Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Membrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.

BackgroundVariability in genes implicated in drug pharmacokinetics or drug response can modulate treatment efficacy or predispose to adverse drug reactions. Besides common genetic polymorphisms, recent sequencing projects revealed a plethora of rare genetic variants in genes encoding proteins involved in drug metabolism, transport, and response.ResultsTo understand the global importance of rare pharmacogenetic gene variants, we mapped the variability in 208 pharmacogenes by analyzing exome sequencing data from 60,706 unrelated individuals and estimated the importance of rare and common genetic variants using a computational prediction framework optimized for pharmacogenetic assessments. Our analyses reveal that rare pharmacogenetic variants were strongly enriched in mutations predicted to cause functional alterations. For more than half of the pharmacogenes, rare variants account for the entire genetic variability. Each individual harbored on average a total of 40.6 putatively functional variants, rare variants accounting for 10.8% of these. Overall, the contribution of rare variants was found to be highly gene- and drug-specific. Using warfarin, simvastatin, voriconazole, olanzapine, and irinotecan as examples, we conclude that rare genetic variants likely account for a substantial part of the unexplained inter-individual differences in drug metabolism phenotypes.ConclusionsCombined, our data reveal high gene and drug specificity in the contributions of rare variants. We provide a proof-of-concept on how this information can be utilized to pinpoint genes for which sequencing-based genotyping can add important information to predict drug response, which provides useful information for the design of clinical trials in drug development and the personalization of pharmacological treatment.Electronic supplementary materialThe online version of this article (10.1186/s40246-018-0157-3) contains supplementary material, which is available to authorized users.

Prediction of phenotypic consequences of mutations constitutes an important aspect of precision medicine. Current computational tools mostly rely on evolutionary conservation and have been calibrated on variants associated with disease, which poses conceptual problems for assessment of variants in poorly conserved pharmacogenes. Here, we evaluated the performance of 18 current functionality prediction methods leveraging experimental high-quality activity data from 337 variants in genes involved in drug metabolism and transport and found that these models only achieved probabilities of 0.1-50.6% to make informed conclusions. We therefore developed a functionality prediction framework optimized for pharmacogenetic assessments that significantly outperformed current algorithms. Our model achieved 93% for both sensitivity and specificity for both loss-of-function and functionally neutral variants, and we confirmed its superior performance using cross validation analyses. This novel model holds promise to improve the translation of personal genetic information into biological conclusions and pharmacogenetic recommendations, thereby facilitating the implementation of Next-Generation Sequencing data into clinical diagnostics.

We have isolated, cDNA cloned and characterised a 29-kDa protein (ERp29), containing a C-terminal endoplasmic reticulum(ER)-retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of proteins in the ER [(e.g. culture in presence of the Ca 2ϩ ionophore A23187, inhibitors of Ca 2ϩ -ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N-glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1% of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress-response machinery of the ER.Keywords : ERp29; endoplasmic reticulum; stress protein; retrieval signal; BiP/GRP78.The luminal compartment of the endoplasmic reticulum (ER) of the export of secretory proteins from the ER by brefeldin A, an inhibitor of intracellular transport, may also result in excesin eukaryotic cells is a site where nascent polypeptides, destined for transport to Golgi, lysosomes or cell exterior attain their sive accumulation of malfolded proteins in ER and hence cause induction of stress proteins [7]. native three-dimensional configuration, undergo several posttranslational modifications and are assembled. Molecular chap-ER stress-inducible proteins belong to structurally diverse protein families including glucose-regulated proteins GRP78/ erones facilitate these processes assisting in folding and preventing aggregation of nascent chains by binding to the interactive BiP and GRP94 which are similar to the cytosolic heat-shock proteins (Hsp70 and Hsp90, respectively), calnexin, calreticulin protein surface [1]. Other luminal enzymes, which are involved in protein maturation, catalyze disulphide bond formation and and protein disulfide isomerase (PDI and other thioredoxin-containing proteins such as ERp72 and ERp61). The importance of rearrangement, N-linked glycosylation, participate in maintaining calcium homeostasis and in protein trafficking (reviewed in these proteins for the luminal compartment is accentuated by their ER-retrieval signal, the C-terminal tetrapeptide KDEL [2Ϫ3]).The biosynthesis of chaperones and some folding enzymes [8Ϫ9]. A protein (ERp29) with a similar C-terminal sequence is increased in stressed ER, i.e. under a variety of adverse physiologic or metabolic conditions, whose common denominator is (KEEL) was isolated in our laboratory from rat liver ER and the corresponding cDNA was cloned [10]. An analogous clone was ...