Biology subjects

O'Brien, T.

Publications and source records attributed to O'Brien, T..

17 recordsLinked to original sources

bioRxiv2026

Multi-modal MRI characterisation of vascular remodelling, muscle fibre integrity, and inflammatory recovery in a hindlimb ischaemia mouse model

The hindlimb ischaemia (HLI) mouse model is a widely used preclinical model of chronic limb-threatening ischaemia (CLTI). While CLTI involves complex interactions between impaired perfusion, inflammation and muscle wasting, the standard imaging approach, laser Doppler imaging (LDI), only assesses perfusion. MRI is used clinically to assess neural tracts, inflammation, and perfusion in the brain. We therefore evaluated whether a multimodal MRI approach could longitudinally monitor recovery in the HLI mouse model. Mice underwent MRI three days pre-HLI surgery, and on Days +3 and +7 post-surgery, with histology on Day +7. The MRI detected significant increases in muscle volume and inflammation after HLI surgery, with significant decreases in perfusion, vascular length, and muscle fibre integrity. Overall, MRI can monitor inflammation, muscle fibre integrity, and vascular recovery post-HLI and should be applied in future studies to identify mechanisms of therapeutic recovery in a sequential in vivo analysis without requiring animal sacrifice. O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/739107v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@100e5d1org.highwire.dtl.DTLVardef@1f0188dorg.highwire.dtl.DTLVardef@1dbdf4forg.highwire.dtl.DTLVardef@21d415_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

biophysics↗

bioRxiv2025

An Assessment of the Functional State of Endothelial Colony Forming Cells from Patients with Diabetes Mellitus and Chronic Limb Threatening Ischemia

Chronic limb threatening ischemia (CLTI) is the most severe form of peripheral vascular disease which can lead to amputation with a high associated mortality rate. Endothelial colony forming cells (ECFCs) show potential as a cell therapy to revascularize the limbs of individuals with CLTI. However, autologous ECFCs from patient peripheral blood (PB) have been reported to have a dysfunctional phenotype. We investigated this disease phenotype in individuals with CLTI, with and without diabetes mellitus (DM), to determine ECFC suitability as an autologous cell therapy. PB-ECFCs were isolated from age-matched controls, individuals with DM, and individuals with CLTI, with and without DM. The frequency of isolating ECFCs from this donor cohort was calculated. Furthermore, in vitro characterization assays were performed (growth kinetics, angiogenic properties, and reactive oxygen species (ROS) levels) and compared between donor groups. We report a significantly increased frequency of ECFCs from individuals with CLTI, with and without DM. Furthermore, our results demonstrate no significant disease related effect on the in vitro functional properties of ECFCs between cohorts. However, there is a significantly higher in vitro angiogenic capacity in individuals with DM vs age-matched controls. Our results demonstrate that ECFCs can be isolated in individuals with CLTI, with and without DM, and that ECFC functionality is similar between cohorts. Therefore, if the 70% isolation efficiency from CLTI cohorts is overcome, then autologous PB-ECFCs may be a suitable therapeutic for CLTI. Further analysis is needed to determine the critical quality attributes of ECFCs from this patient population. Significance StatementTo the authors knowledge, this paper shows for the first time that endothelial colony forming cells can be isolated from individuals with chronic limb threatening ischemia, with and without diabetes. Additionally, we show a significantly higher frequency of endothelial colony forming cells isolated from chronic limb threatening ischemia patient cohorts. There is no significant difference in endothelial colony forming cells between age-matched controls and chronic limb threatening ischemia patient with and without diabetes mellitus in vitro, potentially suggesting an autologous approach may be a viable therapeutic option in the future.

cell biology↗

bioRxiv2024

The TRX assay for triplet repeat expansions

Expansion mutations of triplet repeat sequences cause numerous inherited neurological diseases. In some diseases, affected individuals display somatic expansions in affected tissues which have been linked to accelerated disease onset and progression. There is currently considerable interest in developing therapies to slow somatic repeat expansions to delay or block disease onset. In vitro assays are particularly important to evaluate potential therapeutic interventions. Current assays typically use physical methods to monitor triplet repeat lengths within a population of cells. While useful, most of these assays are relatively slow ([~]six weeks) and are somewhat limited in sensitivity to rare events. Here, a new assay, called TRX, is described to monitor CAG*CTG triplet repeat expansions more rapidly and with better sensitivity. TRX uses human tissue culture cells expressing two fluorescent proteins. Red fluorescent protein TagRFP658 is constitutively expressed and serves as an internal control. GFP is expressed in a CAG*CTG repeat length-dependent manner, with longer repeat lengths predicted to give higher green fluorescence intensity. Standard flow cytometry allows quantification of changes in fluorescent signal as a simple readout with <2% sensitivity. Two independently derived cell lines with 63 or 59 CAG repeats yielded similar rates of TRX activity. Cells with increased green fluorescence were observed within one to two weeks of culture, with longer times leading to additional signal. The appearance of green fluorescence was partly dependent on MutS{beta}, the DNA MSH2-MSH3 complex, based on siRNA knockdown of MSH3. However, physical analysis of the CAG*CTG repeat tracts by MiSeq deep sequencing or capillary electrophoresis showed limited changes in the length of the repeat tracts. We conclude that the TRX assay is a promising new tool for monitoring CAG*CTG repeat expansions but that further development of the assay is needed to make it fully useful.

genetics↗

bioRxiv2024

Metformin inhibits nuclear egress of chromatin fragments in senescence and aging

Chronic inflammation is a hallmark of aging and contributes to many age-associated diseases. Metabolic intervention is a strategy to modulate inflammation. However, the connection between inflammation and metabolism during aging remains poorly understood. A mechanism driving chronic inflammation involves cytoplasmic chromatin fragments (CCFs), which appear in senescent cells and aged tissues, activating the cGAS-STING pathway. The size of the CCFs exceeds the capacity of the nuclear pore complex, raising the question of how chromatin fragments enter the cytoplasm. Here, we report that chromatin fragments exit the nucleus via nuclear egress, a membrane trafficking process at the nuclear envelope that shuttles large complexes from the nucleus to the cytoplasm. Inactivating critical nuclear egress ESCRT-III or Torsin proteins results in accumulation of chromatin fragments at the nuclear membrane, thereby impairing the activation of cGAS-STING and senescence-associated inflammation. Notably, nuclear egress of CCFs is inhibited by glucose limitation or metformin treatment. This is due to AMPK phosphorylation and autophagic degradation of the ESCRT-III component, ALIX. Metformin treatment in naturally aged mice downregulates ALIX protein and blocks cGAS activation and chronic inflammation in the small intestine. Together, our study defines a central mechanism linking nutrient sensing and chronic inflammation, two distinct hallmarks of aging, and suggests a new approach to suppress age-associated inflammation by targeting the nuclear egress of chromatin fragments.

cell biology↗

bioRxiv2024

Mapping and characterization of a novel powdery mildew resistance locus (PM2) in Cannabis sativa L.

Breeding genetic resistance to economically important crop diseases is the most sustainable strategy for disease management and enhancing agricultural and horticultural productivity, particularly where the application of synthetic pesticides is prohibited. Powdery mildew disease, caused by the biotrophic fungal pathogen Golovinomyces ambrosiae, is one of the most prevalent threats to the cannabis and hemp industry worldwide. In this study, we used bulk-segregant analysis (BSA) combined with high-throughput sequencing to identify and map a novel single dominant resistance (R) locus (designated PM2), that strongly suppresses powdery mildew infection and sporulation in Cannabis sativa. Histochemical analysis revealed that PM2-induced resistance is mediated by a highly localized hypersensitive response mainly in the epidermal cells of the host. Importantly, genetic markers capable of tracking PM2 resistance in breeding populations were developed using associated SNPs identified in this study.

plant biology↗

bioRxiv2024

Yes, they're all individuals: Hierarchical models for repeat survey data improve estimates of tree growth and size

AbstractO_LIRepeat measurement surveys of tree size are used in forests to estimate growth be-haviour, biomass, and population dynamics. Although size is measured with error, and individuals vary in their growth trajectories, current size-based growth modelling approaches do not usually or fully account for both of these features, and therefore under-utilise available data. C_LIO_LIWe present a new method that leverages the auto-correlation structure of repeat surveys into a hierarchical Bayesian longitudinal growth model. This new structure allows users to correct for measurement error and capture individual-level variation in growth trajectories and parameters. C_LIO_LITo demonstrate the new method we applied it to a sample of tropical tree survey data from long-term monitoring sites at Barro Colorado Island. We were able to reduce estimated error in size and growth, and extract individual-and population-level growth parameter estimates. We used simulation to evaluate the ability of the new method to improve estimates of growth rate and size, and estimate individual and species-level parameters. Our method substantially improved RMSE for growth by an average of 61% compared to existing approaches using pairwise differences; and reduced RMSE in estimated size RMSE compared to observed values in simulated data. Better numerical integration methods (Runge-Kutta 4th order in comparison to Euler and midpoint) provided better estimates of parameters, but did not improve the estimation of size and growth. The choice of a positive growth function eliminated all negative increments without data exclusion. C_LIO_LIOverall, this study shows how we can gain new and improved insights on growth, using repeat forest surveys. Our new method offers improved biomass dynamics estimation through reduced error in sizes over time, coupled with novel information about within-species variation in growth behaviour that is inaccessible with species average models, such as individual parameters for the growth function which allows for relationships between parameters to be considered for the first time. C_LI

ecology↗

bioRxiv2024

Key link between iron and the size structure of three main mesoplanktonic groups (Crustaceans, Rhizarians, and colonial N2-fixers) in the Global Ocean.

In marine ecosystems, critical services like fish production, carbon export, or the delivery of nutrients through N2-fixation rely heavily on the size spectrum of pelagic organisms, particularly mesoplankton (200-20,000 {micro}m). However, how environmental factors shape mesoplankton spectral biogeography remains largely unresolved, as so far only limited datasets exist to understand the large-scale shifts in mesoplankton size. Using global compilations of Rhizarian, colonial N2-fixer, and Crustacean images, we reveal the paramount role of iron in shaping the size structure and related biogeography of these groups. Our findings underscore the importance of atmospheric sources of iron for N2-fixers and Rhizarians while total iron, accounting for organic and inorganic compounds, appeared to explain most of the variance in Crustacean size structure via apparent recycling. With a comprehensive set of explanatory variables, our models reached high R2 (0.93, 0.61, and 0.69 respectively), providing robust predictions of mesoplankton size structure related to elemental cycling and ecosystem services. Our results suggest that future increases in global temperatures will have negative effects on mesoplankton size, possibly limiting carbon export from the productive layers to sequestration depth, that can be offset by expected increases in iron inputs that benefit N2-fixers, Rhizarians, and eventually Crustaceans.

ecology↗

bioRxiv2023

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study

ObjectiveThe intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current cortico-centric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural MRI in 1,602 adults with epilepsy and 1,022 healthy controls across twenty-two sites from the global ENIGMA-Epilepsy working group. MethodsA state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in i) all epilepsies; ii) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS); iii) non-lesional temporal lobe epilepsy (TLE-NL); iv) genetic generalised epilepsy; and (v) extra-temporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. ResultsAcross all epilepsies, reduced total cerebellar volume was observed (d=0.42). Maximum volume loss was observed in the corpus medullare (dmax=0.49) and posterior lobe grey matter regions, including bilateral lobules VIIB (dmax= 0.47), Crus I/II (dmax= 0.39), VIIIA (dmax=0.45) and VIIIB (dmax=0.40). Earlier age at seizure onset ({eta}{rho}2max=0.05) and longer epilepsy duration ({eta}{rho}2max=0.06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. SignificanceWe provide robust evidence of deep cerebellar and posterior lobe subregional grey matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in non-motor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellum subregions into neurobiological models of epilepsy. Key pointsO_LICerebellar involvement in epilepsy is poorly understood within current cortico-centric models of this disease C_LIO_LIWe used a novel, deep learning segmentation tool to parcellate the cerebellum into 28 anatomical subunits using an international MRI dataset of 1,602 individuals with epilepsy (aged between 18 and 65 years old) including temporal lobe epilepsy with hippocampal sclerosis (TLE-HS, n=562), TLE non-lesional (TLE-NL, n=284), generalised genetic epilepsy (GGE, n=186) and extra temporal focal epilepsy (ETLE, n=251) and 1,022 controls. C_LIO_LIAcross all epilepsies (vs. controls) robust changes in the corpus medullare and posterior lobe "non-motor" regions were observed, with maximal differences in bilateral VIIB and Crus II lobules. Lower volume of these regions correlated with longer disease duration. Anterior "motor lobe" regions were relatively spared. C_LIO_LIFindings were most pronounced in TLE-HS and ETLE groups, with distinct neuroanatomical profiles observed. C_LIO_LICortical thinning was associated with pronounced cerebellar volume loss in TLE-HS epilepsy, relative to controls. C_LI ETHICAL PUBLICATION STATEMENTWe confirm that we have read the Journals position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

neuroscience↗

bioRxiv2023

Rapamycin mitigates Valproic Acid-induced teratogenicity in human and animal models by suppressing AP-1-mediated senescence

Valproic acid (VPA) is an effective and widely used anti-seizure medication but is teratogenic when used during pregnancy, affecting brain and spinal cord development for reasons that remain largely unclear. Here we designed a genetic recombinase-based SOX10 reporter system in human pluripotent stem cells that enables tracking and lineage tracing of Neural Crest cells (NCCs) in a human organoid model of the developing neural tube. We found that VPA induces extensive cellular senescence and promotes mesenchymal differentiation of human NCCs at the expense of neural lineages. We next show that the clinically-approved drug, Rapamycin, inhibits AP1-mediated senescence and restores aberrant NCC differentiation trajectory in human organoids exposed to VPA. Notably, in vivo validation in developing zebrafish highlighted the therapeutic promise of this approach. Collectively our data identifies a novel mechanism for VPA-associated neurodevelopmental teratogenicity and a potential pharmacological preventative strategy. The results exemplify the power of genetically modified human stem cell-derived organoid models for drug discovery and safety testing.

neuroscience↗

bioRxiv2023

Cell Type-Specific Regulation by a Heptad of Transcription Factors in Human Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts and TF binding profiles in HSPC subsets (HSC, CMP, GMP, MEP) and found that heptad occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. These findings suggest that specific heptad-TF combinations play critical roles in regulating hematopoietic differentiation and provide a valuable resource for development of targeted therapies to manipulate specific HSPC subsets.

developmental biology↗

bioRxiv2023

Design and Validation of an Open Loop Controlled Positive-Displacement Pump for Vascular Flow Modelling

Realistic vascular flow-rates are highly patient dependent. There is a need, when evaluating vascular medical devices, to study variable physiological flow-rates corresponding to specific patients. This study presents an experimental analysis of a positive-displacement piston pump designed for generating patient-specific vascular flow-rate wave-forms. Open loop control is used to drive a positive-displacement piston pump according to user-input physiological wave-forms. Transient and frequency analyses are performed for simple and complex inputs. Theoretical analysis of the system shows that it may be modelled as a linear first order system and the response of the system for various inputs shows that the real system approximates a linear first order response, however, higher order effects are noted. These higher order effects do not have any particular detrimental effect on the generation of physiologically realistic flow-rate waveforms.

bioengineering↗

bioRxiv2022

Altered lipid homeostasis underlies selective neurodegeneration in SNX14 deficiency

Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that the cerebellar neurodegeneration caused by SNX14 deficiency is associated with lipid metabolism defects. Recent in vitro and in silico studies indicate that SNX14 is an inter-organelle lipid transfer protein that regulates lipid droplet biogenesis and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates the human SNX14 deficiency at genetic and phenotypic level. Through histological and transcriptomic analyses, we demonstrate that cerebellar Purkinje cells are selectively vulnerable to SNX14 deficiency, while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14 deficient mice. Furthermore, we identify a unique lipid metabolite profile that links the accumulation of acylcarnitines with the selective cerebellar neurodegeneration in SNX14 deficiency. These findings highlight the importance of lipid homeostasis for neuronal function and survival and suggest a mechanism for selective cerebellar vulnerability to altered lipid homeostasis.

neuroscience↗

bioRxiv2022

Multicentre comparison of biological and functional properties of mesenchymal stromal cells from different sources cultivated using a harmonised manufacturing workflow

BackgroundMesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A-to assess the impact on MSC characteristics when different laboratories performed analysis on the same MSC material using harmonised culture conditions and B-to understand source-specific differences. MethodsThree independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). ResultsBy implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. ConclusionThese results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of standardised culture procedures can reduce but not eliminate inter-lab and operator differences. HighlightsIn this study, we have: - Provided a harmonised manufacturing workflow that has demonstrated reproducible results across three independent laboratories when expanding MSCs. - Defined a multi-assay matrix capable of identifying functional differences in terms of angiogenesis, wound healing abilities and immunosuppressive properties. - Demonstrated similar in vivo biodistribution properties regardless of cell origin.

cell biology↗

bioRxiv2022

Disease-modifying effects of sodium selenate in a model of drug-resistant, temporal lobe epilepsy.

There are no pharmacological disease-modifying treatments that can mitigate the seizures and comorbidities associated with established chronic temporal lobe epilepsy (TLE). This study evaluated the effect of sodium selenate in the post-status epilepticus (SE) rat model of chronic drug resistant TLE. Wistar rats underwent kainic acid-induced SE or sham. Ten-weeks post-SE, rats were randomly assigned to receive either sodium selenate, levetiracetam, or vehicle treatments continuously for 4 weeks. To evaluate the effects of the treatments, 1 week of continuous video-EEG was acquired before, during, and 4, 8 weeks post-treatment, followed by behavioral tests. Targeted and untargeted proteomics and metabolomics were performed on post-mortem brain tissue to identify potential pathways associated with modified disease outcomes. Telomere length was investigated as a novel surrogate marker of disease severity. Sodium selenate treatment was able to mitigate disease severity, reducing the number of spontaneous seizures (p< 0.05), cognitive dysfunction (p< 0.05 in both novel object placement and recognition tasks), and sensorimotor deficits (p< 0.01) 8 weeks post-treatment cessation. Moreover, increased protein phosphatase 2A (PP2A) expression, reduced hyperphosphorylated tau, and reversed telomere length shortening caused by SE (p< 0.05). Network medicine integration of multi-omics/ pre-clinical outcomes identified protein-metabolite modules positively correlated with the TLE phenotype. Our results provide evidence that treatment with sodium selenate results in a sustained disease modifying effect in chronically epileptic rats in the post-KA SE model of TLE, including improved comorbid learning and memory deficits.

neuroscience↗

bioRxiv2021

Megapixel camera arrays for high-resolution animal tracking in multiwell plates

Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. Current imaging systems are limited either in spatial resolution or throughput. A system capable of imaging a large number of animals with sufficient resolution to estimate their pose would enable a new class of experiments where detailed behavioural differences are quantified but at a scale where hundreds of treatments can be tested simultaneously. Here we report a new imaging system consisting of an array of six 12-megapixel cameras that can simultaneously record from all the wells of a 96-well plate with a resolution of 80 pixels/mm at 25 frames per second. We show that this resolution is sufficient to estimate the pose of nematode worms including head identification and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines and greatly increases the scale of possible phenotypic screening experiments in C. elegans.

animal behavior and cognition↗

bioRxiv2020

Temporal Changes Guided by Mesenchymal Stem Cells on a 3D Microgel Platform Enhances Angiogenesis In Vivo at a Low-Cell Dose

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival and the non-maintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behaviour including migration, proliferation and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of pre-conditioning human MSCs for 96 hours on a three-dimensional ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the pro-angiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and outside-in integrin signaling. The microgels tested at a low-cell dose (5x104 cells) in a pre-clinical hindlimb ischemia model showed accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were upregulated in the low-cell dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low-cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in no-option patients suffering from peripheral arterial diseases such as Critical Limb Ischemia (CLI).

bioengineering↗

bioRxiv2020

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Nuclei are constantly under external stress - be it during migration through tight constrictions or compressive pressure by the actin cap - and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

biophysics↗