Biology subjects

Brown, H.

Publications and source records attributed to Brown, H..

6 recordsLinked to original sources

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↗

bioRxiv2018

Enhanced Neurite Outgrowth and Regeneration in ALS Resistant Motor Neurons from SOD1 Mutant Mouse Models

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by motor neuron cell death. However, not all motor neurons are equally susceptible. Most of what we know about the surviving motor neurons comes from gene expression profiling, less is known about their functional traits. We found that resistant motor neurons cultured from SOD1 ALS mouse models have enhanced axonal outgrowth and dendritic branching. They also have an increase in the number and size of actin-based structures like growth cones and filopodia. These phenotypes occur in cells cultured from presymptomatic mice and mutant SOD1 models that do not develop ALS, but not in embryonic motor neurons. Enhanced outgrowth and upregulation of filopodia can be induced in wild-type adult cells by expressing mutant SOD1. These results demonstrate that mutant SOD1 can enhance the regenerative capability of ALS resistant motor neurons. Capitalizing on this mechanism could lead to new therapeutic strategies.

neuroscience↗

bioRxiv2018

Loss of charge at solvent exposed Lys residues does not induce the aggregation of superoxide dismutase 1

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce the protein to misfold and aggregate. To date, missense mutations at more than 80 different amino acid positions have been associated with disease. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. One potential mechanism that has been suggested is that when mutations occur at positions occupied by charged amino acids, then repulsive forces that would inhibit aberrant protein:protein interactions would be reduced. Mutations at twenty-one charged residues in SOD1 have been associated with fALS. Here, we examined whether loss of positively charged surface Lys residues would induce the misfolding and aggregation of SOD1. We randomly mutated four different Lys residues (K30, K36, K75, K91) in SOD1 and expressed these variants as fusion proteins with yellow fluorescent protein (YFP). We also assessed whether these mutations induced binding to a conformation-restricted SOD1 antibody, designated C4F6, which recognizes non-natively folded protein. Our findings indicate that SOD1 generally tolerates mutations at surface exposed lysine residues, and that loss of positive charge is insufficient to induce aggregation. Our findings may explain why mutations at these Lys residues have not been identified in ALS patients.

biochemistry↗

bioRxiv2018

Super-multiplexed fluorescence microscopy via photostability contrast

Many areas of biological research rely heavily on fluorescence microscopy to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously with spectrally distinct fluorescent labels. A high degree of multiplexing is desirable as it affords the experiment greater information content, speeding up research timelines. Multiplexing can be increased by imaging a larger number of spectral channels, however, the wide emission spectra of most fluorophores limits multiplexing to four or five labels in standard fluorescence microscopes. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins, and show that the latter are particularly well suited to our method as their bleaching is often reversible. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

bioengineering↗

bioRxiv2017

Age-dependent Pavlovian biases influence motor decision-making

Healthy ageing is associated with decreased risk taking in motor1 and economic2-4 decision-making. However, it is unknown whether a single underlying mechanism explains these changes. Age-related changes in economic risk taking are explained by reduced Pavlovian biases that promote action toward reward2, 5, 6. Although Pavlovian biases also promote inaction in the face of punishment, the role such Pavlovian biases play in motor decision-making, which additionally depends on estimating the probability of successfully executing an action7-10, is unknown. To address this, we developed a novel app-based motor decision-making task to measure sensitivity to reward and punishment when subjects (n=26,532) made a go/no-go motor gamble based on the perceived ability to execut ...

neuroscience↗

bioRxiv2017

Enrichment of the HIV reservoir in CD32+ CD4 T cells occurs early in blood and tissue

The Fc receptor CD32 has been proposed as a marker for CD4 T cells latently infected with HIV. We demonstrate that enrichment for HIV DNA in CD32+ CD4 T cells can be found early in infection in both tissue and blood. However, we find no evidence for a correlation between CD32 expression on CD4 T cells and either HIV DNA levels or time to rebound viraemia following treatment interruption. CD32+ CD4 T cells have a more differentiated memory phenotype, and high levels of expression of immune checkpoint receptors PD-1, Tim-3 and TIGIT as well as the activation marker, HLA DR. There was no difference in the phenotype or frequency of CD32 expressing cells prior to or after the initiation of antiretroviral therapy, or compared with healthy controls, suggesting that preferential infection or survival, rather than up-regulation, may be responsible for the observed enrichment of proviral HIV DNA in CD32+ CD4 T cells.

immunology↗