Biology subjects

Mann, J.

Publications and source records attributed to Mann, J..

16 recordsLinked to original sources

bioRxiv2026

Single-cell RNA-seq reveals conserved and divergent cellular states across wound types and species

Chronic wounds, such as diabetic foot ulcers, fail to progress through the normal healing process and impose a significant burden on healthcare systems. While previous single-cell studies have characterized specific wound conditions, a unified understanding of the shared and distinct cellular landscapes across diverse wound microenvironments has been lacking. Therefore, we integrated over 500,541 cells from patients and mice across multiple wound conditions, including acute wound, diabetic foot ulcer, and venous ulcer as well as their healing outcome. Fibroblast-focused analysis identified a bifurcation in differentiation trajectories and identified STAT3 as a potential regulator of a reparative program in chronic wound. Furthermore, we discovered immune dysfunctions in non-healed chronic wounds, contrasting the quiescent memory-like T cells and TIMP1+ macrophages in healed chronic wounds with the exhausted T cells and foamy SPP1+ macrophage enriched in non-healed chronic wounds. Finally, we translated these results into a clinically applicable three-gene signature (CHI3L1, TIMP1, and SPP1) that accurately predicts chronic wound healing. To support wound biology community, we developed WoundSCAtlas, an interactive web resource for exploring diverse wound pathologies. In conclusion, this work provides a comprehensive and cross-species landscape of chronic wound healing, identifying conversed wound outcome-associated molecular programs, predictive biomarker, and interactive data resource.

bioinformatics↗

bioRxiv2026

How many genes can CRISPR edit to engineer complex adaptations?

Polygenic traits require the coordinated effects of multiple genes. Such complex traits have been a long-term target of study for geneticists, but multiplex CRISPR--the editing of multiple loci in the genome via multiple guide RNAs--is in its infancy. Reviewing 106 plant studies using multiplex CRISPR, we find that the multiplexing capacity has doubled every 5.4 years. Furthermore, a systematic experiment with 8, 16, and 24 simultaneous targets in Arabidopsis thaliana reveals efficiency of up to 75% in 24-plex editing in transformed plants confirmed by sequencing. We surprisingly found that the level of multiplexing, or the number of the targets, causes lesser efficiency reductions than other uncontrolled factors such as gRNA design or natural variation across plants. In fact, mathematical modeling of the decay in editing efficiency as a function of the gRNA numbers, showed a logistic model with sustained efficiency fits the data better than decay due to Cas9 competition or random editing. We then project that editing close to 100 genes in a plant can be feasible with reasonably large plant screens. However, feasible and reliable polygenic genome engineering will need developments outside of the CRISPR editing machinery itself, including innovations in gRNA vector delivery for large cargos, and a broader conceptual shift toward population-level poly-gene editing leveraging large distributions of mutations for breeding, natural selection, or experimental evolution. Author ContributionsM.E.-A. conceived the project and secured funding. M.E.-A. and M.Es. designed the experimental strategy. M.Es. established the multiplex CRISPR and transformation pipelines in the laboratory, propagation through the T1 and T2 generations, and oversaw the first amplicon sequencing. Y.P. established the in-house iSeq amplicon sequencing protocol and contributed to cloning and genotyping pilots. M.Es supervised K.P. to construct cloning, bacterial transformations, plant growth, floral-dip transformations, and selection of T1 plants. E.K. contributed to early amplicon genotyping. J.K. propagated and sampled the T3 and J.K. and J.M. conducted the final amplicon sequencing panel. J.K. and M.E.-A. performed gene editing variant mapping, dataset quality control, and summarized results from published multiplex CRISPR studies. M.E.-A. modeled editing efficiency. J.K and M.E.-A. generated figures and wrote the first draft. All authors revised and improved the manuscript. J.K. and M.Es. contributed equally to this work and are designated as co-first authors.

plant biology↗

bioRxiv2025

Machine Learning Enables Rapid Assessment of Disease Vulnerability in a Threatened Cetacean Population

Cetaceans (whales and dolphins) are important ecosystem sentinels but face growing threats from major disease-related mortality events expected to intensify under climate change. Because both environmental factors (temperature, salinity) and demographics (age, sex) influence health and disease risk, understanding these relationships is essential for effective management. Direct health assessments are challenging in cetaceans, but skin lesions can indicate active infection and tooth-rake marks reflect social stressors that increase transmission risk. Yet, traditional photographic analysis of these indicators is inefficient, creating processing bottlenecks that limit timely evaluation of population health. To address this gap, we applied machine learning to rapidly assess lesions and rake marks in Tamanends bottlenose dolphins (Tursiops erebennus) photographed in the Chesapeake Bay, a known hotspot for disease-related die-offs. This represents the first analysis of environmental and demographic contributions to dolphin health in this region. We found significant negative relationships between lesion prevalence and both temperature and salinity for some lesion types. Adult males also showed higher rake mark coverage than adult females and calves. These patterns suggest dolphins in colder, fresher waters may face elevated disease risk, while adult males may be particularly vulnerable to behavioral stress and related health consequences. Our findings are consistent with prior studies, lending validity to our machine learning models, while also revealing novel patterns of calf and male vulnerability in this threatened population. More broadly, our approach demonstrates the potential of automated image analysis to enable timely, non-invasive health assessments across cetacean populations in an era of rapid global change.

ecology↗

bioRxiv2025

Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cell death

The therapeutic efficacy of anticancer therapeutics is limited by acquired drug resistance of tumor cells. Here, we aim to characterize and overcome resistance mechanisms of ovarian cancer cells to the anthracycline derivative doxorubicin (Doxo). To this end, comparative analyses of Doxo-induced stress responses of parental A2780 and Doxo-resistant A2780ADR variant were performed. A2780ADR cells revealed cross-resistance to multiple compounds, including anticancer drugs (cisplatin (CisPt), etoposide (Eto)) and DNA repair/ DNA damage response (DDR) inhibitors (olaparib, niraparib, entinostat, prexasertib, rabusertib). A2780ADR cells formed significantly less DNA double-strand breaks (DSB) after Doxo exposure as compared to A2780, resulting in a mitigated DDR, reduced proliferation inhibition and attenuated apoptosis. Potential resistance mechanisms identified to contribute to Doxo resistance of A2780ADR cells include increased Doxo efflux due to increased MDR1 expression and reduced topoisomerase II protein expression. Substantial resensitization of A2780ADR cells to Doxo was achieved by both the RAC1 GTPase inhibitor EHT1864, the histone deacetylase inhibitor entinostat (Est) and, most effectively, the calcium channel blocker verapamil (Ver). Notably, Ver-mediated sensitization also pertains to Eto and CisPt. The synergistic effect of Ver in combination with Doxo, which is reflected by low combination index (CI), likely involves inhibition of MDR1-mediated drug transport, leading to increased intracellular steady state levels of Doxo and elevated DNA damage formation, eventually promoting pro-apoptotic DDR. However, combination treatment with Doxo and Ver also increased the cytotoxic response of non-malignant murine cardiomyocytes (HL-1), murine embryonic stem cells (mESC) and human induced pluripotent stem cells (hiPSC). Based on our data we suggest inhibition of MDR1-mediated Doxo efflux by Ver as a useful approach to overcome acquired drug resistance of A2780ADR cells because it promotes DDR-related pro-death mechanisms, yet at the price of a potentially increased risk of normal tissue toxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/650268v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@43c0e5org.highwire.dtl.DTLVardef@2d1c5forg.highwire.dtl.DTLVardef@1a1d97borg.highwire.dtl.DTLVardef@cd4522_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Verapamil-mediated resensitization of Doxo-resistant tumor cells.As concluded from our data we suggest that verapamil increases the anticancer efficacy of doxorubicin (Doxo) in a synergistic manner in anticancer drug resistant ovarian A2780ADR cells. This is likely due to inhibition of MDR1-mediated drug export by Ver, leading to higher intracellular steady-state concentrations of Doxo. In consequence, Doxo-mediated Topo II poisoning is promoted, eventually causing increased DNA damage (DSB) formation and activation of DDR-related signaling mechanims, which in turn impair cell proliferation and stimulate cell death. Apart from verapamil, inhibition of Rac1 GTPase-regulated signaling by EHT1864 and inhibition of HDACs class I by the HDACi entinostat are also useful to overcome Doxo resistance of A2780ADR cells, yet with the exact molecular mechanisms involved being still unclear. C_FIG

pharmacology and toxicology↗

bioRxiv2025

Evaluation of Candidate ''Kill or Cure'' Strategies to Treat MFN2-related Lipodystrophy

The R707W mutation in mitofusin 2, encoded by MFN2, causes a form of Multiple Symmetrical Lipomatosis (MFN2-MSL). This resembles sporadic, alcohol-associated MSL, combining loss of lower body adipose tissue with upper body adipose hyperplasia. Morbidity and sometimes mortality arise both from mechanical complications of head and neck adipose overgrowth, and metabolic complications. We reasoned that interventions that either mitigate the underlying cellular pathology, or that exacerbate it to induce selective death of hyperplastic adipose tissue may be beneficial. We thus assessed the effect of a metabolic or pharmacologic stressors or rapamycin in Mfn2R707W/R707W mice and or derived preadipocytes. 50mmol ethanol had little effect on WT or Mfn2R707W/R707W white preadipocytes, but increased mitochondrial content and blunted mitolysosome formation in Mfn2R707W/R707Wbrown preadipocytes. Daily consumption of 20% EtOH increased brown adipose tissue mass in female Mfn2R707W/R707W mice, and serum lactate in males. 200nM rapamycin - a candidate treatment - increased size and mitolysosome content of WT and Mfn2R707W/R707W white and brown preadipocytes, but these effects were blunted in Mfn2R707W/R707W cells. In male but not female Mfn2R707W/R707W mice, rapamycin reduced or reversed weight gain, reduced brown adipose mass, and increased serum Fgf21. Finally, a panel of other metabolic and pharmacological mitochondrial stressors solicited no selective death or ISR in Mfn2R707W/R707W preadipocytes. We conclude that ethanol mildly exacerbates MFN-MSL in mice, while rapamycin is tolerated. Lack of sensitisation to mitochondrial stressors implies that the MSL-inducing effect of MFN2 R707W may not be exerted through compromised oxidative phosphorylation.

physiology↗

bioRxiv2024

A self-limiting Sterile Insect Technique alternative for Ceratitis capitata

Genetic biocontrol systems have broad applications in population control of insects implicated in both disease spread and food security. In this study we establish and characterise a novel split-CRISPR/Cas9 system we term Sex Conversion Induced by CRISPR (SCIC) in Ceratitis capitata (the Mediterranean fruit fly), a major agricultural pest with a global distribution. Using the white eye gene for toolkit selection, we achieved up to 100% CRISPR/Cas9 efficiency, displaying the feasibility of C. capitata split-CRISPR/Cas9 systems using constitutive promoters. We then induce sex-conversion by targeting the transformer gene in a SCIC approach aimed for SIT-mediated releases upon radiation-based sterilisation. Knock-out of transformer induced partial to full female-to-male sex-conversion, with remaining individuals all being intersex and sterile. SCIC population modelling shows superior performance to traditional population control strategies, allowing for faster population elimination with fewer released sterile males. Our results build the foundation for further genetic pest control methods of C. capitata and related tephritid agricultural pests. Significance statementAgricultural industry faces increasing threat from a multitude of pests including the domineering tephritid fruit flies. Genetic engineering of these pests has been recently tested to develop more efficient and affordable population control strategies. Here, we develop a new approach to improve existing population control measures by testing it in one of the most famous and dangerous tephritids, the Mediterranean fruit fly. Through optimisation, we achieved desired outcomes: female fly absence achieved via semi and full female-to-male sex conversion by CRISPR-mediated genome editing through gene mutations. For the first time in this insect, we used a split, and thus inducible, approach for such genome editing. Our work holds the potential to significantly improve tephritid population control strategies.

genetics↗

bioRxiv2024

Seasonal contact and migration structure mass epidemics and inform outbreak preparedness in bottlenose dolphins

Infectious respiratory diseases have detrimental impacts across wildlife taxa, particularly in marine species. Despite this vulnerability, we lack information on the complex spatial and contact structures of marine populations which reduces our ability to understand disease spread and our preparedness for epidemic response. We leveraged a collated dataset to establish the first data-driven epidemiological model on a cetacean species, the Tamanends bottlenose dolphin (Tursiops erebennus), whose populations are periodically impacted by deadly respiratory disease in the northwest Atlantic. We found their spatial distribution and contact is heterogeneous along the coastline and varies by ecotype, which explains differences in infection burdens observed in past outbreaks. We also determined that outbreaks beginning in northern parts of their habitat during migratory seasons have the highest epidemic risk and that dolphins in North Carolina estuaries would be the best sentinels for disease surveillance. Our mathematical model provides a generalizable, non-invasive tool that takes advantage of routinely collected marine mammal data to mechanistically understand disease transmission and inform disease surveillance tactics for marine sentinels. Our findings highlight the heterogeneities that play a crucial role in shaping the impacts of infectious diseases in wildlife, and how a data-driven understanding of these mechanisms can enhance epidemic preparedness.

ecology↗

bioRxiv2024

Automated Skin Lesion Detection and Prevalence Estimation in Tamanend's Bottlenose Dolphins

Anthropogenic global change is occurring at alarming rates, leading to increased urgency in the ability to monitor wildlife health in real time. Monitoring sentinel marine species, such as bottlenose dolphins, is particularly important due to extensive anthropogenic modifications to their habitats. The most common non-invasive method of monitoring cetacean health is documentation of skin lesions, often associated with poor health or disease, but the current methodology is inefficient and imprecise. Recent advancements in technology, such as machine learning, can provide researchers with more efficient ecological monitoring methods to address health questions at both the population and the individual levels. Our work develops a machine learning model to classify skin lesions on the understudied Tamanends bottlenose dolphins (Tursiops erebennus) of the Chesapeake Bay, using manual estimates of lesion presence in photographs. We assess the models performance and find that our best model performs with a high mean average precision (65.6%-86.8%), and generally increased accuracy with improved photo quality. We also demonstrate the models ability to address ecological questions across scales by generating model-based estimates of lesion prevalence and testing the effect of gregariousness on health status. At the population level, our model accurately estimates a prevalence of 72.1% spot and 27.3% fringe ring lesions, with a slight underprediction compared to manual estimates (82.2% and 32.1%). On the other hand, we find that individual-level analyses from the model predictions may be more sensitive to data quality, and thus, some individual scale questions may not be feasible to address if data quality is inconsistent. Manually, we do find that lesion presence in individuals suggests a positive relationship between lesion presence and gregariousness. This work demonstrates that object detection models on photographic data are reasonably successful, highly efficient, and provide initial estimates on the health status of understudied populations of bottlenose dolphins.

ecology↗

bioRxiv2024

A Human Brain Map of Mitochondrial Respiratory Capacity and Diversity

Mitochondrial oxidative phosphorylation (OxPhos) powers brain activity1,2, and mitochondrial defects are linked to neurodegenerative and neuropsychiatric disorders3,4, underscoring the need to define the brains molecular energetic landscape5-10. To bridge the cognitive neuroscience and cell biology scale gap, we developed a physical voxelization approach to partition a frozen human coronal hemisphere section into 703 voxels comparable to neuroimaging resolution (3x3x3 mm). In each cortical and subcortical brain voxel, we profiled mitochondrial phenotypes including OxPhos enzyme activities, mitochondrial DNA and volume density, and mitochondria-specific respiratory capacity. We show that the human brain contains a diversity of mitochondrial phenotypes driven by both topology and cell types. Compared to white matter, grey matter contains >50% more mitochondria. We show that the more abundant grey matter mitochondria also are biochemically optimized for energy transformation, particularly among recently evolved cortical brain regions. Scaling these data to the whole brain, we created a backward linear regression model integrating several neuroimaging modalities11, thereby generating a brain-wide map of mitochondrial distribution and specialization that predicts mitochondrial characteristics in an independent brain region of the same donor brain. This new approach and the resulting MitoBrainMap of mitochondrial phenotypes provide a foundation for exploring the molecular energetic landscape that enables normal brain functions, relating it to neuroimaging data, and defining the subcellular basis for regionalized brain processes relevant to neuropsychiatric and neurodegenerative disorders.

neuroscience↗

bioRxiv2023

Breathing in sync: how a social behavior structures respiratory epidemic risk in bottlenose dolphins

Dolphin morbillivirus has caused mass mortalities in dolphin populations globally. Given their role as ecosystem sentinels, mass mortalities among these populations can be detrimental. Morbillivirus is transmitted through respiratory droplets and occurs when dolphins breathe synchronously, a variable social behavior. To assess the role of variable social behavior on disease risk empirically, we collected behavioral data from two wild bottlenose dolphins populations (Tursiops spp.), developed network models that synthesize transmission contacts, and used an epidemiological model to predict disease consequences. We find that juveniles have more contacts than adults, adult males have more contacts than adult females, and that individuals preferentially contact others in their own demographic group. These patterns translate to higher infection risk for juveniles and adult males, which we support using data from a morbillivirus outbreak. Our work characterizes the impact of bottlenose dolphin social dynamics on infectious disease risk and informs the structure of vulnerability for future epizootics.

ecology↗

bioRxiv2023

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

biochemistry↗

bioRxiv2023

UCHL1-dependent control of Hypoxia-Inducible Factor Transcriptional Activity in Liver Disease.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver diseases. Fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast like phenotype that is contractile, proliferative and profibrogenic. Hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. HIF activation during HSC activation can by either due to localised decreases in oxygen levels, or through oxygen-independent mechanisms that are not completely understood. Here we describe a role for the deubiquitinase UCHL1 in regulating HIF levels and activity during HSC activation and liver fibrosis. Increased HIF1 expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1 levels. UCHL1 specifically cleaves the degradative ubiquitin chains from HIF1 leading to increased HIF1 levels, even in sufficiently oxygenated cells. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. These results demonstrate how small molecule inhibitors of DUBs can modulate the activity of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity via modulation of the ubiquitin-proteasomal degradation pathway may represent a therapeutic opportunity with other HIF-related pathologies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/523142v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d82ee9org.highwire.dtl.DTLVardef@1eff21aorg.highwire.dtl.DTLVardef@1ffb0eforg.highwire.dtl.DTLVardef@1b93dc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Changes in Social Position Predict Survival in Bottlenose Dolphins

1Social bonds and social structure are important features of animal systems that impact individual fitness. Few studies have examined how temporal dynamics in individual social bonds predict fitness outcomes. This is critical to understand given the high variation in types of social structures and strategies within populations of social mammals, both across time and among individuals. If individually-differentiated social bonds are important, it might be the change in bonds which has fitness consequences, not the absolute number of bonds nor their strength. We investigated how network dynamics predict survival in a wild population of bottlenose dolphins using a 35-year longitudinal study. In particular, we were interested in two sex-specific measures of the "widowhood effect", as well as a more general investigation into the predictability of mortality from changes in higher-order social network metrics. We used two inferential frameworks to provide complimentary evidence for or against hypotheses; namely, a gradient-boosting predictivist approach with relative importance measures, and an inclusion probabilities approach based on stability-selection. We found evidence of a widowhood effect among males but not females. Surprisingly, the most robust predictor of survival was closeness-centrality, whereby the loss of closeness centrality preceded death. This finding, that absolute network position may not be as relevant to survival as changes in network status, is consistent with a large social-psychological literature in humans on the impacts of loss of social capital, dissolution of close friendships, and loss through death of partners (widowhood effect). This study highlights the critical nature of social connections and how its disruption can be a matter of life and death.

animal behavior and cognition↗

bioRxiv2019

mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis.

During postprandial state, the liver is exposed to high levels of dietary fatty acids (FAs) and carbohydrates. FAs are re-esterified into triglycerides, which can be stored in lipid droplets (LDs) in the liver. Aberrant accumulation of LDs can lead to diseases such as alcoholic liver disease and non-alcoholic fatty liver disease, the latter being the most common liver pathology in western countries. Improved understanding of LD biology has potential to unlock new treatments for these liver diseases. The Perilipin (Plin) family is the group of proteins that coat LDs, controlling their biogenesis, stabilization, and preventing their degradation. Recent studies have revealed that autophagy is involved in LD degradation and, therefore, may be crucial to avoid lipid accumulation. Here, we show that a phosphorylated form of Plin3 is required for selective degradation of LDs in fibroblasts, primary hepatocytes and human liver slices. We demonstrate that oleic acid treatment induces the recruitment of the autophagy machinery to the surface of LDs. When Plin3 is silenced, this recruitment is suppressed resulting in accumulation of lipid. Plin3 pulldowns revealed interactions with the autophagy initiator proteins Fip200 and Atg16L indicating that Plin3 may function as a docking protein involved in lipophagy activation. Of particular importance, we define Plin3 as a substrate for mTORC1-dependent phosphorylation and show that this event is decisive for lipophagy. Our study therefore reveals that Plin3, and its phosphorylation by mTORC1, is crucial for degradation of LDs by autophagy. We propose that stimulating this pathway to enhance LD autophagy in hepatocytes will help protect the liver from lipid-mediated toxicity thus offering new therapeutic opportunities in human steatotic liver diseases.

cell biology↗

bioRxiv2018

Length-independent telomere damage drives cardiomyocyte senescence

Ageing is the biggest risk factor for cardiovascular health and is associated with increased incidence of cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate if clearance of senescent cells attenuates age related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction, and crucially can occur independently of cell-division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a and results in a non-canonical senescence-associated secretory phenotype. Pharmacological or genetic clearance of senescent cells in mice alleviates myocardial hypertrophy and fibrosis, detrimental features of cardiac ageing, and promotes cardiomyocyte regeneration. Our data describes a mechanism by which senescence can occur and contribute to ageing in post-mitotic tissues.

cell biology↗

bioRxiv2018

A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices.

Summary boxO_LIWhat is already known about this subject?\nO_LICurrently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.\nC_LIO_LIPreclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.\nC_LIO_LIPrecision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.\nC_LI\nC_LIO_LIWhat are the new findings?\nO_LIWe have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.\nC_LIO_LIBioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.\nC_LIO_LIAdministration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.\nC_LI\nC_LIO_LIHow might it impact on clinical practice in the foreseeable future?\nO_LIThe extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.\nC_LIO_LIBioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.\nC_LI\nC_LI\n\nAbstractO_ST_ABSObjectiveC_ST_ABSPrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.\n\nDesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGF{beta}1 and PDGF{beta}{beta} stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and Smooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.\n\nResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGF{beta}1 and PDGF{beta}{beta} stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGF{beta}1 and PDGF{beta}{beta} stimulated PCLS and fibrotic PCLS.\n\nConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.

cell biology↗