👤 Justin A Bishop

Lipoprotein(a) [Lp(a)] is a genetically determined cardiovascular risk factor. Additionally, Lp(a) levels are affected by dietary saturated fat (SFA) reduction. We previously reported an Lp(a) increase in response to SFA reduction in both white and black cohorts. However, less is known whether diets impact Lp(a)'s oxidized phospholipids (OxPL) and lipid components. We assessed responses of Lp(a)-OxPL concentration, Lp(a)-OxPL subspecies abundance, and the Lp(a)-lipidome to SFA reduction [from 16% energy with the average American diet (AAD) to 6% energy with a DASH-type diet] in 166 African-Americans. Responses by variability in Lp(a) levels and apolipoprotein(a) [apo(a)] sizes were tested. Mean age was 35 years; 70% were women; mean BMI was 28 kg/m Show less

BackgroundMultiple sclerosis (MS) is a prevalent, frequently progressive condition of the central nervous system that can significantly affect employment and career participation. Although researchers have extensively catalogued the factors that people with MS face in maintaining employment, the priorities of people working with MS in terms of career resources and information needs have not been extensively evaluated.ObjectiveWe sought (a) to identify the types of career information and resources that employed or recently-employed people with MS prioritize, and (b) to assess the extent to which the need for these career resources may vary among identifiable subgroups.MethodDescriptive statistics and latent profile analysis (LPA) were applied to the responses of 376 iConquerMS members who were either employed ( Show less

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N, and Conn.D were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging. Show less

The Cancer Genome Atlas (TCGA) sequencing analysis of head and neck squamous cell carcinoma (HNSCC) recently reported on gene fusions, however, few human papillomavirus (HPV) positive samples were included, and the functional relevance of identified fusions was not explored. We therefore performed an independent analysis of gene fusions in HPV-positive oropharyngeal SCC (OPSCC). RNA sequencing was performed on 47 HPV-positive OPSCC primary tumors and 25 normal mucosal samples from cancer unaffected controls on an Illumina TruSeq platform. MapSplice2 was used for alignment and identification of fusion candidates. Putative fusions with less than five spanning reads, detected in normal tissues, or that mapped to the same gene were filtered out. Selected fusions were validated by RT-PCR and Sanger sequencing. Within 47 HPV-positive OPSCC tumors, 282 gene fusions were identified. Most fusions (85.1%) occurred in a single tumor, and the remaining fusions recurred in 2-16 tumors. Gene fusions were associated with significant up regulation of 16 genes (including EGFR and ERBB4) and down regulation of four genes (PTPRT, ZNF750, DLG2, SLCO5A1). Expression of these genes followed similar patterns of up regulation and down regulation in tumors without these fusions compared to normal tissue. Five of six gene fusions selected for validation were confirmed through RT-PCR and sequencing. This integrative analysis provides a method of prioritizing functionally relevant gene fusions that may be expanded to other tumor types. These results demonstrate that gene fusions may be one mechanism by which functionally relevant genes are altered in HPV-positive OPSCC. Show less

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Show less

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a, and less sympathetic innervation compared to wild-type (+/ +)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hours), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2, and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wild-type. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wild-type and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild-type. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling. Show less

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet-Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity. Show less

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning. Show less

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy. Show less