Biology subjects

Crane, M.

Publications and source records attributed to Crane, M..

8 recordsLinked to original sources

bioRxiv2024

Aspartic proteases are abundant and active in acidified wound fluid

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. Studies have shown that treatment of poorly healing wounds with acid results in improved healing. In this study, we systematically evaluated changes in proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0 and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, aspartic acid proteases emerged as the dominant active enzymes at pH 3.5 and their activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified aspartic protease activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as one day post-injury and persisted over the following ten days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes.

biochemistry↗

bioRxiv2023

Interleukin-1α links peripheral CaV2.2 channel activation to rapid adaptive increases in heat sensitivity in skin

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated CaV2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1 (IL-1), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1 was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1 levels were also dependent on peripheral CaV2.2 channel activity. Neutralizing IL-1 in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1 enhances behavioral responses to stimuli and, in culture, IL-1 enhances the responsiveness of Trpv1-expressing sensory neurons. Together, our data suggest that IL-1 is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin.

neuroscience↗

bioRxiv2023

Prevotella copri-linked effects of a therapeutic food for malnutrition

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the metagenome assembled genomes (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed reverse translation experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

microbiology↗

bioRxiv2021

How do King Cobras move across a major highway? Unintentional wildlife crossing structures may facilitate movement.

O_LIGlobal road networks continue to expand, and the wildlife responses to these landscape-level changes need to be understood to advise long-term management decisions. Roads have high mortality risk to snakes because snakes typically move slowly and can be intentionally targeted by drivers. C_LIO_LIWe investigated how radio-tracked King Cobras (Ophiophagus hannah) traverse a major highway in northeast Thailand, and if reproductive cycles were associated with road hazards. C_LIO_LIWe surveyed a 15.3km stretch of Highway 304 to determine if there were any locations where snakes, and other wildlife, could safely move across the road (e.g., culverts, bridges). We used recurse analysis to detect possible road-crossing events, and used subsets of King Cobra movement data to create dynamic Brownian Bridge Movement Models (dBBMM) in an attempt to show movement pathways association with possible unintentional crossing structures. We further used Integrated Step Selection Functions (ISSF) to assess seasonal differences in avoidance of major roads for adult King Cobras in relation to reproductive state. C_LIO_LIWe discovered 32 unintentional wildlife crossing locations capable of facilitating King Cobra movement across the highway. Our dBBMMs failed to show if underpasses were being used by telemetered individuals; however, the tracking locations pre- and post-crossing provided strong evidence of underpass use. Our ISSF suggested a lower avoidance of roads during the breeding season, though the results were inconclusive. With the high volume of traffic, large size of King Cobras and a 98.8% success rate of crossing the road in our study, we strongly suspect that individuals are using the unintentional crossing structures to safely traverse the road. C_LIO_LIFurther research is needed to determine the extent of wildlife underpass use at our study site and globally, alongside using previously proven fencing to facilitate their use. We propose that more consistent integration of drainage culverts and bridges could help mitigate the impacts of roads on some terrestrial wildlife, particularly in areas where roads fragment forests and wildlife corridors. C_LI

ecology↗

bioRxiv2020

No room to roam: King Cobras reduce movement in agriculture

Studying animal movement provides insights into how animals react to land-use changes, specifically how animals can change their behaviour in agricultural areas. Recent reviews show a tendency for species to reduce movements in response to increased human landscape modification, but the study of movement has not been extensively explored in reptiles. We examined movements of a large reptilian predator, the King Cobra (Ophiophagus hannah), in Northeast Thailand. We used a consistent regime of radio-telemetry tracking to document movements across protected forest and adjacent agricultural areas. We then adapted GPS-targeting analytic methods to examine the movement using metrics of site reuse and dynamic Brownian Bridge Movement Model derived motion variance. Examination of motion variance demonstrated that King Cobra movements increased in forested areas and tended to decrease in agricultural areas. Our Integrated Step-Selection Functions indicated that when moving in agricultural areas King Cobras restricted their movements, thereby remaining within vegetated semi-natural areas, often located along the banks of irrigation canals. Site reuse metrics of residency time and number of revisits remained unaffected by distance to landscape features. Neither motion variance nor reuse metrics were consistently affected by the presence of threatening landscape features (e.g. roads, human settlements); suggesting that King Cobras will remain in close proximity to threats, provided habitat patches are available. Although King Cobras displayed heterogeneity in their response to agricultural landscapes, the overall trend suggested a reduction in movements when faced with fragmented habitat patches embedded in an otherwise inhospitable land-use matrix. Reductions in movements are consistent with findings for mammals and forest specialist species.

ecology↗

bioRxiv2020

Revisiting reptile home ranges: moving beyond traditional estimators with dynamic Brownian Bridge Movement Models

Animal movement, expressed through home ranges, can offer insights into spatial and habitat requirements. However, home range estimation methods vary, directly impacting conclusions. Recent technological advances in animal tracking (GPS and satellite tags), have enabled new methods for home range estimation, but so far have primarily targeted mammal and avian movement patterns. Most reptile home range studies only make use of two older estimation methods: Minimum Convex Polygons (MCP) and Kernel Density Estimators (KDE), particularly with the Least Squares Cross Validation (LSCV) and reference (href) bandwidth selection algorithms. The unique characteristics of reptile movement patterns (e.g. low movement frequency, long stop-over periods), prompt an investigation into whether newer movement-based methods -such as dynamic Brownian Bridge Movement Models (dBBMMs)- are applicable to Very High Frequency (VHF) radio-telemetry tracking data. To assess home range estimation methods for reptile telemetry data, we simulated animal movement data for three archetypical reptile species: a highly mobile active hunter, an ambush predator with long-distance moves and long-term sheltering periods, and an ambush predator with short-distance moves and short-term sheltering periods. We compared traditionally used home range estimators, MCP and KDE, with dBBMMs, across eight feasible VHF field sampling regimes for reptiles, varying from one data point every four daylight hours, to once per month. Although originally designed for GPS tracking studies, we found that dBBMMs outperformed MCPs and KDE href across all tracking regimes, with only KDE LSCV performing comparably at some higher-frequency sampling regimes. The performance of the LSCV algorithm significantly declined with lower-tracking-frequency regimes, whereas dBBMMs error rates remained more stable. We recommend dBBMMs as a viable alternative to MCP and KDE methods for reptile VHF telemetry data: it works under contemporary tracking protocols and provides more stable estimates, improving comparisons across regimes, individuals and species.

ecology↗

bioRxiv2019

Phenotypic and genotypic consequences of CRISPR/Cas9 editing of the replication origins in the rDNA of Saccharomyces cerevisiae

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

genetics↗

bioRxiv2017

Morphologically Constrained and Data Informed Cell Segmentation of Budding Yeast

MotivationAlthough high-content image cytometry is becoming increasingly routine, processing the large amount of data acquired during time-lapse experiments remains a challenge. The majority of approaches for automated single-cell segmentation focus on flat, uniform fields of view covered with a single layer of cells. In the increasingly popular microfluidic devices that trap individual cells for long term imaging, these conditions are not met. Consequently, most segmentation techniques perform poorly. Incorporating information about the microfluidic features, media flow and morphology of the cells can substantially improve performance, though it may constrain the generalizability of software.\n\nResultsHere we present DISCO (Data Informed Segmentation of Cell Objects), a framework for using the physical constraints imposed by microfluidic traps, the shape based morphological constraints of budding yeast and temporal information about cell growth and motion, to allow tracking and segmentation of cells in micrflouidic devices. Using manually curated data sets, we demonstrate substantial improvements in both tracking and segmentation for this approach when compared with existing software.\n\nAvailabilityThe MATLAB(R) code for the algorithm and for measuring performance is available at https://github.com/pswain/segmentation-software. The test images and the curated ground truth results used for comparing the algorithms are available at http://swainlab.bio.ed.ac.uk/.

bioinformatics↗