Jiayin Li

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Exosomes are important for intercellular communication, but the role of exosomes in the communication between adipose tissue (AT) and the liver remains unknown. The aim of this study is to determine the contribution of AT-derived exosomes in nonalcoholic fatty liver disease (NAFLD). Exosome components, liver fat content, and liver function were monitored in AT in mice fed a high-fat diet (HFD) or treated with metformin or GW4869 and with AMPKα1-floxed (Prkaα1fl/fl/wild-type [WT]), Prkaα1−/−, liver tissue-specific Prkaα1−/−, or AT-specific Prkaα1−/− modification. In cultured adipocytes and white AT, the absence of AMPKα1 increased exosome release and exosomal proteins by elevating tumor susceptibility gene 101 (TSG101)–mediated exosome biogenesis. In adipocytes treated with palmitic acid, TSG101 facilitated scavenger receptor class B (CD36) sorting into exosomes. CD36-containing exosomes were then endocytosed by hepatocytes to induce lipid accumulation and inflammation. Consistently, an HFD induced more severe lipid accumulation and cell death in Prkaα1−/− and AT-specific Prkaα1−/− mice than in WT and liver-specific Prkaα1−/− mice. AMPK activation by metformin reduced adipocyte-mediated exosome release and mitigated fatty liver development in WT and liver-specific Prkaα1−/− mice. Moreover, administration of the exosome inhibitor GW4869 blocked exosome secretion and alleviated HFD-induced fatty livers in Prkaα1−/− and adipocyte-specific Prkaα1−/− mice. We conclude that HFD-mediated AMPKα1 inhibition promotes NAFLD by increasing numbers of AT CD36-containing exosomes.

Cancer growth is driven by cancer stem-like cells within a tumor, called cancer stem cells (CSCs). Since miRNAs can regulate cell-fate decisions, we compared miRNA expression in stem-like cells and differentiated cells from small cell lung cancer (SCLC) cell lines to develop further understanding of the molecular mechanisms involved in the pathogenesis of SCLC. First, SCLC stem-like cells were enriched by isolating sphere-forming cells using a defined serum-free medium. Further, microRNA microarrays were used to measure the expression of 1212 miRNAs in sphere-forming cells and parental cells. We found 86 miRNAs that were differentially expressed, including 48 upregulated miRNAs and 38 downregulated miRNAs between sphere-forming cells and parental cells. Among them, five downregulated miRNAs (let-7, miR-20, 21, 27a and 30b) and one upregulated miRNA (miR-149*) were selected for validation in 3 sets of SCLC cell lines by qRT-RCR. The qRT-PCR analysis confirmed that all six miRNAs were indeed differentially expressed. However, only miR-27a was consistently downregulated in sphere-forming cells of all 3 cell lines. Antagonizing miR-27a by inhibitor in parental cells enhanced proliferation, self renewal, and the proportion of undifferentiated cells in vitro. The candidate miRNA and some miRNAs with same seed sequence are predicted to have several target genes related to apoptosis, cell proliferation and cell cycle. Our results suggest that downregulation of miR-27a enhanced the stem-like properties of SCLC cells in vitro and may be critical to maintaining a stem cell function in SCLC.