Disruption of mitochondrial fitness in human preadipocytes by fatty acids

Adipose tissue lies at the core of energy metabolism, by not only releasing and taking up fatty acids (FA) according to the individuals overall surplus or deficit of energy, but also signaling other tissues to regulate their own metabolism. In the event that adipose tissue function is lost, insulin resistance, hypertriglyceridemia and hepatic steatosis can develop. Despite the wealth of knowledge that exists for how mature adipocytes function in both normal and diseased states, preadipocytes, which can safe guard against adipocyte dysfunction by differentiating and joining the adipocyte pool, have been overlooked. Therefore, how human preadipocytes function in the presence of a mixture of FA (oleate, palmitate, stearate and linoleate) that resembles a healthy individual's adipose tissue microenvironment was investigated. Experiments using a novel real-time flow cytometry device show that the maximal rate of mitochondrial superoxide accumulation, hydrogen peroxide (H2O2) production, and lipid peroxidation occurred within seconds of exposure to FA. Although mitochondrial ROS occurred in a transient fashion (within 2 hours), preadipocytes still experienced a loss in respiration through depletion of NAD+ and NADH that manifested into overt oxidative stress and cell death after 24 hours of FA exposure. Despite observing cell death in 35% of our population, the majority of preadipocytes were able to mount a protective response through increases in the mitochondrial antioxidant buffering capacity. Investigation into FOXO1, one of the transcription factors that govern mitochondrial antioxidant gene transcription, revealed an increase in deacetylation as soon as six hours post FA exposure. Inhibition of FOXO1 or SIRT1, which deacetylates FOXO1, exacerbated the toxic effects of FA suggesting that the SIRT1-FOXO1 axis may play a beneficial role in protecting preadipocytes from constant exposure to FA. Furthermore, SIRT1, FOXO1 and mitochondrial antioxidant gene expression from human adipose tissue was correlated to insulin sensitivity further highlighting the potential importance of mitochondrial antioxidant buffering systems in adipose tissue function. Altogether, this study suggests that the preadipocyte survivability is centered on mitochondrial resistance to FA-induced ROS through up regulation of mitochondrial antioxidant systems.  Ph.D

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism

Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin

The mouse CD8α+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8α+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8α+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy