Biology subjects

Hargreaves, D.

Publications and source records attributed to Hargreaves, D..

5 recordsLinked to original sources

bioRxiv2026

Development of species-specific real-time PCR assays for the identification of five European Rhinolophus bats.

The detection and monitoring of bat species using non-invasive sampling and molecular techniques has become increasingly popular in recent years. In Europe, these approaches have been applied to identify horseshoe bats of the genus Rhinolophus, which includes five species: R. hipposideros, R. ferrumequinum, R. euryale, R. mehelyi and R. blasii. While species-specific real-time PCR assays exist for R. ferrumequinum and R. hipposideros, no unified panel of real-time PCR assays currently enables the identification of all five European Rhinolophus species from non-invasively collected samples. Here, we developed five species-specific real-time PCR assays, each targeting interspecies nucleotide variation within the mitochondrial cytochrome b gene. To enhance single-base discrimination, RNase H-dependent PCR (rhPCR) primers were employed, incorporating cleavable blocked primers that require perfect complementarity for extension. The assays were applied to droppings non-invasively collected from 18 caves and one church in Serbia and Romania. Of the 149 samples analysed, 131 (88%) yielded successful amplification of Rhinolophus DNA. Detection probabilities for the three species identified in the field ranged from 0.49 to 0.82. Occupancy estimates varied, with R. euryale showing the highest (0.86; UI: 0.69-0.97) and R. mehelyi the lowest (0.23; UI: 0.08-0.43). The assays were capable of detecting up to three species concurrently within a single pooled sample (approximately 15 droppings). These assays are especially valuable for detecting R. mehelyi, given its rarity and uncertain distribution, and offer a robust tool for monitoring Rhinolophus populations across Europe.

ecology↗

bioRxiv2024

Mitotic spindle orientation and dynamics are fine-tuned by anisotropic tissue stretch via NuMA localisation

Cell division orientation, which is key to cell fate and tissue morphogenesis, is influenced by mechanical forces. However, uncoupling whether divisions orient in direct response to force or indirectly via interphase cell shape remains challenging. By applying an external stretch to epithelial tissue and monitoring mitotic spindle dynamics, we show that the spindle orientation protein, NuMA, is recruited to the cell cortex earlier during mitosis in stretched tissue. This stretch-induced recruitment of NuMA coincides with the onset and subsequent amplification of dynamic spindle oscillations. We show by mathematical modelling that increased spindle oscillation indicates increased cortical force generation to orient the spindle. Additionally, we show that knockdown of NuMA reduces spindle oscillations and disrupts division orientation according to stretch and cell shape. Our results indicate that a mechanosensitive re-localisation of NuMA provides a direct response to mechanical force, fine-tuning spindle dynamics and orientation during mitosis and ensuring the accurate alignment of divisions with cell shape and anisotropic tension.

cell biology↗

bioRxiv2023

Multiomics atlas-assisted discovery of transcription factors enables specific cell state programming.

CD8+ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection1-4. To systematically define the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8+ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints, uncovering regulatory mechanisms governing selective cell state differentiation. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states critical in tumor and viral contexts: terminally exhausted T cells (TEXterm), which are dysfunctional5-8, and tissue-resident memory T cells (TRM), which are protective9-13. Global TF community analysis revealed distinct biological pathways and TF-driven networks underlying protective versus dysfunctional states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing (in vivo Perturb-seq), we delineated several TFs that selectively govern TEXterm differentiation. We also identified HIC1 and GFI1 as shared regulators of TEXterm and TRM differentiation and KLF6 as a unique regulator of TRM. Importantly, we discovered novel TEXterm- selective TFs, including ZSCAN20 and JDP2, with no prior known function in T cells. Targeted deletion of these TFs enhanced tumor control and synergized with immune checkpoint blockade but did not interfere with TRM formation. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion TEX-selective from protective TRM programs, our platform enables more precise engineering of T cell states, accelerating the rational design of more effective cellular immunotherapies.

immunology↗

bioRxiv2022

Not getting in too deep: a practical deep learning approach to routine crystallisation image classification

Using a relatively small training set of [~]16 thousand images from macromolecular crystallisation experiments, we compare classification results obtained with four of the most widely-used convolutional deep-learning network architectures that can be implemented without the need for extensive computational resources. We show that the classifiers have different strengths that can be combined to provide an ensemble classifier achieving a classification accuracy comparable to that obtained by a large consortium initiative. We use eight classes to effectively rank the experimental outcomes, thereby providing detailed information that can be used with routine crystallography experiments to automatically identify crystal formation for drug discovery and pave the way for further exploration of the relationship between crystal formation and crystallisation conditions. Author summaryX-ray crystallography can provide structural information on the molecules that play important roles in cell function and allow interactions vital for drug design to be elucidated. However, the technique requires the molecules to be crystallised and obtaining sufficiently high-quality crystals can require hundreds of experiments under different conditions. Robots have been designed to perform the microscopic experiments with imaging systems to record the results over time but automated classification of all these images is becoming essential. In this work we compare different classifiers that can be used without the need for vast computational resources and show that an ensemble classifier which combines the different strengths of four deep-learning networks is able to correctly classify the experimental results into one of eight different categories 94% of the time.

molecular biology↗

bioRxiv2018

Predictors of emergency department attendance following NHS 111 calls for children and young people: analysis of linked data

ObjectivesTo assess whether clinical input during calls to the NHS 111 telephone-based advice service is associated with lower rates of subsequent emergency department attendance and hospital admission.\n\nDesignAlthough NHS 111 largely employs non-clinical call handling staff to triage calls using computerised clinical decision support software, some support is available from clinical supervisors, and additionally some calls are referred to out-of-hours General Practitioners (GP). We used linked data sets to examine GP and secondary care activity following calls to NHS 111, adjusting for the patient characteristics, signs and symptoms recorded during the NHS 111 call.\n\nSettingOut-of-hours care in three areas of North West London that have an integrated approach to delivering NHS 111 and out-of-hours GP care.\n\nParticipantsNHS 111 calls for children and young people aged 15 years or under. We excluded calls that were diverted to the emergency ( 999) service or where patients were advised to go to an emergency department. This left callers who were either referred to a GP or advised to manage their health needs at home.\n\nPrimary and secondary outcome measuresThe percentage of callers attending any emergency departments, major emergency department, or minor injury unit within ten hours of the NHS 111 call, and the percentage admitted to hospital following visits to emergency departments.\n\nResultsOf the 10,356 callers, 2,898 (28.0%) were advised by NHS 111 to manage their health needs at home, with an appointment with an out-of-hours GP made for the remaining 7,458 (72.0%). 14.9% (432/2,898) of the callers who were advised by NHS 111 to manage their health needs at home attended an emergency department with ten hours, compared with 16% (1,207/7,458) of callers who had an out-of-hours appointment with an out-of-hours GP. After adjusting for patient characteristics, GP out-of-hours appointment was associated with lower rates of emergency department attendance (adjusted odds ratio, 0.86, 95% CI, 0.75-0.99),). When we subset emergency department types, a GP out-of-hours appointment was associated with lower rates of minor injury unit attendance (adjusted odds ratio, 0.32, 95% CI, 0.23 - 0.44) but not major emergency department attendance (adjusted odds ratio 1.06, 95% CI 0.90-1.24). There was no association with hospital admission. Review by an NHS 111 clinical supervisor was associated with fewer emergency department attendances (adjusted OR 0.77, 95% CI, 0.62-0.97).\n\nConclusionsClinical input during or following out-of-hours calls to NHS 111 was associated with lower rates of emergency department utilisation for children and young people, though the reduction may be concentrated in lower intensity care settings. Thus, there may be potential to reduce the use of emergency care by providing access to clinical advice or out-of-hour services in other settings through the NHS 111 telephone service.

epidemiology↗