Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.     

Adenoma formation following limited ablation of p120-catenin in the mouse intestine

p120 loss destabilizes E-cadherin and could therefore result in tumor and/or metastasis-promoting activities similar to those caused by E-cadherin downregulation. Previously, we reported that p120 is essential in the intestine for barrier function, epithelial homeostasis and survival. Conditional p120 ablation in the mouse intestine induced severe inflammatory bowel disease, but long-term cancer-related studies were impossible because none of the animals survived longer than 21 days. Here, we used a tamoxifen-inducible mouse model (Vil-Cre-ER(T2);p120(fl/fl)) to limit the extent of p120 ablation and thereby enable long-term studies. Reducing p120 KO to ～10% of the intestinal epithelium produced long-lived animals outwardly indistinguishable from controls. Effects of prolonged p120 absence were then evaluated at intervals spanning 2 to 18 months. At all time points, immunostaining revealed microdomains of p120-null epithelium interspersed with normal epithelium. Thus, stochastic p120 ablation is compatible with crypt progenitor cell function and permitted lifelong renewal of the p120-null cells. Consistent with previous observations, a barrier defect and frequent infiltration of neutrophils was observed, suggesting that focal p120 loss generates a microenvironment disposed to chronic inflammation. We report that 45% of these animals developed tumors within 18 months of tamoxifen induction. Interestingly, β-catenin was upregulated in the majority, but none of the tumors were p120 null. Although further work is required to directly establish mechanism, we conclude that limited p120 ablation can promote tumorigenesis by an indirect non-cell autonomous mechanism. Given that byproducts of inflammation are known to be highly mutagenic, we suggest that tumorigenesis in this model is ultimately driven by the lifelong inability to heal chronic wounds and the substantially increased rates of stochastic gene mutation in tissue microenvironments subjected to chronic inflammation. Indeed, although technical issues precluded direct identification of mutations, β-catenin upregulation in human colon cancer almost invariably reflects mutations in APC and/or β-catenin.     

Tests of two methods for identifying founder effects in metapopulations reveal substantial type II error

Genetic analysis has been promoted as a way to reconstruct recent historical dynamics (“historical demography”) by screening for signatures of events, such as bottlenecks, that disrupt equilibrium patterns of variation. Such analyses might also identify “metapopulation” processes like extinction and recolonization or source-sink dynamics, but this potential remains largely unrealized. Here we use simulations to test the ability of two currently used strategies to distinguish between a set of interconnected subpopulations (demes) that have undergone bottlenecks or extinction and recolonization events (metapopulation dynamics) from a set of static demes. The first strategy, decomposed pairwise regression, provides a holistic test for heterogeneity among demes in their patterns of isolation-by-distance. This method suffered from a type II error rate of 59–100 %, depending on parameter conditions. The second strategy tests for deviations from mutation-drift equilibrium on a deme-by-deme basis to identify sites likely to have experienced recent bottlenecks or founder effects. Although bottleneck tests have good statistical power for single populations with recent population declines, their validity in structured populations has been called into question, and they have not been tested in a metapopulation context with immigration (or colonization) and population recovery. Our simulations of hypothetical metapopulations show that population recovery can rapidly eliminate the statistical signature of a bottleneck, and that moderate levels of gene flow can generate a false signal of recent population growth for demes in equilibrium. Although we did not cover all possible metapopulation scenarios, the performance of the tests was disappointing. Our results indicate that these methods might often fail to identify population bottlenecks and founder effects if population recovery and/or gene flow are influential demographic features of the study system.     

Retinitis Pigmentosa Mutants Provide Insight into the Role of the N-terminal Cap in Rhodopsin Folding,Structure, and Function

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.     

Functional Analysis of Claudin-6 and Claudin-9 as Entry Factors for Hepatitis C Virus Infection of Human Hepatocytes by Using Monoclonal Antibodies

The relevance of claudin-6 and claudin-9 in hepatitis C virus (HCV) entry remains elusive. We produced claudin-6- or claudin-9-specific monoclonal antibodies that inhibit HCV entry into nonhepatic cells expressing exogenous claudin-6 or claudin-9. These antibodies had no effect on HCV infection of hepatoma cells or primary hepatocytes. Thus, although claudin-6 and claudin-9 can serve as entry factors in cell lines, HCV infection into human hepatocytes is not dependent on claudin-6 and claudin-9.     

Black mothering,paid work and identity

Recent figures suggest that in Britain today, 77.3% of black women are engaged in full-time paid work, a figure which has remained fairly consistent since the introduction of statistical data assessing the work activity of African-Caribbean women from post-war years (CRE 1997). The study addresses the extent to which black women's high work rate derives from a combination of historical cultural and structural economic factors. Historical and cultural, because the experiences of slavery, colonialism and economic migration have had a direct impact on black women's relationship to full-time paid work today in contemporary Britain. In addition, structural economic factors such as high rates of unemployment for black men and lower rates of pay for black men and women compared to their white male and female counterparts, actively encourage a high proportion of black women towards full-time paid work in order to make up for this economic shortfall. A primary consequence of these inter-locking factors is that full-time paid work becomes central to black women's mothering and black mothers' work status is part of their everyday family experience.     

Influence of ligand presentation density on the molecular recognition of mannose-functionalised glyconanoparticles by bacterial lectin BC2L-A

Polyvalent carbohydrate-protein interactions play a key role in bio- and pathological processes, including cell-cell communication and pathogen invasion. In order to study, control and manipulate these interactions gold nanoparticles have been employed as a 3D scaffold, presenting carbohydrate ligands in a multivalent fashion for use as high affinity binding partners and a model system for oligosaccharide presentation at biomacromolecular surfaces. In this study, the binding of a series of mannose-functionalised gold nanoparticles to the dimeric BC2L-A lectin from Burkholderia cenocepacia has been evaluated. BC2L-A is known to exhibit a high specificity for (oligo)mannosides. Due to the unique structure and binding nature of this lectin, it provides a useful tool to study (oligo)saccharides presented on multivalent scaffolds. Surface plasmon resonance and isothermal titration calorimetric assays were used to investigate the effect of ligand presentation density towards binding to the bacterial lectin. We show how a combination of structural complementarities between ligand presentation and lectin architecture and statistical re-binding effects are important for increasing the avidity of multivalent ligands for recognition by their protein receptors; further demonstrating the application of glyconanotechnology towards fundamental glycobiology research as well as a potential towards biomedical diagnostics and therapeutic treatments.     

Interaction of YOYO-1 with guanine-rich DNA

The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids. While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect of unorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt to correlate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in the number of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadruplexes in presence of sodium (Na⁺) or potassium (K⁺) ions. The fluorescence behavior of YOYO-1 upon binding such G-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quadruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleotides 5′-G4T2G4-3′ and 5′-G3TG3TG3-3′, sequences possessing longer G-rich regions and shorter loop sequences formed stronger quadruplexes in presence of K⁺ which translated to weaker binding of YOYO-1. The dependence of binding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such studies are expected to aid in more effective interpretation of applications involving the fluorophore.     

From the delta to Detroit: Packaging a folk epic for a new folk

This paper provides a narrative account of the making of the video, Tales from Arab Detroit: Abu Zayd Comes to America. It focuses upon the original project which involved bringing Egyptian epic‐singers to Detroit to perform selections from the epic of the Banî Hilâl tribe and their hero, Abu Zayd, in September 1993. The paper offers a brief background to the epic tradition itself, an analysis of two of the Detroit performances, a critique of the portrayal of the singers found in the final version of the video, and concludes with a textual ethnographer's thoughts about ethnographic filmmaking.