"Liver-on-a-Chip" cultures of primary hepatocytes and Kupffer cells for hepatitis B virus infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2-D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3-D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis

Candida glabrata encodes a longer variant of the mating type (MAT) alpha2 gene in the mating type-like MTL3 locus, which can form homodimers

"The fungal pathogen Candida glabrata is a haploid asexual yeast. Candida glabrata contains orthologs of the genes that control mating and cell-type identity in other fungi, which encode putative transcription factors localized in the MAT locus in Saccharomyces cerevisiae or MTL in other fungi. Candida glabrata contains three copies of the CgMTL locus but only CgMTL1 correctly expresses the information encoded in it. CgMTL1 can encode the Cga1 gene (a information), or the Cgalpha1 and Cgalpha2 genes (alpha information). CgMTL2 contains an identical copy of the Cga1 gene. CgMTL3 contains an identical copy of the Cgalpha1 gene but a longer variant of the Cgalpha2 gene that we termed Cgalpha3. In S. cerevisiae diploid cells, that express Sca and Scalpha information, Sca1 and Scalpha2 proteins form a heterodimer, which represses genes expressed only in haploid cells and some genes involved in stress response. We constructed C. glabrata strains that simultaneously express Cga1 and Cgalpha2 or Cga1 and Cgalpha3 genes. We did not find any phenotype in these strains when grown under a large variety of stress and nutritional conditions. However, we detected an interaction between Cga1 and Cgalpha2 but not between Cga1 and Cgalpha3 by Bimolecular Fluorescence Complementation and co-immunoprecipitation assays.