Faculty Bibliography

Residue levels and patterns of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), their hydroxylated metabolites (OH-PCBs, OH-PBDEs), and methoxylated PBDEs (MeO-PBDEs) in the blood of various terrestrial mammals in Japan, including cats, raccoon dogs, dogs, masked palm civets, foxes, raccoons, badgers, and mongooses were determined. Tri- through penta-chlorinated OH-PCBs were predominant in cat blood, whereas hexa- through octa-chlorinated OH-PCBs were found in other species. High proportion of BDE209 was found in all species, suggesting exposure to municipal waste and soil containing higher levels of deca-BDE products. 6OH-/MeO-BDE47 and 2'OH-/MeO-BDE68 were dominant in all terrestrial mammals. This is first report on the detection of OH-/MeO-PBDEs in the blood of terrestrial mammals. High concentrations of OH-/MeO-PBDEs were found in cats, suggesting the intake of these compounds from seafood. Cats exhibited higher accumulation and specific patterns of OH-PCBs, OH-PBDEs, and MeO-PBDEs, they may be at a high risk from these metabolites.

OBJECTIVE: Several secreted phospholipases A2 (sPLA2s), including group IIA, III, V, and X, have been linked to the development of atherosclerosis, which led to the clinical testing of A-002 (varespladib), a broad sPLA2 inhibitor for the treatment of coronary artery disease. Group X sPLA2 (PLA2G10) has the most potent hydrolyzing activity toward phosphatidylcholine and is believed to play a proatherogenic role. METHODS AND RESULTS: Here, we show that Ldlr(-/-) mice reconstituted with bone marrow from mouse group X-deficient mice (Pla2g10(-/-)) unexpectedly display a doubling of plaque size compared with Pla2g10(+/+) chimeric mice. Macrophages of Pla2g10(-/-) mice are more susceptible to apoptosis in vitro, which is associated with a 4-fold increase of plaque necrotic core in vivo. In addition, chimeric Pla2g10(-/-) mice show exaggerated T lymphocyte (Th)1 immune response, associated with enhanced T-cell infiltration in atherosclerotic plaques. Interestingly, overexpression of human PLA2G10 in murine bone marrow cells leads to significant reduction of Th1 response and to 50% reduction of lesion size. CONCLUSIONS: PLA2G10 expression in bone marrow cells controls a proatherogenic Th1 response and limits the development of atherosclerosis. The results may provide an explanation for the recently reported inefficacy of A-002 (varespladib) to treat patients with coronary artery disease. Indeed, A-002 is a nonselective sPLA2 inhibitor that inhibits both proatherogenic (groups IIA and V) and antiatherogenic (group X) sPLA2s. Our results suggest that selective targeting of individual sPLA2 enzymes may be a better strategy to treat cardiovascular diseases.

Avian malaria is of significant ecological importance and serves as a model system to study broad patterns of host switching and host specificity. The erythrocyte invasion mechanism of the malaria parasite Plasmodium is mediated, in large part, by proteins of the erythrocyte-binding-like (ebl) family of genes. However, little is known about how these genes are conserved across different species of Plasmodium, especially those that infect birds. Using bioinformatical methods in conjunction with polymerase chain reaction (PCR) and genetic sequencing, we identified and annotated one member of the ebl family, merozoite apical erythrocyte-binding ligand (maebl), from the chicken parasite Plasmodium gallinaceum. We then detected the expression of maebl in P. gallinaceum by PCR analysis of cDNA isolated from the blood of infected chickens. We found that maebl is a conserved orthologous gene in avian, mammalian, and rodent Plasmodium species. The duplicate extracellular binding domains of MAEBL, responsible for erythrocyte binding, are the most conserved regions. Our combined data corroborate the conservation of maebl throughout the Plasmodium genus and may help elucidate the mechanisms of erythrocyte invasion in P. gallinaceum and the host specificity of Plasmodium parasites.

STUDY QUESTION: Does resveratrol counteract age-associated infertility in a mouse model of reproductive aging? SUMMARY ANSWER: Long-term-oral administration of resveratrol protects against the reduction of fertility with reproductive aging in mice. WHAT IS KNOWN ALREADY: Loss of oocytes and follicles and reduced oocyte quality contribute to age-associated ovarian aging and infertility. Accumulation of free radicals with age leads to DNA mutations, protein damage, telomere shortening, apoptosis and accelerated ovarian aging. Increasing evidence shows that resveratrol, enriched in certain foods, for example red grapes and wine, has anti-tumor and anti-aging effects on somatic tissues by influencing various signaling pathways, including anti-oxidation, as well as activating Sirt1 and telomerase. We investigated the potential of resveratrol to stave off ovarian aging in the inbred C57/BL6 mouse model. STUDY DESIGN, SIZE, DURATION: Young C57/BL6 females (aged 2-3 months) were fed with resveratrol added to drinking water at 30 mg/l (providing approximately 7.0 mg/kg/day) for 6 or 12 months, and the fertility and ovarian functions were compared among mice treated with or without resveratrol, and young mice served as reproductive controls. Experiments were repeated three times, with an average of 25 females randomly allocated to each treatment group for each repeat. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive performance of female mice was determined by litter size, ovarian follicles and oocyte quantity and quality, and compared with age-matched controls. The impact of resveratrol on telomeres and telomerase activity, and expression of genes associated with cell senescence also was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Young mice fed with resveratrol for 12 months retained the capacity to reproduce, while age-matched controls produced no pups. Consistently, mice fed with resveratrol for 12 months exhibited a larger follicle pool than controls (P < 0.05). Furthermore, telomerase activity, telomere length and age-related gene expression in ovaries of mice fed with resveratrol resembled those of young mice, but differed (P < 0.05) from those of age-matched old mice. Resveratrol improved (P < 0.05) the number and quality of oocytes, as evidenced by spindle morphology and chromosome alignment. Also, resveratrol affected embryo development in vitro in a dose-dependent manner. LIMITATIONS, REASONS FOR CAUTION: The doses of resveratrol and the experimental conditions used by different research groups have varied considerably, and the dosage influences both the effectiveness and toxicity of resveratrol. Fine-tuning the dosage of resveratrol likely will optimize its anti-aging effects on ovarian function. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide a proof of principle of the fertility-sparing effect of resveratrol in female mice. Although depletion of the ovarian reserve of high-quality oocytes also contributes to increased infertility with reproductive aging in women, the data obtained using a mouse model may not extrapolate directly to human reproduction, and more extensive research is needed if any clinic trials are to be attempted. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by MOST of China National Basic Research Program (grant number: 2010CB94500 and 2012CB911200). The authors have no competing interests to declare.

To understand the molecular anatomy of myelin membranes, we performed a large-scale, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based lipidome and proteome screen on freshly purified human and murine myelin fractions. We identified more than 700 lipid moieties and above 1,000 proteins in the two species, including 284 common lipids and 257 common proteins. This study establishes the first comprehensive map of myelin membrane components in human and mice. Although this study demonstrates many similarities between human and murine myelin, several components have been identified exclusively in each species. Future quantitative validation studies focused on interspecies differences will authenticate the myelin membrane anatomy. The combined lipidome and proteome map presented here can nevertheless be used as a reference library for myelin health and disease.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

MicroRNAs (miRNAs) regulate gene expression by binding to their targets and promoting RNA degradation and/or inhibiting protein translation. In recent years, miRNAs have revolutionized our understanding of gene regulatory networks, providing new prospective tools to manage disease. Atherosclerosis and other cardiovascular diseases are a leading cause of disability and death in the US and in other western populations and pose an enormous burden on our healthcare system. Altered lipid homeostasis in liver or in the artery wall, and disruption of endothelial and smooth muscle cell function have been shown to contribute to the onset and progression of cardiovascular disease. This review focuses on recent advances in the field of vascular biology- and lipid metabolism-related miRNomics.

N-RAS is one member of a family of oncoproteins that are commonly mutated in cancer. Activating mutations in NRAS occur in a subset of colorectal cancers, but little is known about how the mutant protein contributes to the onset and progression of the disease. Using genetically engineered mice, we find that mutant N-RAS strongly promotes tumorigenesis in the context of inflammation. The protumorigenic nature of mutant N-RAS is related to its antiapoptotic function, which is mediated by activation of a noncanonical mitogen-activated protein kinase pathway that signals through STAT3. As a result, inhibition of MAP-ERK kinase selectively induces apoptosis in autochthonous colonic tumors expressing mutant N-RAS. The translational significance of this finding is highlighted by our observation that NRAS mutation correlates with a less favorable clinical outcome for patients with colorectal cancer. These data show for the first time the important role that N-RAS plays in colorectal cancer.

OBJECTIVE: Regression of atherosclerosis is a vital treatment goal of atherosclerotic vascular disease. Inhibitors of the microsomal triglyceride transfer protein (MTP) have been shown to reduce apolipoprotein B (apoB)-containing lipoproteins in animals and humans effectively. Therefore, the major aim of our study is to evaluate the effect of MTP inhibition on atherosclerotic plaque regression. METHODS: LDL-receptor-deficient (LDLr(-/-)) mice were fed a Western diet for 16 weeks and then harvested for baseline (n = 8), switched to chow diet (n = 8) or chow diet containing MTP inhibitor (BMS 212122; n = 8) for 2 weeks before harvesting. RESULTS: Treatment with MTP inhibitor led to rapid reduction in plasma lipid levels, which were accompanied by a significant decrease in lipid content and monocyte-derived (CD68+) cells in atherosclerotic plaques compared to baseline and chow diet control groups. MTP inhibitor-treated mice had increased collagen content, a marker associated with increased stability in human plaques. Furthermore, plaques of these mice showed a significant decrease in tissue factor and pro-inflammatory M1 macrophage marker monocyte chemoattractant protein-1 (MCP-I) and an increase in anti-inflammatory M2 macrophage markers arginase-I and mannose receptor 1 compared to mice in the baseline group. CONCLUSION: Reversal of hyperlipidemia in atherosclerotic mice by inhibition of MTP leads to rapid and beneficial changes in the composition and inflammatory state of the plaque.