👤 Nan-Haw Chow

Hepatocellular carcinoma (HCC) exhibits diverse aetiologies and molecular heterogeneity, with a median 5-year overall survival of <70% due to high recurrence rates following curative-intent surgery. This study investigated the complex tumour microenvironment (TME) in HCC and explored interactions between various cell types and their roles in disease recurrence. Using a multi-omics approach on multi-region samples of surgically resected HCC from the PLANet 1.0 cohort (NCT03267641), we performed spatial transcriptomics on 17 tissue samples from four patients and bulk RNA sequencing on 329 sectors from 90 patients. Findings were validated using immunofluorescence and multiplex immunohistochemistry. Our analysis revealed extensive intra- and intertumour gene expression heterogeneity and identified a specific subset of endothelial cells (ECs), INTS6 INTS6 The spatial co-localisation of cell types plays a significant role in the recurrence of hepatocellular carcinoma. In this study, we have pinpointed a particular group of endothelial cells, known as INTS6+ endothelial cells, which are spatially colocalised with tumour cells and enriched in microvascular invasion regions in patients experiencing recurrence. These discoveries highlight novel therapeutic targets that focus on endothelial cell interactions within the tumour microenvironment to prevent recurrence and enhance overall patient survival. Show less

Glucagon-like peptide-1 receptor (GLP-1R)/glucose-dependent insulinotropic peptide receptor (GIPR) agonistic analogs have yielded superior results in enhancing glycemic control and weight management compared to GLP-1R agonism alone. Intriguingly, GIPR agonism appears to induce antiemetic effects, potentially alleviating part of the nausea and vomiting side effects common to GLP-1R agonists like semaglutide. Here, we show in rats and shrews that GIPR agonism blocks emesis and attenuates other malaise behaviors elicited by GLP-1R activation while maintaining reduced food intake and body weight loss and improved glucose tolerance. The GLP-1R/GIPR agonist tirzepatide induced significantly fewer side effects than equipotent doses of semaglutide. These findings underscore the therapeutic potential of combined pharmaceutical strategies activating both incretin systems, leading to enhanced therapeutic index and reduced occurrence of nausea and vomiting for obesity and diabetes treatments. Show less

Dengue virus infection can cause severe complications due to vascular leakage. Angiopoietin-like protein 4 (ANGPTL4) regulates vascular permeability, but its role in dengue pathogenesis is unclear. This study investigated the association between plasma ANGPTL4 levels and dengue severity in Singapore adults. Plasma samples from 48 dengue patients (24 severe and 24 non-severe) during acute and convalescent phases were selected from the prospective COhort study on progression of DENgue severity in Singapore adults (CODEN) cohort. The CODEN was conducted at the National Centre for Infectious Diseases, Tan Tock Seng Hospital, from June 2016 to January 2020. ANGPTL4 levels were measured and compared to 152 healthy controls. Logistic regression assessed the relationship between plasma ANGPTL4 concentrations and disease severity. There were no statistically significant differences in ANGPTL4 levels between severe and non-severe dengue patients during acute (677.4 vs. 909.1 pg/mL, Show less

Patients with schizophrenia show a disproportionally increased risk of cardiovascular disease. Hypertriglyceridemia is prevalent in this population; however, how this relates to levels of remnant cholesterol, triglyceride (TG)-rich lipoprotein (TRL) particle size and composition, TG turnover, and apolipoprotein (apo) and angiopoietin-like protein (ANGPTL) concentrations is unknown. Fasting levels of cholesterol (total [TC], LDL-C, HDL-C, non-HDL-C and remnant cholesterol) and TG were determined in 110 patients diagnosed with schizophrenia, and 46 healthy controls. TRL particle size, concentration and composition, and β-hydroxybutyrate (TG turnover marker) were assessed by NMR. Levels of apoCII, apoCIII, apoE, ANGPTL3, ANGPTL4, and ANGPTL8 were measured by ELISA, and apoCII, apoCIII and apoE were further evaluated in HDL and non-HDL fractions. Patients with schizophrenia had significantly elevated TG, TG:apoB ratio, non-HDL-C, remnant cholesterol, non-HDL-apoCII and non-HDL-apoCIII, and HDL-apoE (all P < 0.05), lower HDL-C and apoA-I (all P < 0.001), and comparable apoB, TC, TC:apoB ratio, LDL-C, β-hydroxybutyrate, ANGPTL3, ANGPTL4 and ANGPTL8 to healthy controls. Patients had a 12.0- and 2.5-fold increase in the concentration of large and medium TRL particles respectively, but similar cholesterol:TG ratio within each particle. Plasma TG, remnant cholesterol, and large and medium TRL particle concentrations correlated strongly with apoCII, apoCIII, and apoE in the non-HDL fraction, and with apoCIII and apoE in the HDL fraction in patients with schizophrenia. Differences in TG, HDL-C, TRL particle concentrations, apoCIII, and apoE persisted after adjustment for conventional risk factors. These results are consistent with impaired TRL lipolysis and clearance in patients with schizophrenia which may be responsive to targeting apoCIII. Show less

RNA G-quadruplex (rG4) structures in the 5' untranslated region (5'UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double-strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5'UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4-specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G-quadruplex-triggered fluorogenic hybridization) probes and modulates translation in an rG4-dependent manner. Our results further substantiate the rG4 structure-DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation. Show less

It is unclear to what extent intrathecal inflammation contributes to the pathogenesis in primary progressive multiple sclerosis (PPMS). We conducted an exploratory study to investigate the degree of intrathecal inflammation and its association with biomarkers of disease activity and severity in patients with PPMS. We included patients with PPMS who participated in a randomized controlled trial conducted at the Danish Multiple Sclerosis Center, patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls. We analyzed concentrations of a panel of cytokines in CSF using electrochemiluminescence assays. We then explored the relationship between cytokines found in increased CSF concentrations in patients with PPMS (compared with healthy controls) with CSF concentrations of neurofilament light chain (NFL) and myelin basic protein (MBP), IgG-index, and magnetic resonance imaging (MRI) metrics (volume, magnetization transfer ratio and diffusion tensor imaging) from lesions, normal-appearing white matter, and cortical grey matter. We included 59 patients with PPMS, 40 patients with RRMS, and 21 healthy controls. In patients with PPMS, CSF concentrations of CC chemokine ligand 3 (CCL-3), CXC chemokine ligand 8 (CXCL-8), CXCL-10, interleukin (IL)-10, IL-15, and vascular endothelial growth factor (VEGF)-A were increased compared with healthy controls and comparable with CSF concentrations in patients with RRMS. In addition, patients with PPMS had increased CSF concentrations of IL-12p40, IL-17A, tumor necrosis factor (TNF)-α, and lymphotoxin (LT)-α compared with healthy controls, but concentrations of these cytokines were even higher in patients with RRMS. For the remaining seven cytokines (CCL22, interferon-γ, IL-5, IL-7, IL-16, IL-22, IL-27), we found no difference between patients with PPMS and healthy controls. CSF concentrations of NFL and MBP correlated weakly with concentrations of IL-15, while the remaining proinflammatory cytokines were not associated with CSF concentrations of NFL or MBP. The IgG-index correlated with four cytokines (IL-10, IL-12p40, TNF-α, and LT-α). We did not observe any significant associations between MRI metrics and CSF biomarkers of inflammation. In this exploratory study, we found few and weak associations between intrathecal inflammation and the extent of neuroaxonal damage and demyelination, and no associations between intrathecal inflammation and MRI metrics, in patients with PPMS. Our findings suggest that, for patients with PPMS, these measures of intrathecal inflammation are not associated with the extent of neuroaxonal injury, demyelination, and disease severity, and these processes may therefore have less relevance in PPMS than in relapsing forms of MS. Show less

Men diagnosed with low-risk prostate cancer (PC) are increasingly electing active surveillance (AS) as their initial management strategy. While this may reduce the side effects of treatment for prostate cancer, many men on AS eventually convert to active treatment. PC is one of the most heritable cancers, and genetic factors that predispose to aggressive tumors may help distinguish men who are more likely to discontinue AS. To investigate this, we undertook a multi-institutional genome-wide association study (GWAS) of 5,222 PC patients and 1,139 other patients from replication cohorts, all of whom initially elected AS and were followed over time for the potential outcome of conversion from AS to active treatment. In the GWAS we detected 18 variants associated with conversion, 15 of which were not previously associated with PC risk. With a transcriptome-wide association study (TWAS), we found two genes associated with conversion ( Show less

Pituitary adenylate cyclase-activating peptide (PACAP) receptor (PAC1R) is a class B Gprotein-coupled receptor (GPCR) that is widely expressed in the human body and is involved in neuronal differentiation. As class B GPCRs are known to form heterocomplexes with family members, we hypothesized that PAC1R mediates neuronal differentiation through interaction with a class B GPCR. We used the BRET assay to identify potential interactions between PAC1R and 11 class B GPCRs. Gastric inhibitory polypeptide receptor (GIPR) and secretin receptor were identified as putative binding partners of PAC1R. The effect of heterocomplex formation by PAC1R on receptor activation was evaluated with the cyclic (c)AMP, luciferase reporter, and calcium signaling assays; and the effects on receptor internalization and subcellular localization were examined by confocal microscopy. The results suggested he PAC1R/GIPR heterocomplex suppressed signaling events downstream of PAC1R, including cAMP production, serum response element and calcium signaling, and β-arrestin recruitment. Protein-protein interaction was analyzed in silico, and induction of neuronal differentiation by the PAC1R heterocomplex was assessed in SH-SY5Y neuronal cells by measure the morphological changes and marker genes expression by real-time quantitative PCR and western blot. Over-expression of GIPR suppressed PACAP/PAC1R-mediated neuronal differentiation and the differentiation markers expression in SH-SY5Y cells. GIPR regulates neuronal differentiation through heterocomplex formation with PAC1R. Show less

We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs in a mouse model of acute lung injury (ALI). Purified pericyte-like cells from lung digests using PDGFRβ as a selection marker were expanded in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 hours. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 hours, but this response persisted for 24 hours only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed the activation of pro-inflammatory, cell migration, and angiogenesis-related pathways, whereas processes associated with tissue development and cell differentiation were down-regulated. We validated select upregulated targets in the inflammatory, angiogenic, and cell migratory pathways using functional biological assays in vitro and in vivo. We conclude that lung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury. Show less

Phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), the mammalian ortholog of yeast vesicular protein sorting 34 (Vps34), belongs to the phosphoinositide 3-kinase (PI3K) family. PIK3C3 can phosphorylate phosphatidylinositol (PtdIns) to generate phosphatidylinositol 3-phosphate (PI3P), a phospholipid central to autophagy. Inhibition of PIK3C3 successfully inhibits autophagy. Autophagy maintains cell survival when modifications occur in the cellular environment and helps tumor cells resist metabolic stress and cancer treatment. In addition, PIK3C3 could induce oncogenic transformation and enhance tumor cell proliferation, growth, and invasion through mechanisms independent of autophagy. This review addresses the structural and functional features, tissue distribution, and expression pattern of PIK3C3 in a variety of human tumors and highlights the underlying mechanisms involved in carcinogenesis. The implications in cancer biology, patient prognosis prediction, and cancer therapy are discussed. Altogether, the discovery of pharmacological inhibitors of PIK3C3 could reveal novel strategies for improving treatment outcomes for PIK3C3-mediated human diseases. Show less

Stroke is the second leading cause of death and a major cause of disability worldwide. Both modifiable and non-modifiable risk factors can affect the occurrence of ischemic stroke at varying degrees. Among them, atherosclerosis has been well-recognized as one of the main culprits for the rising incidence of stroke-related mortality. Hence, the current review aimed to summarize the prominent role of lipid metabolism genes such as PCSK9, ApoB, ApoA5, ApoC3, ApoE, and ABCA1 in mediating ischemic stroke occurrence. Show less

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7 Show less

Arginine deprivation is currently being evaluated for its efficacy and safety in clinical trials aimed at combating tumors. However, the cellular signaling and molecular changes in response to such deprivation have not been systematically deciphered. Here, we evaluate the effect of arginine deprivation on human pancreatic cancer cells, with respect to their migratory and invasive potentials and their ability to undergo epithelial-mesenchymal transition (EMT). The transcription factors Snail, Slug, and Twist are regulators of EMT, as indicated by the suppression of E-cadherin and other epithelial markers and adhesion molecules. Our data indicated that arginine starvation inhibited the migration and impaired the adhesion and invasion of the pancreatic cancer cells, decreased Snail, Slug, and Twist expression, and increased E-cadherin expression without altering the expression of vimentin. It is well known that matrix metalloproteinases (MMPs) are important for the events that underlie tumor dissemination. Arginine starvation inhibited the expression of MMP-1 and MMP-9. Furthermore, the PI3K/Akt pathway was altered when the pancreatic cancer cells underwent arginine deprivation as exhibited by the decreased Akt phosphorylation. Thus, these data reveal that arginine deprivation has the potential to decrease the metastatic ability of pancreatic cancer cells. Show less

Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease that is thought to be reversible by changing the diet. To examine the impact of dietary changes on progression and cure of NAFLD, we fed mice a high-fat diet (HFD) or high-fructose diet (HFrD) for 9 weeks, followed by an additional 9 weeks, where mice were given normal chow diet. As predicted, the diet-induced NAFLD elicited changes in glucose tolerance, serum cholesterol, and triglyceride levels in both diet groups. Moreover, the diet-induced NAFLD phenotype was reversed, as measured by the recovery of glucose intolerance and high cholesterol levels when mice were given normal chow diet. However, surprisingly, the elevated serum triglyceride levels persisted. Metagenomic analysis revealed dietary-induced changes of microbiome composition, some of which remained altered even after reversing the diet to normal chow, as illustrated by species of the Odoribacter genus. Genome-wide DNA methylation analysis revealed a "priming effect" through changes in DNA methylation in key liver genes. For example, the lipid-regulating gene Apoa4 remained hypomethylated in both groups even after introduction to normal chow diet. Our results support that dietary change, in part, reverses the NAFLD phenotype. However, some diet-induced effects remain, such as changes in microbiome composition, elevated serum triglyceride levels, and hypomethylation of key liver genes. While the results are correlative in nature, it is tempting to speculate that the dietary-induced changes in microbiome composition may in part contribute to the persistent epigenetic modifications in the liver. Show less

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a distinct variant of HCC that is characterized by dense tumor-infiltrating lymphocytes (TILs). Patients with LEL-HCC also show better clinical outcomes compared to conventional HCC (c-HCC), which is commonly presented with low TIL. Emerging evidence has begun to highlight tumor-intrinsic genetic abnormalities in the tumor-host immune interfaces. However, genome-wide characterization of LEL-HCC remains largely unexplored. Here, we defined the genomic landscape of 12 LEL-HCC using whole-exome sequencing, and further underpinned those genetic alterations related to an immune active microenvironment by comparing findings to 15 c-HCC that were sequenced in parallel. Overall, the mutational load between LEL-HCC and c-HCC was similar. Interestingly, SNV incidences of specific genes (CTNNB1, AXIN1, NOTCH1, and NOTCH2) were significantly higher in c-HCC than LEL-HCC, suggesting a plausible link between activated Wnt/β-catenin and Notch signaling pathways and immune avoidance. Marked focal amplification of chromosome 11q13.3 was prevalent in LEL-HCC. Using The Cancer Genome Atlas dataset, we further established oncogenes expressed from chromosome 11q13.3 (CCND1, FGF19, and FGF4) to be strongly associated with the immune checkpoint signature (CD274, PDCD1, BTLA, CTLA4, HAVCR2, IDO1, and LAG3). Our results have illustrated for the first time the somatic landscape of LEL-HCC, and highlighted molecular alterations that could be exploited in combinatory therapy with checkpoint inhibitors in targeting HCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Show less

In our preliminary screening, expression of miR-338-5p was found to be higher in primary colorectal cancer (CRC) with metastasis. The autophagy related gene- phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3) appeared to be targeted by miR-338-5p. Here, we provide solid evidence in support of PIK3C3 involved in miR-338-5p related metastasis of CRC in vitro and in vivo. The potential clinical relevance of miR-338-5p and its target gene was analysed on benign colorectal polyps and primary CRCs by QPCR. Mouse spleen xenograft experiment was performed to examine the importance of miR-338-5p for metastasis. PIK3C3 was one of target genes of miR-338-5p. In primary CRCs, expression of miR-338-5p is positively related to tumour staging, distant metastasis and poor patient survival. Patients with higher ratios of miR-338-5p/PIK3C3 also had significantly poor overall survival, supporting their significance in the progression of CRC. Over-expression of miR-338-5p promotes CRC metastasis to the liver and lung in vivo, in which PIK3C3 was down-regulated in the metastatic tumours. In contrast, overexpression of PIK3C3 in miR-338-5p stable cells inhibited the growth of metastatic tumours. Both migration and invasion of CRC in vitro induced by miR-338-5p are mediated by suppression of PIK3C3. Using forward and reverse approaches, autophagy was proved to involve in CRC migration and invasion induced by miR-338-5p. MiR-338-5p induces migration, invasion and metastasis of CRC in part through PIK3C3-related autophagy pathway. The miR-338-5p/PIK3C3 ratio may become a prognostic biomarker for CRC patients. FUND: NCKU Hospital, Taiwan, Ministry of Science and Technology, Taiwan. Show less

Fibroblast growth factor 21 (FGF21) is increasingly recognized as an important metabolic regulator of glucose homeostasis. Here, we conducted an exome-chip association analysis by genotyping 5,169 Chinese individuals from a community-based cohort and two clinic-based cohorts. A custom Asian exome-chip was used to detect genetic determinants influencing circulating FGF21 levels. Single-variant association analysis interrogating 70,444 single nucleotide polymorphisms identified a novel locus, Show less

The strong male predominance in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remains inadequately explained, but sex hormones might be involved. We hypothesized that single nucleotide polymorphisms (SNPs) in the androgen pathway influence risk of developing BE and EAC. This genetic-epidemiological analysis included 14 studies from Australia, Europe and North America. Polymorphisms in 16 genes coding for the androgen pathway were analyzed using a gene-based approach: versatile gene-based test association study. This method evaluates associations between a trait and all SNPs within a specific gene rather than each SNP marker individually as in a conventional GWAS. The data were stratified for sex, body-mass index, waist-to-hip ratio, tobacco smoking and gastroesophageal reflux status. Included were data from 1,508 EAC patients, 2,383 BE patients and 2,170 control participants. SNPs within the gene CYP17A1 were associated with risk of BE in the sexes combined (p = 0.002) and in males (p = 0.003), but not in females separately (p = 0.3). This association was found in tobacco smokers (p = 0.003) and in BE patients without reflux (p = 0.004), but not in nonsmokers (p = 0.2) or those with reflux (p = 0.036). SNPs within JMJD1C were associated with risk of EAC in females (p = 0.001). However, none of these associations replicated in a subsequent sample. Fourteen other genes studied did not reach statistically significant levels of association with BE, EAC or the combination of BE and EAC, after correcting for the number of genes included in the analysis. In conclusion, genetic variants in the androgen-related genes CYP17A1 and JMJD1C might be associated with risk of BE and EAC, respectively, but replication data with larger sample sizes are needed. Show less

Dengue results in a significant public health burden in endemic regions. The World Health Organization (WHO) recommended the use of warning signs (WS) to stratify patients at risk of severe dengue disease in 2009. However, WS is limited in stratifying adult dengue patients at early infection (Day 1-3 post fever), who require close monitoring in hospitals to prevent severe dengue. The aim of this study is to identify and validate prognostic models, built with differentially expressed biomarkers, that enable the early identification of those with early dengue infection that require close clinical monitoring. RNA microarray and protein assays were performed to identify differentially expressed biomarkers of severity among 92 adult dengue patients recruited at early infection from years 2005-2008. This comprised 47 cases who developed WS after first presentation and required hospitalization (WS+Hosp), as well as 45 controls who did not develop WS after first presentation and did not require hospitalization (Non-WS+Non-Hosp). Independent validation was conducted with 80 adult dengue patients recruited from years 2009-2012. Prognostic models were developed based on forward stepwise and backward elimination estimation, using multiple logistic regressions. Prognostic power was estimated by the area under the receiver operating characteristic curve (AUC). The WS+Hosp group had significantly higher viral load (P<0.001), lower platelet (P<0.001) and lymphocytes counts (P = 0.004) at early infection compared to the Non-WS+Non-Hosp group. From the RNA microarray and protein assays, the top single RNA and protein prognostic models at early infection were CCL8 RNA (AUC:0.73) and IP-10 protein (AUC:0.74), respectively. The model with CCL8, VPS13C RNA, uPAR protein, and with CCL8, VPS13C RNA and platelets were the best biomarker models for stratifying adult dengue patients at early infection, with sensitivity and specificity up to 83% and 84%, respectively. These results were tested in the independent validation group, showing sensitivity and specificity up to 96% and 54.6%, respectively. At early infection, adult dengue patients who later presented WS and require hospitalization have significantly different pathophysiology compared with patients who consistently presented no WS and / or require no hospitalization. The molecular prognostic models developed and validated here based on these pathophysiology differences, could offer earlier and complementary indicators to the clinical WHO 2009 WS guide, in order to triage adult dengue patients at early infection. Show less

We have previously mapped a major susceptibility locus influencing familial lung cancer risk to chromosome 6q23-25. However, the causal gene at this locus remains undetermined. In this study, we further refined this locus to identify a single candidate gene, by fine mapping using microsatellite markers and association studies using high-density single nucleotide polymorphisms (SNP). Six multigenerational families with five or more affected members were chosen for fine-mapping the 6q linkage region using microsatellite markers. For association mapping, we genotyped 24 6q-linked cases and 72 unrelated noncancer controls from the Genetic Epidemiology of Lung Cancer Consortium resources using the Affymetrix 500K chipset. Significant associations were validated in two independent familial lung cancer populations: 226 familial lung cases and 313 controls from the Genetic Epidemiology of Lung Cancer Consortium, and 154 familial cases and 325 controls from Mayo Clinic. Each familial case was chosen from one high-risk lung cancer family that has three or more affected members. A region-wide scan across 6q23-25 found significant association between lung cancer susceptibility and three single nucleotide polymorphisms in the first intron of the RGS17 gene. This association was further confirmed in two independent familial lung cancer populations. By quantitative real-time PCR analysis of matched tumor and normal human tissues, we found that RGS17 transcript accumulation is highly and consistently increased in sporadic lung cancers. Human lung tumor cell proliferation and tumorigenesis in nude mice are inhibited upon knockdown of RGS17 levels. RGS17 is a major candidate for the familial lung cancer susceptibility locus on chromosome 6q23-25. Show less

Retinal Müller glial cells, in addition to providing homeostatic support to retinal neurons, have been shown to engage in modulation of neuronal activity and regulate vasomotor responses in the retina, among other functions. Calcium-mediated signaling in Müller cells has been implicated to play a significant role in the intracellular and intercellular interactions necessary to carry out these functions. Although the basic molecular mechanisms of calcium signaling in Müller cells have been described, the dynamics of calcium responses in Müller cells have not been fully explored. Here, we provide a quantitative characterization of calcium signaling in an in vitro model of Müller cell signaling using the rMC-1 cell line, a well-established line developed from rat Müller cells. rMC-1 cells displayed robust intracellular calcium transients and the capacity to support calcium transient-mediated intercellular calcium waves with signaling dynamics similar to that reported for Müller cells in in situ retinal preparations. Furthermore, pharmacological perturbations of intracellular calcium transients with thapsigargin and intercellular calcium waves with purinergic receptor antagonists and gap junction blockers (PPADS and FFA, respectively) suggest that the molecular mechanisms that underlie calcium signaling in rMC-1 cells has been conserved with those of Müller cells. This model provides a robust in vitro system for investigating specific mechanistic hypotheses of intra- and intercellular calcium signaling in Müller cells. Show less

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity. Show less