to open up new display functions. Combined with large-scale microfluidic integration, our dynamic-metasurface flat-optics platform could open up the possibility of dynamic display, imaging, holography and sensing applications.