Biology subjects

Marshall, A.

Publications and source records attributed to Marshall, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Therapeutic signature mapping of paired direct and indirect LPS injury in an ex vivo human lung perfusion platform reveals injury-specific druggable programs

Acute Respiratory Distress Syndrome (ARDS) remains highly morbid and lacks approved disease-modifying pharmacotherapies. Direct (pulmonary) and indirect (extrapulmonary) insults may initiate biologically distinct early injury programs, but human tissue-level evidence from the first hours is scarce. Here we establish a paired, acellular ex vivo lung perfusion (EVLP) platform using human donor lungs unsuitable for transplantation to model direct (endobronchial) and indirect (perfusate) lipopolysaccharide (LPS) injury within the same donor. We profiled lung tissue proteomes at 4 h post-insult and performed therapeutic nomination by querying proteomics-derived injury signatures against the CLUE L1000 perturbational compendium with independent cross-platform validation. Both models developed histological injury and robust cytokine release. Direct injury preferentially enriched neutrophil degranulation, extracellular matrix remodelling and metabolic reprogramming modules, whereas indirect injury showed prominent complement/coagulation perturbation with greater endothelial activation markers in perfusate. Cross-platform prioritisation converged on tractable signalling and epigenetic axes, including JAK/STAT, PI3K/AKT/mTOR, SYK, CDK and HDAC inhibitor classes - yielding a tiered shortlist for EVLP intervention testing. This intact human lung perturbation platform enables injury-stratified mechanistic inference and therapeutic prioritisation in early lung injury relevant to ARDS.

immunology↗

bioRxiv2026

Rapid peripheral reprogramming of myelinated afferents drives human hyperalgesia

A small burn can render a large area of skin painfully tender. This widespread hyperalgesia protects injured tissue and is typically attributed to altered spinal cord mechanisms. Whether peripheral sensory afferents directly interact to contribute to hyperalgesia remains unclear. Using single-unit microneurography, we recorded cutaneous afferents before and after inducing localized TRPV1-mediated inflammatory flare. This triggered minutes-scale reweighting of transcriptomically defined TRPV1- afferents, with divergent effects among myelinated (A{beta}-range) classes: tactile receptors showed reduced responsiveness, whereas mechano-nociceptors underwent sensitization. These changes paralleled diminished tactile sensitivity and intensified mechanical pain. Recruitment of mechanically silent branches in A{beta}-range mechano-nociceptors produced wide-field amplification of peripheral nociceptive signaling beyond the inflamed site. These findings suggest that rapid peripheral crosstalk from TRPV1+ afferents reprograms TRPV1- A{beta}-afferents and drives human hyperalgesia.

neuroscience↗

bioRxiv2026

Light Microscopy-Based Organelle Quantification: A Comprehensive Protocol

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

cell biology↗

bioRxiv2025

Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

Traumatic brain injury (TBI) is a leading cause of chronic neurological disability, yet no disease-modifying therapy exists. Emerging evidence indicates that TBI activates cellular aging programs, including telomere erosion and persistent inflammation, that contribute to progressive neurodegeneration. Telomerase reverse transcriptase (TERT) preserves telomere homeostasis and provides cytoprotective effects in the central nervous system, but has not been therapeutically targeted after TBI. Here, we developed an mRNA nanotherapy consisting of mouse TERT mRNA encapsulated in lipid nanoparticles (mTERT-LNPs) and evaluated it in a controlled cortical impact model of moderate TBI. We first established that TBI transiently disrupts TERT biology, with reduced cortical TERT mRNA and shortened telomeres at 3 days post-injury (dpi), followed by partial recovery by 14 dpi. mTERT-LNPs were well tolerated in vitro and in vivo. Following intravenous delivery in the acute post-injury window, LNPs localized to the injured brain and displayed expected peripheral biodistribution. A single systemic dose increased cortical TERT mRNA and protein and partially restored telomere length at 3 dpi. TERT mRNA delivery significantly reduced Iba1+ microglial activation and suppressed pro-inflammatory cytokines, with modest increases in anti-inflammatory markers. Systemically, mTERT-LNPs lowered serum C-reactive protein and malondialdehyde, indicating reduced peripheral inflammation and oxidative stress, without adverse effects on body weight or peripheral organ histology. Several outcomes showed sex-dependent patterns. Collectively, these data provide the first in vivo evidence that telomerase therapy can modulate telomere biology and neuroinflammation after TBI, supporting mRNA-LNP-mediated TERT restoration as a scalable, mechanistically grounded strategy for disease modification in TBI and related disorders. Significant statementTraumatic brain injury (TBI) can initiate progressive brain changes that worsen long after the initial impact, and no therapy directly slows or prevents this decline. A key contributor may be "accelerated aging" in injured tissue, including telomere damage (protective chromosome ends) and persistent neuroinflammation driven by activated immune cells. This study is significant because it tests a targeted way to disrupt these processes by restoring telomerase reverse transcriptase (TERT), which helps maintain telomeres and cellular resilience. Using a clinically validated mRNA-lipid nanoparticle platform, a single intravenous dose increased brain TERT, partially restored telomere length, and reduced neuroinflammation and systemic oxidative stress without obvious toxicity. These findings connect telomere dysfunction to a scalable disease-modifying strategy for TBI and related neurodegenerative conditions.

neuroscience↗

bioRxiv2025

Resolution of the circulating pathogenic phenotype of alpha-1 antitrypsin deficiency following liver transplantation

Alpha-1 antitrypsin is a member of the serpin family of protease inhibitors and normally found in monomeric form at high concentration in the circulation. Certain pathogenic variants of this protein self-assemble into flexible chains (polymers) that undergo toxic accumulation in the liver, underlying the development of liver disease, and a corresponding functional deficiency in circulation results in a susceptibility to chronic damage to the lungs and thereby COPD. A proportion of these polymers are detectable in the circulation. This study aimed to provide data on the dynamics of AAT monomers and polymers in the circulation after correction of a deficiency state by liver transplantation. The evolution of the circulating AAT phenotype was characterised quantitatively over time by analysis of blood samples taken pre- and post-liver transplant from three PiZZ AAT deficiency patients, using a panel of conformer-specific monoclonal antibodies and a novel ELISA with enhanced sensitivity for AAT polymers. Circulating wild-type M AAT was found to increase with a half-time of 29-39 h, attaining a clinically-defined putative protective threshold level within 24-50 h post-transplant. Baseline circulating polymer levels ranged from 5-35 g/mL with a half-time clearance of 3-12 h following transplant, and levels were indistinguishable from reference wild-type plasma 22 months post-surgery. The glycosylation profile and disappearance of detectable circulating polymers of AAT were consistent with their origin in, and canonical secretion by, the liver. To our knowledge this is the first post-transplant evaluation of the hybrid conformational profile of endogenous and donor tissue-derived AAT.

pathology↗

bioRxiv2025

Sex-specific effects of fecal microbiota transplantation on TBI-exacerbated Alzheimer's pathology in mice

BackgroundTraumatic brain injury (TBI) accelerates Alzheimers disease (AD) pathology and neuroinflammation, potentially via gut-brain axis disruptions. Whether restoring gut microbial homeostasis mitigates TBI-exacerbated AD features remains unclear, particularly with respect to sex differences. ObjectiveThe goal of our study was to test whether fecal microbiota transplantation (FMT) modifies amyloid pathology, neuroinflammation, gut microbial composition, metabolites, and motor outcomes in male and female 5xFAD mice subjected to TBI. MethodsMale and female 5xFAD mice received sham treatments or controlled cortical impact, followed 24 hours later by vehicle (VH) or sex-matched FMT from C57BL/6 donors. Assessments at baseline, 1, and 3 days post-injury included Thioflavin-S and 6E10 immunostaining for A{beta}, Iba-1 and GFAP for glial activation, lesion volume, rotarod performance, 16S rRNA sequencing for microbiome profiling, serum short-chain fatty acids (SCFAs), and gut histology. ResultsTBI increased cortical and dentate gyrus A{beta} burden, with females showing greater vulnerability. FMT reduced A{beta} deposition in sham animals and shifted plaque morphology but did not attenuate TBI-induced amyloid escalation. FMT differentially modulated glial responses by sex and region (reduced microgliosis in males) without altering lesion volume. Rotarod performance was better in sham females compared to males and declined in FMT-treated TBI females. Fecal microbiome alpha diversity and richness were unchanged, while beta diversity revealed marked, time-dependent community shifts after TBI that were slightly altered by FMT. Gut morphology remained broadly intact, but crypt width increased after TBI, particularly in males. ConclusionIn 5xFAD mice, TBI drives sex-dependent worsening of amyloid pathology, neuroinflammation, and dysbiosis. Acute FMT partially restores microbial composition and plaque features in sham animals but fails to reverse TBI-induced neuroinflammation or motor deficits. These findings underscore the context- and sex-dependence of microbiome interventions and support longer-term, sex-specific strategies for AD with comorbid TBI.

neuroscience↗

bioRxiv2025

White matter reorganization of motor and affective-motivational networks in pain-indifferent carriers of the R221W mutation

Congenital insensitivity to pain (CIP) due to the R221W mutation on the nerve growth factor gene results in reduced peripheral C-nociceptor density and behavioural indifference to painful stimuli. While functional neuroimaging has revealed altered cortical and sub-cortical pain processing in R221W carriers, structural white matter changes remain unexplored and may suggest an anatomical basis of symptoms. Heterozygous R221W carriers (n = 11) and age-, sex-, education-matched controls (n = 11) diffusion tensor imaging data were compared using fixel-based analysis, and complimentary edge and node analyses using graph theory, and network-based statistics. Whole-brain and region of interest (ROI) fixel-based analyses revealed significantly reduced fibre density and fibre-bundle cross-section in brainstem motor tracts of R221W carriers, encompassing the corticospinal pathways, corona radiata, external capsule, cerebellar peduncles, and pontine crossing (p < 0.05). Graph theory analysis of pain-processing ROIs demonstrated reduced local efficiency in right anterior cingulate cortex (ACC) and altered betweenness centrality in bilateral insula and left ACC of R221W carriers. Despite R221W carriers showing higher node degrees in the somatosensory cortex and ACC, these connections had reduced efficiency and integration with cortical network regions. Network-based statistics identified a possible compensatory subnetwork with stronger connectivity from right thalamus to left ACC and left insula in R221W carriers (p < 0.019). These findings suggest that congenitally reduced peripheral nociception could lead to abnormalities in the thalamocortical and motor efferent pathway, but not sensory afferent pathways. The combination of reduced brainstem motor tract integrity and altered cortical network efficiency, alongside potentially compensatory thalamo-cortical connectivity, could support a model of R221W CIP as motor under-reactivity rather than sensory insensitivity.

neuroscience↗

bioRxiv2025

The sensitising effect of IgG in fibromyalgia syndrome is mediated by Mrgprb2 in mast cells

Fibromyalgia syndrome (FMS) is characterized by elevated levels of immunoglobulin G (IgG), altered bowel habits, and increased pain sensitivity, suggesting immune dysregulation, but the exact mechanism remains unclear. Here, we found that FMS-IgG binds to mast cells in a MRGPRX2/b2-dependent manner, leading to mast cell recruitment and IL-6 secretion. Transferring serum-IgG from FMS patients to mice induced FMS-like symptoms and increased skin mast cells, indicating that FMS-IgG acts through mast cell activation. The ablation of mice Mrgprb2 mast cells or deleting Mrgprb2 receptors prevented IgG-induced heightened sensitivity to mechanical and cold stimuli. Stimulating human LAD2 cells with FMS IgG elicited MRGPRX2-dependent IL-6 production. Consistent with mice findings, mast cell density and tryptase levels increased in human FMS skin samples compared to healthy controls. Taken together our results suggests that FMS IgG mediates hypersensitivity via activation of mast cells bearing the MRGPRX2 receptor and that these cells are a potential therapeutic target.

immunology↗

bioRxiv2025

Hierarchical Lineage Tracing Reveals Diverse Pathways of AML Treatment Resistance

Cancer cells adapt to treatment, leading to the emergence of clones that are more aggressive and resistant to anti-cancer therapies. We have a limited understanding of the development of treatment resistance as we lack technologies to map the evolution of cancer under the selective pressure of treatment. To address this, we developed a hierarchical, dynamic lineage tracing method called FLARE (Following Lineage Adaptation and Resistance Evolution). We use this technique to track the progression of acute myeloid leukemia (AML) cell lines through exposure to Cytarabine (AraC), a front-line treatment in AML, in vitro and in vivo. We map distinct cellular lineages in murine and human AML cell lines predisposed to AraC persistence and/or resistance via the upregulation of cell adhesion and motility pathways. Additionally, we highlight the heritable expression of immunoproteasome 11S regulatory cap subunits as a potential mechanism aiding AML cell survival, proliferation, and immune escape in vivo. Finally, we validate the clinical relevance of these signatures in the TARGET-AML cohort, with a bisected response in blood and bone marrow. Our findings reveal a broad spectrum of resistance signatures attributed to significant cell transcriptional changes. To our knowledge, this is the first application of dynamic lineage tracing to unravel treatment response and resistance in cancer, and we expect FLARE to be a valuable tool in dissecting the evolution of resistance in a wide range of tumor types.

cancer biology↗

bioRxiv2024

Therapeutic window for treatment of prion disease defined by timed depletion of Prion protein

There is no effective treatment preventing the progression of neurodegenerative diseases such as prion and Alzheimers diseases. These fatal diseases of the central nervous system, involve progressive accumulation of a misfolded protein long before overt clinical signs of disease. Removal of prion protein early in the pathological process appears to halt the progression however, it is not known whether intervention at later disease stages could be effective. We investigated the potential for intervention throughout the course of prion disease, by developing a mouse model in which Prnp expression can be manipulated in a tissue specific and time dependent manner. Depleting Prnp from neuronal populations in CNS throughout the preclinical phase substantially prolonged incubation. The pathology was dramatically altered to a pattern of astrocytic associated prion deposition. However once overt clinical symptoms of disease were apparent Prnp depletion did not alter disease progression. This study establishes a wide window for intervention, and suggests timely treatment could delay the onset of clinical disease potentially well beyond the lifetime of an individual.

neuroscience↗

bioRxiv2024

Antibiotic treatment induces microbiome dysbiosis and reduction of neuroinflammation following traumatic brain injury in mice.

Traumatic brain injury (TBI) induces both neuroinflammation and gut microbiome dysbiosis, yet the influence of antibiotics (ABX) on TBI-related neuropathology remains unclear. We administered a broad-spectrum oral ABX regimen to deplete the gut microbiome in single and repeated TBI mouse models. In male mice, ABX treatment significantly reduced neuroinflammation and neurodegeneration post-TBI, with no effects observed in uninjured controls. ABX also altered microbiome composition and decreased serum and fecal short-chain fatty acid levels, while intestinal damage and dysbiosis were further exacerbated by TBI severity. Notably, germ-free male mice exhibited heightened neuroinflammation and larger lesion volumes following TBI, underscoring the microbiomes essential role in recovery. Metagenomic analyses revealed Parasutterella excrementihominis and Lactobacillus johnsonii as potential ABX-resistant taxa post-injury. These findings suggest that short-term ABX treatment may attenuate TBI-induced neuroinflammation by reshaping the gut microbiome, offering directions for microbiome-targeted therapies in TBI.

neuroscience↗

bioRxiv2024

Probiotic treatment causes sex-specific neuroprotection after traumatic brain injury in mice

BackgroundRecent studies have shed light on the potential role of gut dysbiosis in shaping traumatic brain injury (TBI) outcomes. Changes in the levels and types of Lactobacillus bacteria present might impact the immune system disturbances, neuroinflammatory responses, anxiety and depressive-like behaviors, and compromised neuroprotection mechanisms triggered by TBI. ObjectiveThis study aimed to investigate the effects of a daily pan-probiotic (PP) mixture in drinking water containing strains of Lactobacillus plantarum, L. reuteri, L. helveticus, L. fermentum, L. rhamnosus, L. gasseri, and L. casei, administered for either two or seven weeks before inducing TBI on both male and female mice. MethodsMice were subjected to controlled cortical impact (CCI) injury. Short-chain fatty acids (SCFAs) analysis was performed for metabolite measurements. The taxonomic profiles of murine fecal samples were evaluated using 16S rRNA V1-V3 sequencing analysis. Histological analyses were used to assess neuroinflammation and gut changes post-TBI, while behavioral tests were conducted to evaluate sensorimotor and cognitive functions. ResultsOur findings suggest that PP administration modulates the diversity and composition of the microbiome and increases the levels of SCFAs in a sex-dependent manner. We also observed a reduction of lesion volume, cell death, and microglial and macrophage activation after PP treatment following TBI in male mice. Furthermore, PP-treated mice show motor function improvements and decreases in anxiety and depressive-like behaviors. ConclusionOur findings suggest that PP administration can mitigate neuroinflammation and ameliorate motor and anxiety and depressive-like behavior deficits following TBI. These results underscore the potential of probiotic interventions as a viable therapeutic strategy to address TBI-induced impairments, emphasizing the need for gender-specific treatment approaches.

neuroscience↗

bioRxiv2024

Strategies for Inducing and Validating Zinc Deficiency and Zinc Repletion

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases. New and NoteworthyThis methods paper details 1) dietary approaches to alter zinc homeostasis in rodents, and 2) qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/582542v3_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@15d4cddorg.highwire.dtl.DTLVardef@617214org.highwire.dtl.DTLVardef@188b38forg.highwire.dtl.DTLVardef@1c47526_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

PI3K-dependant reprogramming of hexokinase isoforms controls glucose metabolism and functional responses of B lymphocytes

B lymphocyte metabolic reprogramming is essential for B cell differentiation and mounting a healthy immune response. The PI3K signaling pathway regulates B cell metabolism, but the mechanisms involved are not well understood. Here we report that signaling via PI3K8 can impact B cell glucose metabolism and immune functions via selective upregulation of hexokinase 2 (HK2). Three HK enzymes can catalyze the critical first step for glucose utilization and may selectively direct glucose into specific catabolic and anabolic pathways. While HK1 is constitutively expressed in B cells, HK2 is strikingly upregulated during B cell activation in a PI3K8-dependent manner. HK2 shows a unique distribution between mitochondrial and cytoplasmic pools that is also regulated by PI3K. Genetic deletion of HK2 significantly impairs extracellular acidification rate and glycolytic ATP production despite strong expression of HK1. B cell-specific deletion of HK2 in mice caused mild perturbations in B cell development but did not prevent generation of mature B cell subsets. HK2-deficient B cells show altered functional responses in vitro and evidence of metabolic adaptation to become less dependent on glucose and more dependent on glutamine. HK2-deficient B cells exhibit impaired glycolysis, altered metabolite profiles and altered flux of labeled glucose carbons into the pentose phosphate pathway. Upon immunization, HK2-deficient mice exhibit impaired generation of germinal centre B cells, plasmablasts and antibody responses. We further found that HK2 expression in primary human chronic lymphocytic leukemia (CLL) cells was associated with recent proliferation and could be reduced by PI3K inhibition. Our study identifies hexokinase 2 upregulation as a functionally important component of B cell metabolic reprogramming dependent on the PI3K pathway.

immunology↗

bioRxiv2023

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/566502v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1d2be0eorg.highwire.dtl.DTLVardef@bba6caorg.highwire.dtl.DTLVardef@1c335e5org.highwire.dtl.DTLVardef@10789c9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Age-related skeletal muscle atrophy shows morphological alterations in mitochondrial structure associated with declining function. Our findings propose that exercise intervention may counteract these structural declines by reinstating levels of mitofusin 2, thus highlighting a potential mechanism by which exercise attenuates age-induced mitochondrial dysfunction. C_FIG

biophysics↗

bioRxiv2023

CFL1-dependent dynamicity of surface ectoderm filopodia-like protrusions increases neurulation zippering speed in mice

Epithelial fusion is critical for formation of many embryonic tissues. The developing mammalian spinal cord fuses by zippering, through cell behaviours dependent on F-actin turnover. We propose a caudal-to-rostral sequence of surface ectoderm cell behaviours which drive zippering progression in mice, and test the requirement for the F-actin severing protein CFL1 in this process. Key zipper-advancing behaviours are i. constriction of supracellular actomyosin cables; ii. extension of short-lived (<2 min) filopodial or more persistent lamellipodial protrusions, which we live-image establishing pioneering contacts across the midline; remodelling of iii. cell-cell and iv. cell-ECM adhesions; v. zipper advancement through constriction of leading-edge cell borders. In wildtype embryos CFL1 is enriched at the leading edge of surface ectoderm cells adjacent to the zippering point and localised within some protrusions. Conditional Cfl1 deletion in the surface ectoderm produces more prominent F-actin stress fibres, makes filopodial protrusions excessively stable, diminishes their exploratory movements which may establish nascent contacts, and impairs junctional constriction of cells at the point of fusion. Zippering speed is reduced [~]30% in conditional Cfl1-deleted embryos and [~]30% develop spina bifida. We therefore propose that impaired filopodial dynamicity limits the sequence of cell behaviours driving spinal zippering in mice, predisposing to spina bifida.

developmental biology↗

bioRxiv2023

Towards establishing a 24-hour, microplate-based, transcriptomics assay for rainbow trout embryos

There is interest in the development of early-life stage (ELS) tests with fish embryo models that are high-throughput and can generate transcriptomics point of departure (tPOD) values. The objective of this study was to establish a method in rainbow trout (Oncorhynchus mykiss) hatchlings that could satisfy both of these interests. We based our pilot method on recent efforts by U.S. EPA researchers to establish a larval fathead minnow high throughput transcriptomics assay. Here, 1-2 day post hatch trout were assayed in 24-well plates in which they were exposed for 24 hours to 12 different concentrations of test chemicals, including a negative control (DMSO, culture water). Test concentrations were anchored with a chemicals LC50 data from the US EPA ECOTOX database and EnviroTox database, and from this, concentrations were spaced on a half-log basis that spanned 6-7 orders of magnitude. In pilot study 1 we tested 3,4-dichloroaniline, CuSO4 (0.34 mg/L), and ethinylestradiol. In pilot study 2 we tested 3,4-dichloroaniline (58.5 mg/L), CuSO4 (0.34 and 0.41 mg/L), ethinylestradiol (>10 {micro}g/L), permethrin (>10 {micro}g/L), malathion (0.61 mg/L), 6PPD quinone (5.6 {micro}g/L), acetaldehyde (41.2 mg/L), 4-fluoroaniline (242.7 mg/L), glyphosate ([~]150 mg/L), ethanol (>1 g/L), thiamethoxam (>300 mg/L), and allyl alcohol (>30 mg/L). In both pilot studies derived LC50 values are provided in parentheses. Repeated studies of CuSO4 yielded consistent LC50 values (0.34, 0.34, 0.41 mg/L). The correlation between LC50s from the current study for rainbow trout embryos versus those from the literature on adult rainbow trout for 7 chemicals was r2 = 0.91. Work is underway to optimize transcriptomics assays from these samples using EcoToxChips and UPXome, with the ultimate goal to be able to derive transcriptomics points of departure. Taken together these results provide a foundation towards establishing a novel testing platform for chemical and environmental risk assessment that is much quicker (24 hrs), ethical (non-protected life stages), resource efficient (e.g., microplate-based, small volumes of chemicals), and more informative (molecular clues into MOA) than traditional bioassay approaches.

pharmacology and toxicology↗

bioRxiv2023

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/540609v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@96099borg.highwire.dtl.DTLVardef@50b4f4org.highwire.dtl.DTLVardef@983092org.highwire.dtl.DTLVardef@19ceb9b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Overview of serial block facing-scanning electron microscopy (SBF-SEM) workflow, data segmentation, and 3D analysis of mitochondria using Amira software for murine interscapular BAT. C_FIG

biophysics↗