240 research outputs found
Metabolic Profiling Based Quantitative Evaluation of Hepatocellular Metabolism in Presence of Adipocyte Derived Extracellular Matrix
The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine
Metabolic Patterning on a Chip: Towards in vitro Liver Zonation of Primary Rat and Human Hepatocytes
An important number of healthy and diseased tissues shows spatial variations in their metabolic capacities across the tissue. The liver is a prime example of such heterogeneity where the gradual changes in various metabolic activities across the liver sinusoid is termed as “zonation” of the liver. Here, we introduce the Metabolic Patterning on a Chip (MPOC) platform capable of dynamically creating metabolic patterns across the length of a microchamber of liver tissue via actively enforced gradients of various metabolic modulators such as hormones and inducers. Using this platform, we were able to create continuous liver tissues of both rat and human origin with gradually changing metabolic activities. The gradients we have created in nitrogen, carbohydrate and xenobiotic metabolisms recapitulated an in vivo like zonation and zonal toxic response. Beyond its application in recapitulation of liver zonation in vitro as we demonstrate here, the MPOC platform can be used and expanded for a variety of purposes including better understanding of heterogeneity in many different tissues during developmental and adult stages
Progressive Hypoxia-on-a-chip: An In Vitro Oxygen Gradient Model for Capturing the Effects of Hypoxia on Primary Hepatocytes in Health and Disease
Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia‐inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia‐on‐a‐chip platform can be used to study the role of oxygen and hypoxia‐associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen‐limited conditions
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Bioinformatics analysis of the early inflammatory response in a rat thermal injury model
BACKGROUND: Thermal injury is among the most severe forms of trauma and its effects are both local and systemic. Response to thermal injury includes cellular protection mechanisms, inflammation, hypermetabolism, prolonged catabolism, organ dysfunction and immuno-suppression. It has been hypothesized that gene expression patterns in the liver will change with severe burns, thus reflecting the role the liver plays in the response to burn injury. Characterizing the molecular fingerprint (i.e., expression profile) of the inflammatory response resulting from burns may help elucidate the activated mechanisms and suggest new therapeutic intervention. In this paper we propose a novel integrated framework for analyzing time-series transcriptional data, with emphasis on the burn-induced response within the context of the rat animal model. Our analysis robustly identifies critical expression motifs, indicative of the dynamic evolution of the inflammatory response and we further propose a putative reconstruction of the associated transcription factor activities. RESULTS: Implementation of our algorithm on data obtained from an animal (rat) burn injury study identified 281 genes corresponding to 4 unique profiles. Enrichment evaluation upon both gene ontologies and transcription factors, verifies the inflammation-specific character of the selections and the rationalization of the burn-induced inflammatory response. Conducting the transcription network reconstruction and analysis, we have identified transcription factors, including AHR, Octamer Binding Proteins, Kruppel-like Factors, and cell cycle regulators as being highly important to an organism's response to burn response. These transcription factors are notable due to their roles in pathways that play a part in the gross physiological response to burn such as changes in the immune response and inflammation. CONCLUSION: Our results indicate that our novel selection/classification algorithm has been successful in selecting out genes with play an important role in thermal injury. Additionally, we have demonstrated the value of an integrative approach in identifying possible points of intervention, namely the activation of certain transcription factors that govern the organism's response
Reversible pH-controlled DNA-binding peptide nanotweezers: An in-silico study
We describe the molecular dynamics (MD)-aided engineering design of mutant
peptides based on the α-helical coiled-coil GCN4 leucine zipper
peptide (GCN4-p1) in order to obtain environmentally-responsive nanotweezers.
The actuation mechanism of the nanotweezers depends on the modification of
electrostatic charges on the residues along the length of the coiled coil.
Modulating the solution pH between neutral and acidic values results in the
reversible movement of helices toward and away from each other and creates a
complete closed-open-closed transition cycle between the helices. Our results
indicate that the mutants show a reversible opening of up to 15 Å
(1.5 nm; approximately 150% of the initial separation) upon pH
actuation. Investigation on the physicochemical phenomena that influence
conformational properties, structural stability, and reversibility of the
coiled-coil peptide-based nanotweezers revealed that a rationale- and
design-based approach is needed to engineer stable peptide or macromolecules
into stimuli-responsive devices. The efficacy of the mutant that demonstrated
the most significant reversible actuation for environmentally responsive
modulation of DNA-binding activity was also demonstrated. Our results have
significant implications in bioseparations and in the engineering of novel
transcription factors
Moloney murine leukemia virus decay mediated by retroviral reverse transcriptase degradation of genomic RNA
AbstractRetroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 °C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme
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Resuscitation of Ischemic Donor Livers with Normothermic Machine Perfusion: A Metabolic Flux Analysis of Treatment in Rats
Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use
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Development of Metabolic Indicators of Burn Injury: Very Low Density Lipoprotein (VLDL) and Acetoacetate Are Highly Correlated to Severity of Burn Injury in Rats
Hypermetabolism is a significant sequela to severe trauma such as burns, as well as critical illnesses such as cancer. It persists in parallel to, or beyond, the original pathology for many months as an often-fatal comorbidity. Currently, diagnosis is based solely on clinical observations of increased energy expenditure, severe muscle wasting and progressive organ dysfunction. In order to identify the minimum number of necessary variables, and to develop a rat model of burn injury-induced hypermetabolism, we utilized data mining approaches to identify the metabolic variables that strongly correlate to the severity of injury. A clustering-based algorithm was introduced into a regression model of the extent of burn injury. As a result, a neural network model which employs VLDL and acetoacetate levels was demonstrated to predict the extent of burn injury with 88% accuracy in the rat model. The physiological importance of the identified variables in the context of hypermetabolism, and necessary steps in extension of this preliminary model to a clinically utilizable index of severity of burn injury are outlined
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A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation
In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs
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