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1.  Nuclear Lamins and Neurobiology 
Molecular and Cellular Biology  2014;34(15):2776-2785.
Much of the work on nuclear lamins during the past 15 years has focused on mutations in LMNA (the gene for prelamin A and lamin C) that cause particular muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These disorders, often called “laminopathies,” mainly affect mesenchymal tissues (e.g., striated muscle, bone, and fibrous tissue). Recently, however, a series of papers have identified important roles for nuclear lamins in the central nervous system. Studies of knockout mice uncovered a key role for B-type lamins (lamins B1 and B2) in neuronal migration in the developing brain. Also, duplications of LMNB1 (the gene for lamin B1) have been shown to cause autosome-dominant leukodystrophy. Finally, recent studies have uncovered a peculiar pattern of nuclear lamin expression in the brain. Lamin C transcripts are present at high levels in the brain, but prelamin A expression levels are very low—due to regulation of prelamin A transcripts by microRNA 9. This form of prelamin A regulation likely explains why “prelamin A diseases” such as Hutchinson-Gilford progeria syndrome spare the central nervous system. In this review, we summarize recent progress in elucidating links between nuclear lamins and neurobiology.
PMCID: PMC4135577  PMID: 24842906
2.  Feedback regulation of cholesterol uptake by the LXR-IDOL-LDLR axis 
Inducible Degrader Of the Low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the low-density lipoprotein receptor (LDLR). IDOL expression is controlled at the transcriptional level by the cholesterol-sensing nuclear receptor LXR. In response to rising cellular sterol levels, activated LXR induces IDOL production, thereby limiting further uptake of exogenous cholesterol through the LDLR pathway. The LXR–IDOL–LDLR mechanism for feedback inhibition of cholesterol uptake is independent of and complementary to the SREBP pathway. Since the initial description of the LXR–IDOL pathway, biochemical studies have helped to define the structural basis for both IDOL target recognition and LDLR ubiquitin transfer. Recent work has also suggested links between IDOL and human lipid metabolism.
PMCID: PMC4280256  PMID: 22936343
3.  Pathomx: an interactive workflow-based tool for the analysis of metabolomic data 
BMC Bioinformatics  2014;15(1):396.
Metabolomics is a systems approach to the analysis of cellular processes through small-molecule metabolite profiling. Standardisation of sample handling and acquisition approaches has contributed to reproducibility. However, the development of robust methods for the analysis of metabolomic data is a work-in-progress. The tools that do exist are often not well integrated, requiring manual data handling and custom scripting on a case-by-case basis. Furthermore, existing tools often require experience with programming environments such as MATLAB® or R to use, limiting accessibility. Here we present Pathomx, a workflow-based tool for the processing, analysis and visualisation of metabolomic and associated data in an intuitive and extensible environment.
The core application provides a workflow editor, IPython kernel and a HumanCyc™-derived database of metabolites, proteins and genes. Toolkits provide reusable tools that may be linked together to create complex workflows. Pathomx is released with a base set of plugins for the import, processing and visualisation of data. The IPython backend provides integration with existing platforms including MATLAB® and R, allowing data to be seamlessly transferred. Pathomx is supplied with a series of demonstration workflows and datasets. To demonstrate the use of the software we here present an analysis of 1D and 2D 1H NMR metabolomic data from a model system of mammalian cell growth under hypoxic conditions.
Pathomx is a useful addition to the analysis toolbox. The intuitive interface lowers the barrier to entry for non-experts, while scriptable tools and integration with existing tools supports complex analysis. We welcome contributions from the community.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0396-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4271363  PMID: 25490956
Metabolomics; Omics; nmr; Analysis; Visualisation; Workflow; Automation; Python
4.  Palmoplantar keratoderma along with neuromuscular and metabolic phenotypes in Slurp1-deficient mice 
Mutations in SLURP1 cause mal de Meleda, a rare palmoplantar keratoderma (PPK). SLURP1 is a secreted protein that is expressed highly in keratinocytes but has also been identified elsewhere (e.g., spinal cord neurons). Here, we examined Slurp1-deficient mice (Slurp1−/−) created by replacing exon 2 with β-gal and neo cassettes. Slurp1−/− mice developed severe PPK characterized by increased keratinocyte proliferation, an accumulation of lipid droplets in the stratum corneum, and a water barrier defect. In addition, Slurp1−/− mice exhibited reduced adiposity, protection from obesity on a high-fat diet, low plasma lipid levels, and a neuromuscular abnormality (hind limb clasping). Initially, it was unclear whether the metabolic and neuromuscular phenotypes were due to Slurp1 deficiency because we found that the targeted Slurp1 mutation reduced the expression of several neighboring genes (e.g., Slurp2, Lypd2). We therefore created a new line of knockout mice (Slurp1X−/− mice) with a simple nonsense mutation in exon 2. The Slurp1X mutation did not reduce the expression of adjacent genes, but Slurp1X−/− mice exhibited all of the phenotypes observed in the original line of knockout mice. Thus, Slurp1 deficiency in mice elicits metabolic and neuromuscular abnormalities in addition to PPK.
PMCID: PMC4214150  PMID: 24499735
6.  Dimensionality of Social Networks Using Motifs and Eigenvalues 
PLoS ONE  2014;9(9):e106052.
We consider the dimensionality of social networks, and develop experiments aimed at predicting that dimension. We find that a social network model with nodes and links sampled from an m-dimensional metric space with power-law distributed influence regions best fits samples from real-world networks when m scales logarithmically with the number of nodes of the network. This supports a logarithmic dimension hypothesis, and we provide evidence with two different social networks, Facebook and LinkedIn. Further, we employ two different methods for confirming the hypothesis: the first uses the distribution of motif counts, and the second exploits the eigenvalue distribution.
PMCID: PMC4154874  PMID: 25188391
7.  Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2 
Molecular Biology of the Cell  2014;25(10):1666-1675.
To assess the redundancy of lamins B1 and B2, knock-in lines were created that produce lamin B2 from the Lmnb1 locus and lamin B1 from the Lmnb2 locus. Both lines developed severe neurodevelopmental abnormalities, indicating that the abnormalities elicited by the loss of one B-type lamin cannot be prevented by increased synthesis of the other.
Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated “reciprocal knock-in mice”—mice that make lamin B2 from the Lmnb1 locus (Lmnb1B2/B2) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2B1/B1). Lmnb1B2/B2 mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1B2/B2 mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2B1/B1 mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.
PMCID: PMC4019497  PMID: 24672053
8.  Incident Hepatitis C Virus Infection in Men Who Have Sex With Men: A Prospective Cohort Analysis, 1984–2011 
In the United States, incident hepatitis C among men who have sex with men has been ongoing since at least 1984. Risk factors included unprotected receptive anal intercourse with multiple partners, HIV infection, and lower CD4 T-cell count among HIV-infected men.
Background Prospective characterization of hepatitis C virus (HCV) transmission in both human immunodeficiency virus (HIV)–infected and –uninfected men who have sex with men (MSM) over the entire HIV epidemic has not been comprehensively conducted.
Methods To determine the trends in and risk factors associated with incident HCV in MSM since 1984, 5310 HCV antibody (anti-HCV)–negative MSM in the Multicenter AIDS Cohort Study were prospectively followed during 1984–2011 for anti-HCV seroconversion.
Results During 55 343 person-years (PYs) of follow-up, there were 115 incident HCV infections (incidence rate, 2.08/1000 PYs) scattered throughout the study period. In a multivariable analysis with time-varying covariates, older age (incidence rate ratio [IRR], 1.40/10 years, P < .001), enrollment in the later (2001–2003) recruitment period (IRR, 3.80, P = .001), HIV infection (IRR, 5.98, P < .001), drinking >13 alcoholic drinks per week (IRR, 1.68, P < .001), hepatitis B surface antigen positivity (IRR, 1.68, P < .001), syphilis (IRR, 2.95, P < .001), and unprotected receptive anal intercourse with >1 male partner (IRR, 3.37, P < .001) were independently associated with incident HCV. Among HIV-infected subjects, every 100 cell/mm3 increase in CD4 count was associated with a 7% (P = .002) decrease in the HCV incidence rate up to a CD4 count of 500 cells/mm3, whereas there was no association with highly active antiretroviral therapy.
Conclusions The spread of HCV among both HIV-infected and -uninfected MSM in the United States has been ongoing since the beginning of the HIV epidemic. In HIV-infected men with <500 CD4+ T cells, the HCV incidence rate was inversely proportional to CD4 T-cell count.
PMCID: PMC3669529  PMID: 23532480
incident HCV; sexual transmission; MSM
9.  Lipins, Lipinopathies, and the Modulation of Cellular Lipid Storage and Signaling 
Progress in lipid research  2013;52(3):10.1016/j.plipres.2013.04.001.
Members of the lipin protein family are phosphatidate phosphatase (PAP) enzymes, which catalyze the dephosphorylation of phosphatidic acid to diacylglycerol, the penultimate step in TAG synthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in that they also promote fatty acid oxidation through their activity as co-regulators of gene expression by DNA-bound transcription factors. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins—lipin-1, lipin-2, and lipin-3. In mice and humans, the levels of lipin activity are a determinant of TAG storage in diverse cell types, and humans with deficiency in lipin-1 or lipin-2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of “lipinopathies” in mouse models and in humans has revealed an important role for lipin activity in the regulation of lipid intermediates (phosphatidate and diacylglycerol), which influence fundamental cellular processes including adipocyte and nerve cell differentiation, adipocyte lipolysis, and hepatic insulin signaling. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.
PMCID: PMC3830937  PMID: 23603613
11.  GPIHBP1 and the intravascular processing of triglyceride-rich lipoproteins 
Journal of internal medicine  2012;272(6):528-540.
Lipoprotein lipase (LPL) is produced by parenchymal cells, mainly adipocytes and myocytes, but its role in hydrolyzing triglycerides in plasma lipoproteins occurs at the capillary lumen. For decades, the mechanism by which LPL reached its site of action in capillaries was unclear, but this mystery was recently solved. GPIHBP1, a GPI-anchored protein of capillary endothelial cells, picks up LPL from the interstitial spaces and shuttles it across endothelial cells to the capillary lumen. When GPIHBP1 is absent, LPL is mislocalized to the interstitial spaces, leading to severe hypertriglyceridemia. Some cases of hypertriglyceridemia in humans are caused by GPIHBP1 mutations that interfere with GPIHBP1's ability to bind LPL, and some are caused by LPL mutations that impair LPL's ability to bind to GPIHBP1. This review will cover recent progress in understanding GPIHBP1's role in health and disease and will discuss some remaining mysteries surrounding the processing of triglyceride-rich lipoproteins.
PMCID: PMC3940157  PMID: 23020258
hypertriglyceridemia; chylomicronemia; GPIHBP1; lipoprotein lipase; endothelial cells; lymphocyte antigen 6 proteins
12.  Targeting GGTase-I Activates RHOA, Increases Macrophage Reverse Cholesterol Transport, and Reduces Atherosclerosis in Mice 
Circulation  2013;127(7):782-790.
Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I–deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor–deficient mice.
Methods and Results
Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b∆/∆) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b∆/∆ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I–deficient macrophages revealed upregulation of the cyclooxygenase 2–peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I– and CD36-mediated basal and high-density lipoprotein–stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo.
Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.
PMCID: PMC3940160  PMID: 23334894
atherosclerosis; cholesterol; hydroxymethylglutaryl-CoA reductase inhibitors; macrophages; prenylation; statins
13.  Nuclear lamins in the brain—new insights into function and regulation 
Molecular neurobiology  2012;47(1):290-301.
The nuclear lamina is an intermediate filament meshwork composed largely of four nuclear lamins—lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Located immediately adjacent to the inner nuclear membrane, the nuclear lamina provides a structural scaffolding for the cell nucleus. It also interacts with both nuclear membrane proteins and the chromatin and is thought to participate in many important functions within the cell nucleus. Defects in A-type lamins cause cardiomyopathy, muscular dystrophy, peripheral neuropathy, lipodystrophy, and progeroid disorders. In contrast, the only bona fide link between the B-type lamins and human disease is a rare demyelinating disease of the central nervous system—adult-onset autosomal-dominant leukoencephalopathy, caused by a duplication of the gene for lamin B1. However, this leukoencephalopathy is not the only association between the brain and B-type nuclear lamins. Studies of conventional and tissue-specific knockout mice have demonstrated that B-type lamins play essential roles in neuronal migration in the developing brain and in neuronal survival. The importance of A-type lamin expression in the brain is unclear, but it is intriguing that the adult brain preferentially expresses lamin C rather than lamin A, very likely due to microRNA-mediated removal of prelamin A transcripts. Here, we review recent studies on nuclear lamins, focusing on the function and regulation of the nuclear lamins in the central nervous system.
PMCID: PMC3538886  PMID: 23065386
Nuclear lamina; brain development; A-type lamins; B-type lamins; differential gene expression
14.  Does Oxidative Inactivation of CD45 Phosphatase in Rheumatoid Arthritis Underlie Immune Hyporesponsiveness? 
Antioxidants & Redox Signaling  2013;19(18):2280-2285.
The protein tyrosine phosphatase (PTP) CD45 is critical in regulating the earliest steps in T-cell-receptor signaling but, similar to all PTPs, it is susceptible to oxidative inactivation. Given the widely reported effects of oxidant damage associated with rheumatoid arthritis (RA), we examined whether CD45 phosphatase activity was altered in CD4+ T cells from RA patients and related this to CD4+ T-cell function and redox status. CD45 phosphatase specific activity in T cells from RA peripheral blood (PB) and synovial fluid was 56% and 59% lower than in healthy control (HC) PB, respectively. In contrast, CD45 activity in T cells from disease controls (DSC) was not significantly different from HC. Both reduced glutathione (GSH) (p<0.001) and oxidized glutathione (GSSG) (p<0.05) were significantly lower in RA PB T cells compared with HC PB T cells. Treatment of RA PB T cells with the GSH precursor N-acetyl cysteine increased CD45 phosphatase activity and proliferation, while it decreased Lck kinase phosphorylation, which is regulated by CD45. Our observations lead to the hypothesis that the largely reversible oxidative inactivation of the CD45 phosphatase may underlie the decreased signaling efficiency and functional responsiveness which are characteristic of RA PB CD4+ T cells. Antioxid. Redox Signal. 19, 2280–2285.
PMCID: PMC3869428  PMID: 23758132
15.  Lipin-1 and lipin-3 together determine adiposity in vivo☆ 
Molecular Metabolism  2013;3(2):145-154.
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
PMCID: PMC3953701  PMID: 24634820
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
16.  Factors Affecting the Prevalence of Strongly and Weakly Carcinogenic and Lower-Risk Human Papillomaviruses in Anal Specimens in a Cohort of Men Who Have Sex with Men (MSM) 
PLoS ONE  2013;8(11):e79492.
MSM are at higher risk for invasive anal cancer. Twelve human papillomaviruses (HPVs) cause cervical cancer in women (Group 1 high-risk HPVs (hrHPVs)) and 13 HPVs are probable/possible causes (Group 2 hrHPVs) of cervical malignancy. HPVs rarely associated with malignancy are classified as lower-risk HPVs (lrHPVs).
Materials and Methods
Dacron-swab anal-cytology specimens were collected from and data complete for 97% (1262/1296) of Multicenter AIDS Cohort Study (MACS) men tested for HPVs using the Linear Array assay. Multivariate Poisson regression analyses estimated adjusted prevalence ratios for Group 1/2 hrHPVs and lrHPVs, controlling for the effects of age, race, ethnicity, sexual partnerships, smoking; HIV-infection characteristics, treatment, and immune status among HIV-infected men.
HIV-infected men showed 35–90% higher prevalence of Group 1/2 hrHPVs and lrHPVs than HIV-uninfected men, and higher prevalence of multi-Type, and multiple risk-group infections. CD4+ T-cell count was inversely associated with HPV Group 2 prevalence (p<0.0001). The number of receptive anal intercourse (RAI) partners reported in the 24 months preceding HPV testing predicted higher prevalence of Group 1/2 hrHPVs. Men reporting ≥30 lifetime male sex partners before their first MACS visit and men reporting ≥1 RAI partners during the 24 months before HPV testing showed 17–24% and 13–17% higher prevalence of lrHPVs (p-values ≤0.05). Men reporting smoking between MACS visit 1 and 24 months before HPV testing showed 1.2-fold higher prevalence of Group 2 hrHPVs (p = 0.03). Both complete adherence to CART (p = 0.02) and HIV load <50 copies/mL (p = 0.04) were protective for Group 1 hrHPVs among HIV-infected men.
HIV-infected men more often show multi-type and multi-group HPV infections HIV-uninfected men. Long-term mutual monogamy and smoking cessation, generally, and CART-adherence that promotes (HIV) viremia control and prevents immunosuppression, specifically among HIV-infected MSM, are important prevention strategies for HPV infections that are relevant to anal cancer.
PMCID: PMC3835810  PMID: 24278140
17.  Development and Characterization of the cobas Human Papillomavirus Test 
Journal of Clinical Microbiology  2013;51(5):1478-1484.
The cobas human papillomavirus (HPV) test, approved by the FDA in April 2011, is a fully automated assay for the detection of 14 high-risk (hr) HPV genotypes from cervical specimens collected in liquid-based cytology medium using real-time PCR amplification of the L1 gene and TaqMan probes. Results are simultaneously reported as positive or negative for the pooled 12 oncogenic HPV types (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68) from channel 1, with HPV16 and HPV18 genotypes read individually from channels 2 and 3. A fourth channel detects the human β-globin gene as a control for sample adequacy and assay inhibition. To optimize clinical sensitivity and specificity, cutoff values (cycle thresholds [CT]) were established for each channel based on the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or greater (≥CIN2). For women aged ≥21 years with cytology results indicating atypical squamous cells of undetermined significance (ASC-US), CT values provided a sensitivity of 90% (95% confidence interval [CI], 81.5% to 94.8%) for the detection of ≥CIN2 and a specificity of 70.5% (95% CI, 68.1% to 72.7%). The analytic sensitivity (limit of detection) ranged from 150 to 2,400 copies/ml, depending on genotype. The analytic specificity, evaluated by comparing the HPV result with a combined comparator of Sanger sequencing and the Qiagen digene HC2 high-risk HPV DNA test (hc2), demonstrated overall positive agreement of 96.3% for 14 hrHPV types in women with ASC-US cytology results who were aged ≥21 years and 86.1% in women with NLIM (negative for intraepithelial neoplasia or malignancy) cytology who were aged ≥30 years. These and other performance validation studies demonstrate that the cobas HPV test is a fully automated and clinically validated robust test.
PMCID: PMC3647918  PMID: 23447640
18.  Germinal center B cells govern their own fate via antibody feedback 
High-affinity antibodies reenter germinal centers (GCs) and limit antigen access, thus causing sustained directional evolution in GCs toward higher-affinity antibody production.
Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells’ own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.
PMCID: PMC3600904  PMID: 23420879
19.  Differential expression of CD148 on leukocyte subsets in inflammatory arthritis 
Arthritis Research & Therapy  2013;15(5):R108.
Monocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease.
We have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays.
We report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity.
CD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.
PMCID: PMC3978474  PMID: 24016860
20.  Adipose subtype–selective recruitment of TLE3 or Prdm16 by PPARγ specifies lipid-storage versus thermogenic gene programs 
Cell metabolism  2013;17(3):423-435.
Transcriptional effectors of white adipocyte-selective gene expression have not been described. Here we show that TLE3 is a white-selective cofactor that acts reciprocally with the brown-selective cofactor Prdm16 to specify lipid storage and thermogenic gene programs. Occupancy of TLE3 and Prdm16 on certain promoters is mutually exclusive, due to the ability of TLE3 to disrupt the physical interaction between Prdm16 and PPARγ. When expressed at elevated levels in brown fat, TLE3 counters Prdm16, suppressing brown-selective genes and inducing white-selective genes, resulting in impaired fatty acid oxidation and thermogenesis. Conversely, mice lacking TLE3 in adipose tissue show enhanced thermogenesis in inguinal white adipose depots and are protected from age-dependent deterioration of brown adipose tissue function. Our results suggest that the establishment of distinct adipocyte phenotypes with different capacities for thermogenesis and lipid storage is accomplished in part through the cell type–selective recruitment of TLE3 or Prdm16 to key adipocyte target genes.
PMCID: PMC3626567  PMID: 23473036
21.  Targeting Protein Prenylation in Progeria 
Science translational medicine  2013;5(171):171ps3.
A clinical trial of a protein farnesyltransferase inhibitor (lonafarnib) for the treatment of Hutchinson-Gilford progeria syndrome (HGPS) was recently completed. Here, we discuss the mutation that causes HGPS, the rationale for inhibiting protein farnesyltransferase, the potential limitations of this therapeutic approach, and new potential strategies for treating the disease.
PMCID: PMC3725554  PMID: 23390246
22.  Chylomicronemia mutations yield new insights into interactions between lipoprotein lipase and GPIHBP1 
Human Molecular Genetics  2012;21(13):2961-2972.
Lipoprotein lipase (LPL) is a 448-amino-acid head-to-tail dimeric enzyme that hydrolyzes triglycerides within capillaries. LPL is secreted by parenchymal cells into the interstitial spaces; it then binds to GPIHBP1 (glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1) on the basolateral face of endothelial cells and is transported to the capillary lumen. A pair of amino acid substitutions, C418Y and E421K, abolish LPL binding to GPIHBP1, suggesting that the C-terminal portion of LPL is important for GPIHBP1 binding. However, a role for LPL's N terminus has not been excluded, and published evidence has suggested that only full-length homodimers are capable of binding GPIHBP1. Here, we show that LPL's C-terminal domain is sufficient for GPIHBP1 binding. We found, serendipitously, that two LPL missense mutations, G409R and E410V, render LPL susceptible to cleavage at residue 297 (a known furin cleavage site). The C terminus of these mutants (residues 298–448), bound to GPIHBP1 avidly, independent of the N-terminal fragment. We also generated an LPL construct with an in-frame deletion of the N-terminal catalytic domain (residues 50–289); this mutant was secreted but also was cleaved at residue 297. Once again, the C-terminal domain (residues 298–448) bound GPIHBP1 avidly. The binding of the C-terminal fragment to GPIHBP1 was eliminated by C418Y or E421K mutations. After exposure to denaturing conditions, the C-terminal fragment of LPL refolds and binds GPIHBP1 avidly. Thus, the binding of LPL to GPIHBP1 requires only the C-terminal portion of LPL and does not depend on full-length LPL homodimers.
PMCID: PMC3373243  PMID: 22493000
23.  Influence of Breastfeeding During the Postpartum Oral Glucose Tolerance Test on Plasma Glucose and Insulin 
Obstetrics and gynecology  2012;120(1):136-143.
To examine the effect of breastfeeding during the postpartum oral glucose tolerance test (OGTT) on maternal blood glucose and insulin among women with recent gestational diabetes mellitus (GDM).
Participants were enrolled in the Study of Women, Infant Feeding, and Type 2 Diabetes (SWIFT), a prospective observational cohort study of Kaiser Permanente Northern California members who had been diagnosed with GDM and subsequently underwent a 2-hour 75 gram OGTT at 6–9 weeks postpartum for the SWIFT enrollment examinations from 2008 to 2011. For this analysis, we selected 835 SWIFT participants who reported any intensity of lactation and were observed either breastfeeding their infant (ie, putting the infant to the breast) or not breastfeeding during the OGTT.
Of 835 lactating women, 205 (25%) breastfed their infant during the 2-hr 75 g OGTT at 6–9 weeks postpartum. Mean (SD) duration of breastfeeding during the OGTT was 15.3 (8.1) minutes. Compared with not having breastfed during the OGTT, having breastfed during the test was associated with lower adjusted mean (95%CI) 2-hr glucose (mg/dl) by −6.2 (−11.5, −1.0), P=0.02, 2-hr insulin (μU/ml) by −15.1 (−26.8, −3.5 ), P=0.01, and natural log 2-hr insulin by −0.15 (−0.25, −0.06), P<0.01, and with higher insulin sensitivity index0, 120 by 0.08 (0.02, 0.15), P=0.02, but no differences in plasma fasting glucose or insulin concentrations.
Among postpartum women with recent GDM, breastfeeding an infant during the 2-hr 75 g OGTT may modestly lower plasma 2-hr glucose (5% lower on average), as well as insulin concentrations in response to ingestion of glucose.
PMCID: PMC3427537  PMID: 22914402
24.  Circumventing Embryonic Lethality with Lcmt1 Deficiency: Generation of Hypomorphic Lcmt1 Mice with Reduced Protein Phosphatase 2A Methyltransferase Expression and Defects in Insulin Signaling 
PLoS ONE  2013;8(6):e65967.
Protein phosphatase 2A (PP2A), the major serine/threonine phosphatase in eukaryotic cells, is a heterotrimeric protein composed of structural, catalytic, and targeting subunits. PP2A assembly is governed by a variety of mechanisms, one of which is carboxyl-terminal methylation of the catalytic subunit by the leucine carboxyl methyltransferase LCMT1. PP2A is nearly stoichiometrically methylated in the cytosol, and although some PP2A targeting subunits bind independently of methylation, this modification is required for the binding of others. To examine the role of this methylation reaction in mammalian tissues, we generated a mouse harboring a gene-trap cassette within intron 1 of Lcmt1. Due to splicing around the insertion, Lcmt1 transcript and LCMT1 protein levels were reduced but not eliminated. LCMT1 activity and methylation of PP2A were reduced in a coordinate fashion, suggesting that LCMT1 is the only PP2A methyltransferase. These mice exhibited an insulin-resistance phenotype, indicating a role for this methyltransferase in signaling in insulin-sensitive tissues. Tissues from these animals will be vital for the in vivo identification of methylation-sensitive substrates of PP2A and how they respond to differing physiological conditions.
PMCID: PMC3688711  PMID: 23840384
25.  Development and validation of a robust automated analysis of plasma phospholipid fatty acids for metabolic phenotyping of large epidemiological studies 
Genome Medicine  2013;5(4):39.
A fully automated, high-throughput method was developed to profile the fatty acids of phospholipids from human plasma samples for application to a large epidemiological sample set (n > 25,000). We report here on the data obtained for the quality-control materials used with the first 860 batches, and the validation process used. The method consists of two robotic systems combined with gas chromatography, performing lipid extraction, phospholipid isolation, hydrolysis and derivatization to fatty-acid methyl esters, and on-line analysis. This is the first report showing that fatty-acid profiling is an achievable strategy for metabolic phenotyping in very large epidemiological and genetic studies.
PMCID: PMC3706814  PMID: 23618465

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