Biology subjects

Bennett, P.

Publications and source records attributed to Bennett, P..

5 recordsLinked to original sources

bioRxiv2026

The generation and validation of a de novo-designed titin binder for use in fluorescence microscopy

Advances in the generation of proteins in silico has enabled the efficient design of such that can bind to a specified target. Here, we demonstrate the use of a fluorescently-labelled de novo-designed protein to bind its target in situ and be imaged using fluorescence microscopy, a widely used experimental technique that typically relies on antibodies or similar evolutionary derived binders to identify the presence and location of targets in their native environment. Our de novo-designed protein binds the C-terminal domain M10 (Ig-169) of the giant muscle protein titin, which spans half a sarcomere, the basic contractile unit of striated muscle. M10 antibodies suitable for fluorescence microscopy are unavailable. Confocal microscopy of muscle sections shows the binder localises to the M-band of the sarcomere - where M10 is found - and fails to label muscle in competition experiments and in mutant muscle where M10 is absent. These results demonstrate the utility of de novo-designed proteins in immunostaining-like experiments and suggest a future where targets can be routinely identified in complex biological samples by in silico-generated binders. Such an approach avoids the need to generate antibodies or similar binders either in vivo or in vitro, which can have technical, financial and ethical challenges.

bioengineering↗

bioRxiv2025

Multi-omic analysis of vaginal bacteria:host immune and metabolic interactions in adverse early pregnancy outcomes

Accurate and timely diagnosis of early pregnancy outcomes remains a clinical challenge, particularly in cases classified as pregnancy of unknown location, where ultrasound fails to confirm the location of the pregnancy. Delayed diagnosis can lead to serious complications, especially in ectopic pregnancies. This prospective cohort study aimed to characterise host-microbe interactions during early pregnancy in the vaginal mucosa by exploring the relationship between the bacterial community composition, immune response, and metabolic phenotype. We analyzed vaginal swabs from 91 women with pregnancy of unknown location, including 22 with viable intrauterine pregnancies and 69 with adverse outcomes. Metataxonomic profiling, immune assays, and direct on-swab metabolomics were performed. Adverse outcomes, especially ectopic pregnancies, were associated with reduced Lactobacillus abundance, increased bacterial diversity, mucosal inflammation, and elevated lipid species. Vaginal bleeding, a common symptom in PUL, significantly impacted immune and metabolic profiles and should be considered as a confounder in studies of the lower female reproductive tract. Multi-omic data integration highlighted metabolic phenotypes of immune response in the context of bacterial community composition, underscoring the influence of microbe-host interactions on mucosal health and pregnancy outcomes. IMPORTANCEEarly detection of complications in pregnancy is essential for improving maternal health outcomes, yet current diagnostic tools often fail to identify ectopic or failing pregnancies in a timely manner. This study highlights how changes in the vaginal microbiome, inflammation, and metabolic signals are closely linked to adverse outcomes in early pregnancy. By showing that metabolic profiling can predict microbial and immune states even in the presence of vaginal bleeding, the research provides a foundation for developing rapid, non-invasive diagnostic tools. These insights could pave the way for more personalized and effective early pregnancy care.

microbiology↗

bioRxiv2024

Characterisation and comparison of semen microbiota and sperm function in men with infertility, recurrent miscarriage, or proven fertility

Several studies have associated seminal microbiota abnormalities with male infertility but have yielded differing results owing to their limited sizes or depths of analyses. The semen microbiota during recurrent pregnancy loss (RPL) has not been investigated. Comprehensively assessing the seminal microbiota in men with reproductive disorders could elucidate its potential role in clinical management. We used semen analysis, terminal-deoxynucleotidyl-transferase-mediated-deoxyuridine-triphosphate-nick-end-labelling, Comet DNA fragmentation, luminol ROS chemiluminescence and metataxonomic profiling of semen microbiota by16S rRNA amplicon sequencing in this prospective, cross-section study to investigate composition and bacterial load of seminal bacterial genera and species, semen parameters, reactive oxidative species (ROS), and sperm DNA fragmentation in men with reproductive disorders and proven fathers. 223 men were enrolled included healthy men with proven paternity (n=63), the male partners in a couple encountering RPL (n=46), men with male factor infertility (n=58), and the male partners of couples unexplained infertility (n=56). Rates of high sperm DNA fragmentation, elevated ROS and oligospermia were more prevalent in the study group compared with control. In all groups, semen microbiota clustered into three major genera-dominant groups (1, Streptococcus; 2, Prevotella; 3, Lactobacillus and Gardnerella); no species clusters were identified. Group 2 had the highest microbial richness (P<0.001), alpha-diversity (P<0.001), and bacterial load (P<0.0001). Overall bacterial composition or load has not found to associate with semen analysis, ROS or DNA fragmentation. Whilst, global perturbation of the seminal microbiota is not associated with male reproductive disorders, men with unidentified seminal Flavobacterium are more likely to have abnormal seminal analysis. Future studies may elucidate if Flavobacterium reduction has therapeutic potential.

microbiology↗

bioRxiv2023

In situ structures from relaxed cardiac myofibrils reveal the organization of the muscle thick filament

The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit of striated muscle1. Mutations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2, 3. Despite this central importance for sarcomere force generation, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction. Here, we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by electron cryo-tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin heads and tails, myosin-binding protein C (MyBP-C) and titin, elucidating the structural basis for their interaction during muscle contraction. The arrangement of myosin heads is variable depending on their position along the filament, suggesting that they have different capacities in terms of strain susceptibility and activation. Myosin tails exhibit a distinct arrangement and pattern of interactions. These are likely orchestrated by three alpha and three beta titin chains that are arranged like a spring, suggesting the existence of specialized roles of thick filament segments in length-dependent activation and contraction. Surprisingly, while the three titin alpha chains run along the entire length of the thick filament, titin beta does not. The structure also demonstrates that the C-terminal region of MyBP-C binds myosin tails and unexpectedly also directly interacts with the myosin heads, suggesting a previously undescribed direct role in the preservation of the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. These findings establish a robust groundwork for forthcoming research endeavors aiming to explore muscle disorders that involve sarcomeric structural components.

biochemistry↗

bioRxiv2018

Assessment of abduction movement in older people with painful shoulder: an analysis based on inertial sensors

Reduced range of motion in the shoulder can be a source of functional limitation. Current quantitative evaluation systems are limited to assessing the functionality or the maximum articular amplitudes in each of the planes of movement, both in isolation. These separate clinical evaluation systems may not allow the identification of the underlying impairments contributing to the functional limitation. The use of inertial sensors to quantify movement in addition to more common clinical assessments of the shoulder may allow clinicians to understand that are potentially unnoticed by the human eye. The main objective of this cross-sectional study was to generate an explanatory model for shoulder abduction based on data from inertial sensors. Shoulder abduction of thirteen older adults suffering from shoulder dysfunction was evaluated using two inertial sensors placed on the humerus and scapula. Movement variables (maximum angular mobility, angular peak of velocity, peak of acceleration) were used to explain the functionality of the upper limb (assessed using the Upper Limb Functional Index). Abduction movement of the shoulder was explained by six variables related to the mobility of the shoulder joint complex. A manova analysis was used to explain the results obtained on the functionality of the upper limb. The MANOVA model based on angular mobility explained 69% of the variance of the ULFI value (r-squared=0.69). The most relevant variables were the abduction-adduction of the humerus and the medial and lateral rotation of the scapula. However, given the limited sample size, none of these individual variables were statistically significant in the decomposition model on their own. The method used in the present study reveals the potential importance of the analysis of the scapular and humeral movements for comprehensive evaluation of the upper limb. Further research should include a wider sample and may seek to use this assessment technique in a range of potential clinical applications.

bioengineering↗