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3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes  [PDF]
Yeonhee Kim,Padmavathy Rajagopalan
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015456
Abstract: Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.
Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures
Jonathan Wu, Núria Marí-Buyé, Teresa Mui?os, Salvador Borrós, Pietro Favia, Carlos E Semino
Journal of Nanobiotechnology , 2010, DOI: 10.1186/1477-3155-8-29
Abstract: The modified sandwich cultures replace collagen with self-assembling peptide, RAD16-I, combined with functional peptide motifs such as the integrin-binding sequence RGD and the laminin receptor binding sequence YIG to create a cell-instructive scaffold. In this work, we show that a plasma-deposited coating can be used to obtain a peptide layer thickness in the nanometric range, which in combination with the incorporation of functional peptide motifs have a positive effect on the expression of adult hepatocyte markers including albumin, CYP3A2 and HNF4-alpha.This study demonstrates the capacity of sandwich cultures with modified instructive self-assembling peptides to promote cell-matrix interaction and the importance of thinner scaffold layers to overcome mass transfer problems. We believe that this bioengineered platform improves the existing hepatocyte culture methods to be used for predictive toxicology and eventually for hepatic assist technologies and future artificial organs.The liver is an important and complex organ that plays a vital role in metabolism and is responsible for many important functions of the body including glycogen storage, plasma protein production, drug detoxification and xenobiotics metabolization. Due to the importance of this organ in many of the body's daily processes, liver malfunction often leads to death. Most of the activity of the liver can be attributed to hepatocytes, which make up 60-80% of the cytoplasmic mass of the liver [1,2]. Loss of hepatocyte function can result in acute or chronic liver disease and, as a result, substantially compromise the rest of the organ and the body. Many previous strategies have been implemented to maintain these hepatocyte functions in vitro, including the use of extracellular matrices such as the current standard, collagen [3-6], Matrigel [7] or liver derived basement membrane matrix [8]. However, the liver carries out and regulates numerous biochemical reactions that require the combined effort
Discovering Patterns in Biological Sequences by Optimal Segmentation  [PDF]
Joseph Bockhorst,Nebojsa Jojic
Computer Science , 2012,
Abstract: Computational methods for discovering patterns of local correlations in sequences are important in computational biology. Here we show how to determine the optimal partitioning of aligned sequences into non-overlapping segments such that positions in the same segment are strongly correlated while positions in different segments are not. Our approach involves discovering the hidden variables of a Bayesian network that interact with observed sequences so as to form a set of independent mixture models. We introduce a dynamic program to efficiently discover the optimal segmentation, or equivalently the optimal set of hidden variables. We evaluate our approach on two computational biology tasks. One task is related to the design of vaccines against polymorphic pathogens and the other task involves analysis of single nucleotide polymorphisms (SNPs) in human DNA. We show how common tasks in these problems naturally correspond to inference procedures in the learned models. Error rates of our learned models for the prediction of missing SNPs are up to 1/3 less than the error rates of a state-of-the-art SNP prediction method. Source code is available at www.uwm.edu/~joebock/segmentation.
Representing perturbed dynamics in biological network models  [PDF]
Gautier Stoll,Jacques Rougemont,Felix Naef
Quantitative Biology , 2007, DOI: 10.1103/PhysRevE.76.011917
Abstract: We study the dynamics of gene activities in relatively small size biological networks (up to a few tens of nodes), e.g. the activities of cell-cycle proteins during the mitotic cell-cycle progression. Using the framework of deterministic discrete dynamical models, we characterize the dynamical modifications in response to structural perturbations in the network connectivities. In particular, we focus on how perturbations affect the set of fixed points and sizes of the basins of attraction. Our approach uses two analytical measures: the basin entropy $H$ and the perturbation size $\Delta$, a quantity that reflects the distance between the set of fixed points of the perturbed network to that of the unperturbed network. Applying our approach to the yeast-cell cycle network introduced by Li \textit{et al.} provides a low dimensional and informative fingerprint of network behavior under large classes of perturbations. We identify interactions that are crucial for proper network function, and also pinpoints functionally redundant network connections. Selected perturbations exemplify the breadth of dynamical responses in this cell-cycle model.
Optimización de cultivos de hepatocitos humanos para estudios de citotoxicidad Optimization of human hepatocyte cultures for citotoxicity studies
Revista Chilena de Cirugía , 2007,
Abstract: Los cultivos de hepatocitos entregan un valioso acercamiento al estudio de las funciones metabólicas específicas del hígado, evaluación de citotoxicidad. No existen líneas humanas inmortales con función normal. La inmortalización de hepatocitos humanos con el método UCHT1(medio de cultivo condicionado por células tumorales de tiroides) permitirá prolongar la sobrevida y función de estos, siendo útil para evaluar funcionalidad y citotoxicidad. Objetivo: Optimizar el cultivo de hepatocitos humanos. Metodología: En cultivos primarios de hepatocitos humanos, se agregó medio UCHT1 cultivando en superficies de colágeno, polilisina, gelatina y matrigel. Como control positivo, se utilizó línea Gherschenson (GER) para evaluar curva de crecimiento y producción de Glucógeno (PAS). Se evaluó citotoxicidad (LIVE/DEAD) en hepatocitos GER expuestos a Metotrexato (10, 100 y 1000 mM) a 24, 48 y 72 hrs. Resultados: Se realizó 3 cultivos primarios. Fue efectiva la utilización de Polilisina y Colágeno. Duración 8 meses. No se ha realizado la curva de crecimiento, ni evaluación de funcionalidad en hepatocitos humanos. La línea GER tiene un crecimiento exponencial (tiempo duplicación: 36 hrs). Se observó producción de glucógeno en condiciones de diferenciación hasta 120 hrs. La citotoxicidad por Metotrexato tiene una curva dosis dependiente, significativa en todas las concentraciones (p<0,001) (CL50 a 1000 mM a 24 hrs). Conclusiones: Se logró establecer una línea primaria de hepatocitos humanos. La polilisina y el colágeno han optimizado el establecimiento de cultivos primarios. El método PAS permitió evaluar producción de glucógeno (diferenciación). Los valores de citotoxicidad demostraron un efecto dosis dependiente en las condiciones experimentales. Logrando estandarizar el método para evaluación futura de líneas celulares humanas Background: Hepatocyte cultures are a valuable tool to study specific metabolic liver functions and cytoxicity. Human hepatocyte cell lines with normal function do not exist. Immortalization of human hepatocytes with a rat thyroid cell line (UCHT1) allows long-term survival and function of these cells, becoming useful to evaluate functionality and cytotoxicity. Aim: To optimize long-term culture of human hepatocytes. Material and Methods: UCHT1 media was added to primary cultures of human hepatocytes, seeding in collagen, gelatin, matrigel and polilisine surfaces. Gherschenson cell line (GER) was used as a positive control to evaluate the growth curve and Glycogen production (PAS). Cytotoxicity was evaluated (LIVE/ DEAD) in GER hepatocytes expos
Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures  [PDF]
Martina Buck
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002660
Abstract: Background Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. Methodology/Principal Findings Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect na?ve human hepatocytes. Conclusions/Significance This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.
High concentration but low biological activity of hepatocyte growth factor in patients with chronic renal failure  [PDF]
Johanna L?nn, Faisal Shahzad, Fredrik Uhlin, Torbj?rn Bengtsson, Gabriel Almroth, Fariba Nayeri
Advances in Bioscience and Biotechnology (ABB) , 2012, DOI: 10.4236/abb.2012.324068
Abstract: Hepatocyte growth factor (HGF) is a renotropic, antifibrotic and regenerative factor with cytoprotective effects that is produced by mesenchymal cells and shows high affinity to components of extra cellular matrix, such as heparan sulphate proteoglycan (HS-PG), in healthy. Patients with chronic renal failure (CRF) suffer from a chronic inflammatory disorder. In order to assess the underlying mechanisms for development of CRF we aimed to assess the amounts and affinity of HGF in this patient group. Elisa, western blot and surface plasmon resonance (SPR) were used to study HGF in blood samples, as well as in isolated neutrophils, in CRF patients compared to healthy controls. Patients with CRF showed higher HGF levels in serum (P < 0.0001), but decreased affinity to HSPG (P < 0.0001), compared to healthy controls. Addition of protease inhibitors decreased the difference between patients with CRF compared to healthy individuals. HGF with potent regenerative function during injury lacks affinity to HSPG in patients with CRF that may depend on production of proteases from activated immune cells. This information might be used to highlight underlying mechanisms for chronicity and leading to new strategies for treatment of chronic injuries.
Biological aspects on the cultures of the entomophthoralean fungusPandora delphacis grown on broomcorn millets
Mingguang Feng,Yong Liang
Chinese Science Bulletin , 2003, DOI: 10.1007/BF03184061
Abstract: A novel method was developed to use glutinous broomcorn millets (Panicum miliaceum L.) as solid substrate to make cultures of the entomophthoralean fungusPandora delphacis specifically pathogenic to planthoppers, leafhoppers and aphids. Steamed millets with water content of 45% were inoculated with a liquid culture ofP. delphacis at a ratio of 20% (v/w) and then incubated at 25°C and L:D 12:12. The millets cultured for 3–17 d exhibited high potential for conidial production. The 5-d-old millet culture sporulated most abundantly, discharging up to 17.12 (±1.31) × 104 conidia/ millet. The cultures incubated for 7–11 d also had a satisfactory sporulation capability, yielding 13.00–13.90 × 104 conidia/millet. Compared to 2.32 (±0.34) × 104 conidia discharged from each ofMyzus persicae adults killed byP. delphacis and a ≤60-h duration of sporulation, each of the millets cultured for 5–11 d produced 5.6–7.4 times more conidia with an over doubled duration for conidial discharge (144 h). Among 106M. persicae adults exposed to the shower of conidia discharged from the cultured millets, a total mortality of 69.8% caused byP. delphacis infection was observed within 7 d after exposure, but no death was attributed to the fungal infection in the aphids unexposed. The results indicate that the millet cultures ofP. delphacis are biologically similar to aphid cadavers killed by the same fungus. Due to the superiority of the cultured millets to the cadavers in sporulation potential and duration, the method for making cultures ofP. delphacis on the broomcorn millets is highly recommended for use in study of entomophthoralean fungi for microbial control. This is the first report on the success of the solid culture ofPandora species on cereals.
Molecular Biological Progress of Hepatocyte Growth Factor (HGF)

YU Shui-Liang,YANG Fu-Hua,

生物工程学报 , 2002,
Abstract: Hepatocyte growth factor (HGF) is a multifunctional growth factor which is a heterodimeric glycoprotein molecule. There are three truncated HGF variants:NK1,NK2,NK4.The structure of the promoter of HGF is very complicated, and the expression of HGF is regulated by various factors and regulatory elements. Because of the importance of HGF to human body, the genetic engineering expression and gene therapy investigation of HGF are being done.
Discovering biological connections between experimental conditions based on common patterns of differential gene expression
Adam C Gower, Avrum Spira, Marc E Lenburg
BMC Bioinformatics , 2011, DOI: 10.1186/1471-2105-12-381
Abstract: To demonstrate the utility of openSESAME, we used gene expression signatures of two biological perturbations to query a set of 75,164 human expression profiles that were generated using Affymetrix microarrays and deposited in GEO. The first query, using a signature of estradiol treatment, identified experiments in which estrogen signaling was perturbed and also identified differences in estrogen signaling between estrogen receptor-positive and -negative breast cancers. The second query, which used a signature of silencing of the transcription factor p63 (a key regulator of epidermal differentiation), identified datasets related to stratified squamous epithelia or epidermal diseases such as melanoma.openSESAME is a tool for leveraging the growing body of publicly available microarray data to discover relationships between different biological states based on common patterns of differential gene expression. These relationships may serve to generate hypotheses about the causes and consequences of specific patterns of observed differential gene expression. To encourage others to explore the utility of this approach, we have made a website for performing openSESAME queries freely available at http://opensesame.bu.edu webcite.Genome-wide gene expression microarrays have found widespread use because of their high throughput and ability to measure the expression of tens of thousands of genes simultaneously. This technology has made it possible to perform genome-wide searches for changes in gene expression in response to perturbations such as gene knockouts [1] and treatment with bioactive compounds [2]. It has also been useful in identifying gene expression differences associated with histologic subtypes of disease [3], clinical diagnosis [4], prognosis [5], or the efficacy of various therapeutic strategies [6]. However, a challenge for scientists performing genome-wide gene expression microarray analysis has been using these data to generate hypotheses about biological pro
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