Gout is a common disease resulting from hyperuricemia. Recently, a genome-wide association study identified an association between gout and a single nucleotide polymorphism (SNP) rs2188380, located on an intergenic region between MYL2 and CUX2 on chromosome 12. However, other genes around rs2188380 could possibly be gout susceptibility genes. Therefore, we performed a fine-mapping study of the MYL2-CUX2 region. From 8,595 SNPs in the MYL2-CUX2 region, 9 tag SNPs were selected, and genotyping of 1,048 male gout patients and 1,334 male controls was performed by TaqMan method. Eight SNPs showed significant associations with gout after Bonferroni correction. rs671 (Glu504Lys) of ALDH2 had the most significant association with gout (P = 1.7 × 10(-18), odds ratio = 0.53). After adjustment for rs671, the other 8 SNPs no longer showed a significant association with gout, while the significant association of rs671 remained. rs671 has been reportedly associated with alcohol drinking behavior, and it is well-known that alcohol drinking elevates serum uric acid levels. These data suggest that rs671, a common functional SNP of ALDH2, is a genuine gout-associated SNP in the MYL2-CUX2 locus and that "A" allele (Lys) of rs671 plays a protective role in the development of gout.

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We test the sensor on HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to anchor the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.

Chronic kidney disease (CKD) is well known as a strong risk factor for both end stage renal disease and cardiovascular disease. To clarify the association of polymorphisms in the PPAR genes (PPARD, PPARG, and PPARGC1A) with the risk of CKD in Japanese, we examined this association among the Japanese subjects using the cross-sectional data of J-MICC (Japan Multi-Institutional Collaborative Cohort) Study. The subjects for this analysis were 3,285 men and women, aged 35-69 years, selected from J-MICC Study participants; genotyping was conducted by multiplex polymerase chain reaction-based Invader assay. The prevalence of CKD was determined for CKD stages 3-5 (defined as eGFR < 60 ml/min/1.73 m(2)). Participants with CKD accounted for 17.3% of the study population. When those with PPARD T-842C T/T were defined as reference, those with PPARD T-842C T/C and C/C demonstrated the OR for CKD of 1.26 (95%CI 1.04-1.53) and 1.31 (95%CI 0.83-2.06), respectively. There were no significant associations between the polymorphisms in other PPAR genes and the risk of CKD. The present study found a significantly increased risk of CKD in those with the C allele of PPARD T-842C, which may suggest the possibility of personalized risk estimation of this life-limiting disease in the near future.

Regulation of connexin43 (Cx43) expression affects cell proliferation, differentiation and apoptosis in a gap junctional intercellular communication (GJIC)-independent manner. However, the underlying mechanisms of Cx43-mediated cell cycle suppression are still poorly understood. To elucidate the molecular mechanism of Cx43-mediated cell cycle suppression, we searched for Cx43 interacting proteins by using a proteomics approach. Here, we have identified a Cx43-interacting protein, heat shock cognate protein 70 (Hsc70). We confirmed that Hsc70 directly binds to the C-terminus of Cx43, whereas Hsc54, a splice variant of Hsc70, does not, that Cx43 competes with cyclin D1 for binding to Hsc70, and that the nuclear accumulation of cyclin D1 is reduced by overexpression of Cx43 in a GJIC-independent manner, which is restored by co-overexpression with Hsc70. As a result, the cell proliferation is regulated by Cx43. Our results suggest that Cx43-Hsc70 interaction probably plays a critical role during G1/S progression.

Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the "stopped-flow," global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm-1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm-1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.

The preventive effect of coffee on cancer at different sites has been reported, but the effect on all-sites cancer incidence has not been extensively investigated. We evaluated the association between frequency of coffee consumption and risk of all-sites cancer incidence and mortality among 39 685 men and 43 124 women (age 40-79 years, at baseline), in the Three-Prefecture Cohort Study. The association between frequency of coffee consumption and risk of all-sites cancer incidence and mortality was assessed by a Cox proportional hazards regression model, adjusted for potential confounders. During 411 341 person-years among men and 472 433 person-years among women, a total of 4244 men and 2601 women developed cancer at different sites and a total of 3021 men and 1635 women died of cancer at different sites. We showed an inverse association between frequency of coffee consumption and all-sites cancer incidence in both men and women. Comparing participants who consumed coffee with those who never drank coffee, the adjusted hazard ratios (95% confidential interval) for all-sites cancer incidence was 0.74 (0.62-0.88) for coffee consumption of ≥5 cups/day in men (P for trend < 0.001) and 0.76 (0.58-1.02) in women (P for trend = 0.020). Coffee consumption frequency was inversely associated with mortality from all-sites cancer. In this population, increasing coffee consumption resulted in a decreased risk of all-sites cancer incidence and mortality.

Stroke is associated closely with vascular homeostasis, and several complex processes and interacting pathways, which involve various genetic and environmental factors, contribute to the risk of stroke. Although adrenomedullin (ADM) has a number of physiological and vasoprotective functions, there are few studies of the ADM receptor system in humans. The ADM receptor comprises a calcitonin-receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMPs). We analyzed single nucleotide polymorphisms (SNPs) in the RAMP2 and CLR genes to determine their association with stroke in the light of gene-environment interactions.

Nonalcoholic fatty liver disease (NAFLD) includes a broad range of liver pathologies from simple steatosis to cirrhosis and fibrosis, in which a subtype accompanying hepatocyte degeneration and fibrosis is classified as nonalcoholic steatohepatitis (NASH). NASH accounts for approximately 10-30% of NAFLD and causes a higher frequency of liver-related death, and its progression of NASH has been considered to be complex involving multiple genetic factors interacting with the environment and lifestyle.

Raman spectroscopy allows label-free, minimally invasive, and accurate detection of peripheral nerves. However, the conventional Raman imaging technique is time-consuming when measuring a large area of a sample. Establishing a method for rapidly acquiring spatial distribution of a bundle of peripheral nerve fibers is an essential step for Raman spectroscopy towards application in clinical surgery. Here we present a multipoint Raman spectroscopic technique for rapid peripheral nerve imaging. In only 5 seconds, spectra at 32 points situated on ex vivo rat peripheral nerve bundles and adjoining connective tissues were acquired. Principal component regression and discriminant analysis of spectra revealed that the sensitivity, specificity and accuracy for nerve detection were 85.8%, 96.0%, and 90.8%, respectively. Of 158 peripheral nerves, 152 (96.2%) showed ratio of the number of nerve-positive prediction points to the total measurement points being 0.4 or larger, whereas 119 (99.2%) connective tissues among 120 showed ratio smaller than 0.4. Based on the ratio and a bright-field image of the sample, accurate visualization of peripheral nerves was implemented. The results indicated that the multipoint Raman spectroscopic technique is capable of rapid and accurate peripheral nerve imaging.

Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5'-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.

Whether abstinence from smoking among cancer patients reduces cancer pain is still unclear. Opioids can act as a surrogate index for evaluating the incidence of severe cancer pain in countries where opioid abuse is infrequent. This study aimed to investigate whether changed smoking behavior after cancer diagnosis influences the incidence of severe cancer pain as determined by strong opioid use.

Elevated serum uric acid (SUA)-hyperuricemia-is caused by overproduction of urate or by its decreased renal and/or intestinal excretion. This disease, which is increasing in prevalence worldwide, is associated with both gout and metabolic diseases. Several studies have reported relationships between apolipoprotein E (APOE) haplotypes and SUA levels in humans; however, their results remain inconsistent. This prompted us to investigate the relationship between APOE polymorphisms and SUA levels. Our subjects were 5,272 Japanese men, premenopausal women, and postmenopausal women. Multiple linear regression analyses revealed the ε2 haplotype of APOE to be independently associated with higher SUA in men (N = 1,726) and postmenopausal women (N = 1,753), but not in premenopausal women (N = 1,793). In contrast, the ε4 haplotype was little related to SUA levels in each group. Moreover, to examine the effect of Apoe deficiency on SUA levels, we conducted animal experiments using Apoe knockout mice, which mimics ε2/ε2 carriers. We found that SUA levels in Apoe knockout mice were significantly higher than those in wild-type mice, which is consistent with the SUA-raising effect of the ε2 haplotype observed in our clinico-genetic analyses. Further analyses suggested that renal rather than intestinal underexcretion of urate could be involved in Apoe deficiency-related SUA increase. In conclusion, we successfully demonstrated that the ε2 haplotype, but not the ε4 haplotype, increases SUA levels. These findings will improve our understanding of genetic factors affecting SUA levels.

Kawasaki disease (KD) is a systemic vasculitis in infants that develops predominantly in the coronary arteries. Despite the clinically transient nature of active inflammation in childhood albeit rare complications (e.g., coronary artery aneurysm), KD has recently been suggested to increase the incidence of ischemic heart diseases in young adulthood. However, little is known about the histopathology of the coronary artery long after development of the acute KD vasculitis. To address this, we conducted histological studies of rabbit coronary arteries in adolescent phase after induction of the KD-like vasculitis induced by horse serum administration. After a transmural infiltration of inflammatory cells in acute phase at day 7, the artery exhibited a gradual decrease in the number of inflammatory cells and thickening of the intima during the chronic phase up to day 90, where proteoglycans were distinctly accumulated in the intima with abundant involvement of α-smooth muscle actin (α-SMA)-positive cells, most of which accompanied expression of VCAM-1 and NF-κB. Distinct from classical atherosclerosis, inflammatory cells, e.g., macrophages, were barely detected during the chronic phase. These observations indicate that the KD-like coronary arteritis is followed by intimal thickening via accumulation of proteoglycans and proliferation of α-SMA-positive cells, reflecting aberrant coronary artery remodeling.

Tooth loss impairs oral function. The aim of the present review was to evaluate the causal association between smoking and tooth loss on the basis of high-quality studies.

Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples.

Intracellular Ca(2+) ([Ca(2+)]i) dynamics in isolated myocytes differ between the atria and ventricles due to the distinct t-tubular distributions. Although cellular aspects of ventricular [Ca(2+)]i dynamics in the heart have been extensively studied, little is known about those of atrial myocytes in situ. Here we visualized precise [Ca(2+)]i dynamics of atrial myocytes in Langendorff-perfused rat hearts by rapid-scanning confocal microscopy. Of 16 fluo-4-loaded hearts imaged during pacing up to 4-Hz, five hearts showed spatially uniform Ca(2+) transients on systole among individual cells, whereas no discernible [Ca(2+)]i elevation developed during diastole. In contrast, the remaining hearts showed non-uniform [Ca(2+)]i dynamics within and among the cells especially under high-frequency (4 Hz) excitation, where subcellular cluster-like [Ca(2+)]i rises or wave-like [Ca(2+)]i propagation occurred on excitation. Such [Ca(2+)]i inhomogeneity was more pronounced at high-frequency pacing, showing beat-to-beat Ca(2+) transient alternans. Despite such non-uniform dynamics, cessation of burst pacing of the atria was not followed by emergence of spontaneous Ca(2+) waves, indicating minor Ca(2+)-releasing potentials of the sarcoplasmic reticulum (SR). In summary, rat atria display a propensity to show non-uniform [Ca(2+)]i dynamics on systole due to impaired Ca(2+)-release from the SR and paucity of t-tubules. Our results provide an important basis for understanding atrial pathophysiology.

Tobacco smoking is a major risk factor for many diseases. We sought to quantify the burden of tobacco-smoking-related deaths in Asia, in parts of which men's smoking prevalence is among the world's highest.

Gout is a common arthritis caused by elevated serum uric acid (SUA) levels. Here we investigated loci influencing SUA in a genome-wide meta-analysis with 121,745 Japanese subjects. We identified 8948 variants at 36 genomic loci (P<5 × 10-8) including eight novel loci. Of these, missense variants of SESN2 and PNPLA3 were predicted to be damaging to the function of these proteins; another five loci-TMEM18, TM4SF4, MXD3-LMAN2, PSORS1C1-PSORS1C2, and HNF4A-are related to cell metabolism, proliferation, or oxidative stress; and the remaining locus, LINC01578, is unknown. We also identified 132 correlated genes whose expression levels are associated with SUA-increasing alleles. These genes are enriched for the UniProt transport term, suggesting the importance of transport-related genes in SUA regulation. Furthermore, trans-ethnic meta-analysis across our own meta-analysis and the Global Urate Genetics Consortium has revealed 15 more novel loci associated with SUA. Our findings provide insight into the pathogenesis, treatment, and prevention of hyperuricemia/gout.

Brown adipocytes play an important role in human energy metabolism and prevention of obesity and diabetes. Induced pluripotent stem cells (iPSCs) represent a promising source for brown adipocytes; however, exogenous gene induction is generally required for iPSCs generation, which might cause undesired effects particularly in long-term treatment after transplantation. We have previously reported a cocktail of six small chemical compounds that enables a conversion of human fibroblasts into chemical compound-induced neuronal cells (CiNCs). Here, we report that modified combinations of the chemical compounds and rosiglitazone, a PPARγ agonist, afforded direct conversion of human fibroblasts into brown adipocytes. The chemical compound-induced brown adipocytes (ciBAs) exhibit induction of human brown adipocyte-specific genes such as Ucp1, Ckmt1, Cited1 and other adipocyte-specific genes such as Fabp4, AdipoQ, and Pparγ. Treatment with either isoproterenol or Forskolin further induced the expression of Ucp1, suggesting that β adrenergic receptor signalling in ciBAs could be functional for induction of thermogenic genes. Moreover, oxygen consumption rates were elevated in ciBAs along with increase of cellular mitochondria. Our findings might provide an easily accessible approach for generating human brown adipocytes from fibroblasts and offer therapeutic potential for the management of obesity, diabetes, and related metabolic disorders.