Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults who have no history of primary liver disease or cirrhosis. We performed RNA sequencing on FL-HCC tumors and identified a chimeric transcript that was expressed in all tumor samples but not in adjacent normal liver. The chimeric RNA was confirmed by RT-PCR and Sanger Sequencing. Based on the results of whole genome sequencing, the chimeric transcript is the result of a ~400 kilobase deletion on chromosome 19. The chimera was predicted to code for a protein with the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. The presence of this chimera protein was established by immunoprecipitation and Western Blot analysis. Expression of the chimera in human cell culture demonstrates that it retains kinase activity. Evidence for a DNAJB1-PRKACA chimeric transcript in 15 out of 15 FL-HCC patients suggests that it contributes to tumor pathogenesis.

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

In humans, epidermal melanocytes are responsible for skin pigmentation, defense against ultraviolet radiation, and the deadliest common skin cancer, melanoma. While there is substantial overlap in melanocyte development pathways between different model organisms, species dependent differences are frequent and the conservation of these processes in human skin remains unresolved 1-3 . Thus, the biology of developing and adult human melanocytes remains largely uncharacterized. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes derived directly from skin, capturing transcriptomes across different anatomic sites, developmental age, sexes, and multiple skin tones. Using donor-matched skin from distinct volar and non-volar anatomic locations, we uncovered subpopulations of melanocytes exhibiting site-specific enrichment that occurs during gestation and persists through adulthood. In addition, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we use these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis. Overall, the characterization of human melanocytes fresh from skin revealed new subpopulations, human-specific transcriptional programs, and valuable insights into melanoma dedifferentiation. INTRODUCTION:Epidermal melanocytes, the pigment producing cells of human skin, are responsible for skin tone and orchestrate the primary defense against damage from ultraviolet (UV) radiation. Some anatomic site-specific differences in pigmentation are due to environmental factors, such as the tanning response to UV exposure. Others, like the hypopigmentation at volar sites (such as palms and soles), are present at birth. In adult skin, mesenchymal -epithelial interactions are known to influence anatomic site-specific melanocyte survival and pigment production 4 but melanocyte intrinsic factors that contribute to site-specific specialization remain unclear.Model organisms are powerful tools for investigating melanocyte development. In chick and mouse, a transient, multipotent neural crest cell population gives rise to committed immature melanocyte precursors, called melanoblasts, via two spatially and temporally distinct pathways 2,3 . Such studies focus primarily on melanocytes in skin appendages (hair follicle, feather, and sweat gland). However, despite constituting the predominate subtype in human skin, resident epidermal melanocytes have not been the subject of analogous investigations into developmental trajectories and anatomic-specializations.Melanocytes can give rise to melanomas which present distinct phenotypic and genomic characteristics correlated with primary tumor location 5,6 . Like many cancers, melanoma progression is coupled to dedifferentiation of the cell of origin 7 . The aggressive nature of melanoma is proposed to be rooted in unique attributes of the melanocytic linage 8 . Decoding the transcriptome of epidermal mela...

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.