👤 Hua-Chen Chan

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91,462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee. Show less

We investigated the kinetics of 4N-acetyl-pentapeptides, Ac-P1, Ac-P2, Ac-P3, and Ac-P4, regarding inhibition of mushroom tyrosinase activity. The peptides sequences of Ac-P1, Ac-P2, Ac-P3, and Ac-P4 were Ac-RSRFK, Ac-KSRFR, Ac-KSSFR, and Ac-RSRFS, respectively. The 4N-acetyl-pentapeptides were able to reduce the oxidation of l-DOPA by tyrosinase in a dose-dependent manner. Of the 4N-acetyl-pentapeptides, only Ac-P4 exhibited lag time (80 s) at a concentration of 0.5 mg/mL. The tyrosinase inhibitory effects of Ac-P4 (IC50 0.29 mg/mL) were more effective than those of Ac-P1, Ac-P2, and Ac-P3, in which IC50s were 0.75 mg/mL, 0.78 mg/mL, and 0.81 mg/mL, respectively. Kinetic analysis demonstrated that all 4N-acetyl-pentapeptides were mixed-type tyrosinase inhibitors. Furthermore, 0.1 mg/mL of Ac-P4 exhibited significant melanogenesis inhibition on B16F10 melanoma cells and was more effective than kojic acid. The melanogenesis inhibition of Ac-P4 was dose-dependent and did not induce any cytotoxicity on B16F10 melanoma cells. Show less

Glioblastoma multiforme (GBM), a highly invasive primary brain tumour, remains an incurable disease. Rho GTPases and their activators, guanine nucleotide exchange factors (GEFs), have central roles in GBM invasion. Anti-angiogenic therapies may stimulate GBM invasion via HGF/c-Met signalling. We aim to identify mediators of HGF-induced GBM invasion that may represent targets in a combination anti-angiogenic/anti-invasion therapeutic paradigm. Guanine nucleotide exchange factor expression was measured by microarray analysis and western blotting. Specific depletion of proteins was accomplished using siRNA. Cell invasion was determined using matrigel and brain slice assays. Cell proliferation and survival were monitored using sulforhodamine B and colony formation assays. Guanine nucleotide exchange factor and GTPase activities were determined using specific affinity precipitation assays. We found that expression of Dock7, a GEF, is elevated in human GBM tissue in comparison with non-neoplastic brain. We showed that Dock7 mediates serum- and HGF-induced glioblastoma cell invasion. We also showed that Dock7 co-immunoprecipitates with c-Met and that this interaction is enhanced upon HGF stimulation in a manner that is dependent on the adaptor protein Gab1. Dock7 and Gab1 also co-immunoprecipitate in an HGF-dependent manner. Furthermore, Gab1 is required for HGF-induced Dock7 and Rac1 activation and glioblastoma cell invasion. Dock7 mediates HGF-induced GBM invasion. Targeting Dock7 in GBM may inhibit c-MET-mediated invasion in tumours treated with anti-angiogenic regimens. Show less

Known genetic loci explain only a small proportion of the familial relative risk of colorectal cancer (CRC). We conducted a genome-wide association study of CRC in East Asians with 14,963 cases and 31,945 controls and identified 6 new loci associated with CRC risk (P = 3.42 × 10(-8) to 9.22 × 10(-21)) at 10q22.3, 10q25.2, 11q12.2, 12p13.31, 17p13.3 and 19q13.2. Two of these loci map to genes (TCF7L2 and TGFB1) with established roles in colorectal tumorigenesis. Four other loci are located in or near genes involved in transcriptional regulation (ZMIZ1), genome maintenance (FEN1), fatty acid metabolism (FADS1 and FADS2), cancer cell motility and metastasis (CD9), and cell growth and differentiation (NXN). We also found suggestive evidence for three additional loci associated with CRC risk near genome-wide significance at 8q24.11, 10q21.1 and 10q24.2. Furthermore, we replicated 22 previously reported CRC-associated loci. Our study provides insights into the genetic basis of CRC and suggests the involvement of new biological pathways. Show less

Multi-causality and heterogeneity of phenotypes and genotypes characterize complex diseases. In a database with comprehensive collection of phenotypes and genotypes, we compared the performance of common machine learning methods to generate mathematical models to predict diabetic kidney disease (DKD). In a prospective cohort of type 2 diabetic patients, we selected 119 subjects with DKD and 554 without DKD at enrolment and after a median follow-up period of 7.8 years for model training, testing and validation using seven machine learning methods (partial least square regression, the classification and regression tree, the C5.0 decision tree, random forest, naïve Bayes classification, neural network and support vector machine). We used 17 clinical attributes and 70 single nucleotide polymorphisms (SNPs) of 54 candidate genes to build different models. The top attributes selected by the best-performing models were then used to build models with performance comparable to those using the entire dataset. Age, age of diagnosis, systolic blood pressure and genetic polymorphisms of uteroglobin and lipid metabolism were selected by most methods. Models generated by support vector machine (svmRadial) and random forest (cforest) had the best prediction accuracy whereas models derived from naïve Bayes classifier and partial least squares regression had the least optimal performance. Using 10 clinical attributes (systolic and diastolic blood pressure, age, age of diagnosis, triglyceride, white blood cell count, total cholesterol, waist to hip ratio, LDL cholesterol, and alcohol intake) and 5 genetic attributes (UGB G38A, LIPC -514C > T, APOB Thr71Ile, APOC3 3206T > G and APOC3 1100C > T), selected most often by SVM and cforest, we were able to build high-performance models. Amongst different machine learning methods, svmRadial and cforest had the best performance. Genetic polymorphisms related to inflammation and lipid metabolism warrant further investigation for their associations with DKD. Show less

Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis) is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney) cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm), human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress. Show less

Neuronal ceroid lipofuscinosis (NCL), commonly referred to as Batten disease, is a group of autosomal recessive neurodegenerative diseases of childhood characterized by seizures, blindness, motor and cognitive decline and premature death. Currently, there are over 400 known mutations in 14 different genes, leading to five overlapping clinical variants of NCL. A large portion of these mutations lead to premature stop codons (PTCs) and are predicted to predispose mRNA transcripts to nonsense-mediated decay (NMD). Nonsense-mediated decay is associated with a number of other genetic diseases and is an important regulator of disease pathogenesis. We contend that NMD targets PTCs in NCL gene transcripts for degradation. A number of PTC mutations in CLN1, CLN2 and CLN3 lead to a significant decrease in mRNA transcripts and a corresponding decrease in protein levels and function in patient-derived lymphoblast cell lines. Inhibiting NMD leads to an increased transcript level, and where protein function is known, increased activity. Treatment with read-through drugs also leads to increased protein function. Thus, NMD provides a promising therapeutic target that would allow read-through of transcripts to enhance protein function and possibly ameliorate Batten disease pathogenesis. Show less

CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133(+) -C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133(+) -C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133(+) -C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133(+) -C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133(+) -C6. A decline in the invasion of hCD133(+) -C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133(+) -C6. In vivo study further showed that hCD133(+) -C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133(+) -C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133(+) -C6 in vitro, as well as progressive tumor formation in vivo. Show less

Glucose-dependent insulinotropic peptide (GIP) exerts multiple biological effects via the G-protein-coupled receptor GIPR, including glucose-stimulated insulin production and secretion, cell proliferation, and antiapoptosis in pancreatic β-cells. In an obese state, the circulating level of GIP is elevated. GIPR-knockout mice are resistant to high-fat-diet-induced obesity. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism. In our study, we overexpressed GIPR in 3T3-L1 CAR adipocytes and demonstrated that GIP impaired the physiological functions of adipocytes as a consequence of increased production of inflammatory cytokines and chemokines and phosphorylation of IkB kinase (IKK)-β through activation of the cAMP-PKA pathway. Activation of Jun N-terminal kinase (JNK) pathway was also observed during GIP-induced inflammatory responses in adipocytes. The inhibition of JNK blocked GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. In addition, GIP-induced inflammatory response increased basal glucose uptake but inhibited insulin-stimulated glucose uptake. Moreover, GIP-induced adipocyte inflammation impaired insulin signaling in adipocytes as demonstrated by a reduction of AKT phosphorylation. Our results suggest that GIP might be one of the stimuli attributable to obesity-induced insulin resistance via the induction of adipocyte inflammation. Show less

BTN1, the yeast homolog to human CLN3 (which is defective in Batten disease), has been implicated in the regulation of vacuolar pH, potentially by modulating vacuolar-type H(+)-ATPase (V-ATPase) activity. However, we report that Btn1p and the V-ATPase complex do not physically interact, suggesting that any influence that Btn1p has on V-ATPase is indirect. Because membrane lipid environment plays a crucial role in the activity and function of membrane proteins, we investigated whether cells lacking BTN1 have altered membrane phospholipid content. Deletion of BTN1 (btn1-Δ) led to a decreased level of phosphatidylethanolamine (PtdEtn) in both mitochondrial and vacuolar membranes. In yeast there are two phosphatidylserine (PtdSer) decarboxylases, Psd1p and Psd2p, and these proteins are responsible for the synthesis of PtdEtn in mitochondria and Golgi-endosome, respectively. Deletion of both BTN1 and PSD1 (btn1-Δ psd1-Δ) led to a further decrease in levels of PtdEtn in ER membranes associated to mitochondria (MAMs), with a parallel increase in PtdSer. Fluorescent-labeled PtdSer (NBD-PtdSer) transport assays demonstrated that transport of NBD-PtdSer from the ER to both mitochondria and endosomes and/or vacuole is affected in btn1-Δ cells. Moreover, btn1-Δ affects the synthesis of PtdEtn by the Kennedy pathway and impairs the ability of psd1-Δ cells to restore PtdEtn to normal levels in mitochondria and vacuoles by ethanolamine addition. In summary, lack of Btn1p alters phospholipid levels and might play a role in regulating their subcellular distribution. Show less

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC. Show less

Molecular and cellular signaling pathways are involved in the process of neural differentiation from human embryonic stem cells (hESC) to terminally differentiated neurons. The Sonic hedgehog (SHH) morphogen is required to direct the differentiation of hESC to several neural subtypes, for example, dopaminergic (DA) or motor neurons. However, the roles of SHH signaling and the pathway target genes that regulate the diversity of cellular responses arising from SHH activation during neurogenesis of hESC have yet to be elucidated. In this study, we report that overexpression of SHH in hESC promotes the derivation of neuroprogenitors (NP), increases proliferation of NP, and subsequently increases the yield of DA neurons. Next, gene expression changes resulting from the overexpression of SHH in hESC-derived NP were examined by genome-wide transcriptional profiling. Categorizing the differentially expressed genes according to the Gene Ontology biological processes showed that they are involved in numerous cellular processes, including neural development, NP proliferation, and neural specification. In silico GLI-binding sites analysis of the differentially expressed genes also identified a set of putative novel direct target genes of SHH in hESC-derived NP, which are involved in nervous system development. Electrophoretic mobility shift assays and promoter-luciferase assays confirmed that GLI1 binds to the promoter region and activates transcription of HEY2, a NOTCH signaling target gene. Taken together, our data provide evidence for the first time that there is cross-talk between the NOTCH and SHH signaling pathways in hESC-derived NP and also provide significant new insights into transcriptional targets in SHH-mediated neural differentiation of hESC. Show less

This study was aimed at the elucidation of the pathogenesis of glucotoxicity, i.e. the mechanism whereby hyperglycaemia damages pancreatic beta cells. The identification of pathways in the process may help identify targets for beta cell-protective therapy. Carbohydrate response element-binding protein (ChREBP), a transcription factor that regulates the expression of multiple hyperglycaemia-induced genes, is produced in abundance in pancreatic beta cells. We hypothesise that ChREBP plays a pivotal role in mediating beta cell glucotoxicity. We assessed the role of ChREBP in glucotoxicity in 832/13 beta cells, isolated mouse islets and human pancreas tissue sections using multiple complementary approaches under control and high-glucose-challenge conditions as well as in adeno-associated virus-induced beta cell-specific overexpression of Chrebp (also known as Mlxipl) in mice. Under both in vitro and in vivo conditions, ChREBP activates downstream target genes, including fatty acid synthase and thioredoxin-interacting protein, leading to lipid accumulation, increased oxidative stress, reduced insulin gene transcription/secretion and enhanced caspase activity and apoptosis, processes that collectively define glucotoxicity. Immunoreactive ChREBP is enriched in the nucleuses of beta cells in pancreatic tissue sections from diabetic individuals compared with non-diabetic individuals. Finally, we demonstrate that induced beta cell-specific Chrebp overexpression is sufficient to phenocopy the glucotoxicity manifestations of hyperglycaemia in mice in vivo. These data indicate that ChREBP is a key transcription factor that mediates many of the hyperglycaemia-induced activations in a gene expression programme that underlies beta cell glucotoxicity at the molecular, cellular and whole animal levels. Show less

Lutein protects retinal neurons by its anti-oxidative and anti-apoptotic properties in ischemia/reperfusion (I/R) injury while its anti-inflammatory effects remain unknown. As Müller cells play a critical role in retinal inflammation, the effect of lutein on Müller cells was investigated in a murine model of I/R injury and a culture model of hypoxic damage. Unilateral retinal I/R was induced by a blockade of internal carotid artery using the intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein (0.2 mg/kg) or vehicle was administered. Flash electroretinogram (flash ERG) and glial fibrillary acidic protein (GFAP) activation were assessed. Lutein's effect on Müller cells was further evaluated in immortalized rat Müller cells (rMC-1) challenged with cobalt chloride-induced hypoxia. Levels of IL-1β, cyclooxygenase-2 (Cox-2), TNFα, and nuclear factor-NF-kappa-B (NF-κB) were examined by Western blot analysis. Lutein treatment minimized deterioration of b-wave/a-wave ratio and oscillatory potentials as well as inhibited up-regulation of GFAP in retinal I/R injury. In cultured Müller cells, lutein treatment increased cell viability and reduced level of nuclear NF-κB, IL-1β, and Cox-2, but not TNFα after hypoxic injury. Reduced gliosis in I/R retina was observed with lutein treatment, which may contribute to preserved retinal function. Less production of pro-inflammatory factors from Müller cells suggested an anti-inflammatory role of lutein in retinal ischemic/hypoxic injury. Together with our previous studies, our results suggest that lutein protected the retina from ischemic/hypoxic damage by its anti-oxidative, anti-apoptotic, and anti-inflammatory properties. Show less

The abused volatile solvent toluene shares many behavioral effects with classic central nervous system depressants such as ethanol. Similarities between toluene and ethanol have also been demonstrated using in vitro electrophysiology. Together, these studies suggest that toluene and ethanol may be acting, at least in part, via common mechanisms. We used the genetic model, Caenorhabditis elegans, to examine the behavioral effects of toluene in a simple system, and used mutant strains known to have altered responses to other CNS depressants to examine the involvement of those genes in the motor effects induced by toluene. Toluene vapor brings about an altered pattern of locomotion in wild-type worms that is visibly distinct from that generated by ethanol. Mutants of the slo-1, rab-3 and unc-64 genes that are resistant to ethanol or the volatile anesthetic halothane show no resistance to toluene. A mutation in the unc-79 gene results in hypersensitivity to ethanol, halothane and toluene indicating a possible convergence of mechanisms of the three compounds. We screened for, and isolated, two mutations that generate resistance to the locomotor depressing effects of toluene and do not alter sensitivity to ethanol. In C. elegans, ethanol and toluene have distinct behavioral effects and minimal overlap in terms of the genes responsible for these effects. These findings demonstrate that the C. elegans model system provides a unique and sensitive means of delineating both the commonalities as well as the differences in the neurochemical effects of classical CNS depressants and abused volatile inhalants. Show less

Autoantibodies to brain proteins are present in Juvenile Neuronal Ceroid Lipofuscinosis (Batten disease) patients and in the Cln3-/- mouse model of this disease, suggesting an autoimmune component to pathogenesis. Using genetic or pharmaceutical approaches to attenuate this immune response in Cln3-/- mice, we demonstrate decreased neuroinflammation, decreased deposition of immunoglobulin G in the brain and protection of vulnerable neuron populations. Moreover, immune suppression results in a significant improvement in motor performance providing for the first plausible therapeutic approach for juvenile Batten disease. Show less

Although widely prescribed for their potent antiinflammatory actions, glucocorticoid drugs (e.g., dexamethasone) cause undesirable side effects that are features of the metabolic syndrome, including hyperglycemia, fatty liver, insulin resistance, and type II diabetes. Liver x receptors (LXRs) are nuclear receptors that respond to cholesterol metabolites and regulate the expression of a subset of glucocorticoid target genes. Here, we show LXRβ is required to mediate many of the negative side effects of glucocorticoids. Mice lacking LXRβ (but not LXRα) were resistant to dexamethasone-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, but remained sensitive to dexamethasone-dependent repression of the immune system. In vivo, LXRα/β knockout mice demonstrated reduced dexamethasone-induced expression of the key hepatic gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK). In perfused liver and primary mouse hepatocytes, LXRβ was required for glucocorticoid-induced recruitment of the glucocorticoid receptor to the PEPCK promoter. These findings suggest a new avenue for the design of safer glucocorticoid drugs through a mechanism of selective glucocorticoid receptor transactivation. Show less

The association of small, ubiquitin-related modifier-specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat-containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway. Show less

Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties. Show less

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation. Show less

Gprc5b, a retinoic acid-inducible orphan G protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses. We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3' terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line. Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences. Show less

Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP. Show less

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV-infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients. Show less

Recent genome-wide association studies have identified multiple novel loci associated with obesity in Europeans. We hypothesized that these genetic variants may be associated with obesity and type 2 diabetes (T2D) in Chinese. We examined 14 associated single-nucleotide polymorphisms at 12 loci (NEGR1, SEC16B, TMEM18, ETV5/DGKG, GNPDA2, LIN7C/BDNF, MTCH2, BCDIN3D/FAIM2, SH2B1, FTO, MC4R, and KCTD15) in 605 healthy adults, 1087 healthy adolescents and 6013 T2D patients from Hong Kong. The European at-risk alleles at five loci including GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 were significantly associated with increased body mass index (BMI), waist circumference (4.5 x 10(-8) < P < 0.024), and/or obesity risk (odds ratio 1.14-1.22, 2.0 x 10(-5) < P < 0.002) in our Chinese populations. The former four loci as well as LIN7C/BDNF were also modestly associated with T2D risk (odds ratio 1.09-1.22, 0.008 < P < 0.041), but the associations were lost after adjustment for BMI, suggesting their roles in T2D risk are mediated through modulation of adiposity. Joint effect analyses of the five adiposity loci revealed an increase of about 0.29 kg/m(2) in BMI with each additional copy of at-risk allele (P(trend) = 4.2 x 10(-12)). Our findings support the important contribution of GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 in the regulation of adiposity, which in turn affects T2D risk in Chinese. Show less

Juvenile neuronal ceroid lipofuscinoses (JNCL) or juvenile Batten disease is a recessively inherited childhood neurodegenerative disorder resulting from a mutation in CLN3, which encodes a putative lysosomal protein of unknown function. Recent evidence suggests that a disruption in CLN3 function results in altered regulation of arginine transport into lysosomes, and may influence intracellular arginine levels. We sought to investigate the possible consequences of arginine dysregulation in the brain of the Cln3(-/-) mouse model of JNCL. Using a combination of enzyme assays, metabolite profiling, quantitative reverse-transcription polymerase chain reaction and Western blotting, we analysed the activities and expression of enzymes involved in arginine metabolism in the cerebral cortex and cerebellum of Cln3(-/-) mice over several developmental time points. We report subtle, but significant changes in the activities of enzymes involved in the citrulline-NO recycling pathway, and altered regulation of neuronal nitric oxide synthase in the cortex and cerebellum of Cln3(-/-) mice. In addition, a significant decrease in arginine transport into cerebellar granule cells was observed, despite an apparent upregulation of the cationic amino acid transporter-1 transporter at the cell surface. Our results provide further evidence that CLN3 function and arginine homeostasis are intricately related, and that cellular mechanisms may act to compensate for the loss of this protein. This and other studies indicate that CLN3 dysfunction in JNCL may result in multiple disturbances in metabolism that together contribute to the pathophysiological processes underlying this disease. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is a fatal inherited neurodegenerative disorder. The major clinical features of this disease are vision loss, seizures and progressive cognitive and motor decline starting in childhood. Mutations in CLN3 are known to cause the disease, allowing the generation of mouse models that are powerful tools for JNCL research. In this study, we applied behavioural phenotyping protocols to test for early behavioural alterations in Cln3(Deltaex7/8) knock-in mice, a genetic model that harbours the most common disease-causing CLN3 mutation. We found delayed acquisition of developmental milestones, including negative geotaxis, grasping, wire suspension time and postural reflex in both homozygous and heterozygous Cln3(Deltaex7/8) preweaning pups. To further investigate the consequences of this neurodevelopmental delay, we studied the behaviour of juvenile mice and found that homozygous and heterozygous Cln3(Deltaex7/8) knock-in mice also exhibit deficits in exploratory activity. Moreover, when analysing motor behaviour, we observed severe motor deficits in Cln3(Deltaex7/8) homozygous mice, but only a mild impairment in motor co-ordination and ambulatory gait in Cln3(Deltaex7/8) heterozygous animals. This study reveals previously overlooked behaviour deficits in neonate and young adult Cln3(Deltaex7/8) mice indicating neurodevelopmental delay as a putative novel component of JNCL. Show less

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to nonstem spermatogonia. We isolated type A spermatogonia (stem and nonstem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1-positive and GFRA1-negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are overexpressed in the former cell population with a >2-fold change. Interestingly, the highest level of overexpression was observed for Csf1r, encoding the receptor for macrophage colony-stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R that may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18-4. Semiquantitative RT-PCR and immunohistochemistry confirmed the previously mentioned microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation. Show less

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements. Show less

Juvenile neuronal ceroid lipofuscinoses (JNCL), commonly known as Batten disease, is a progressive neurodegenerative disorder of childhood characterized by blindness, seizures, motor and cognitive decline, leading to death in early adulthood. Mutations within the CLN3 gene, which encodes a putative lysosomal protein of unknown function, are the underlying cause of JNCL. Over 85% of JNCL patients harbor a 1 kb deletion that is predicted to result in a truncated CLN3 protein and is presumed to be a null mutation. A recent study by Kitzmuller et al. (1) suggested that the 1 kb deletion-associated truncated protein may have partial function, and proposed that JNCL is a mutation-specific disease. In addition, the validity of the original and most widely utilized JNCL mouse model, the Cln3(Deltaex1-6) mouse, as a true null mutant was questioned. We report a substantial decrease in the transcript level of the truncated CLN3 gene product in cells from 1 kb deletion patients. We contend that the truncated CLN3 protein is unlikely to be expressed in JNCL patients since cellular quality control mechanisms at the RNA and protein levels are likely to degrade the mutant transcript and polypeptides. Moreover, we present analysis identifying the expressed transcripts present in Cln3(Deltaex1-6) mouse brain. From the analysis of expressed Cln3(Deltaex1-6) mouse transcripts, combined with in silico prediction of the expected consequences of the Cln3(Deltaex1-6) mutation on these transcripts, we argue that aberrant Cln3 proteins are unlikely to be expressed in this disease model. Taken together our results indicate that the most common mutation associated with JNCL results in a loss of functional CLN3, that the Cln3(Deltaex1-6) mouse harbors a null Cln3 allele, and that it therefore represents a valid model for this disease. Show less

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family. Show less