Visceral obesity, but not central obesity, is associated with cardiac remodeling in subjects with suspected metabolic syndrome.

BACKGROUND AND AIMS: Metabolic syndrome (MetS) is a cluster of multiple risk factors including central obesity that may lead to cardiac damage and cardiovascular events. We investigated whether visceral obesity induces cardiac structural and functional remodeling independently from central obesity and other risk factors in subjects with suspected MetS. METHODS AND RESULTS: We studied 229 participants with suspected MetS. Visceral fat area (VFA) was measured by bioelectrical impedance analysis. Left ventricular (LV) mass index, early diastolic velocity of mitral annulus (e'), and LV global longitudinal strain (GLS) were measured by echocardiography. Subjects were categorized into high and low VFA group (VFAh and VFAl). MetS was more prevalent in the VFAh than in the VFAl (p = 0.004). The VFAh had a higher waist circumference (WC) than the VFAl (p < 0.001). LV mass index was higher, but e' and GLS were lower in the VFAh than in VFAl (all p < 0.05). VFA was well correlated with blood pressure, fasting blood glucose, triglyceride, high-sensitivity C-reactive protein and adiponectin (all p < 0.05). VFA was correlated to LV mass index, e', and GLS (all p < 0.05) and was independently associated with GLS after adjustment for other risk factors, including WC (p = 0.005). CONCLUSIONS: Visceral obesity assessed by VFA was well correlated with parameters of MetS. Visceral obesity, but not central obesity measured by WC, was independently associated with structural and functional cardiac remodeling in subjects with suspected MetS. It suggests that visceral obesity should be considered as an important risk factor for cardiac damage in dysmetabolic subjects. TRIAL REGISTRATION: NCT02077530 (date of registration: November 1, 2013).

Alitretinoin can be a good treatment option for idiopathic recalcitrant trachyonychia in adults: an open-label study.

BACKGROUND: Trachyonychia can be refractory to conventional treatments including topical, intralesional or systemic corticosteroids, as well as cyclosporine and retinoids. Therefore, new treatment options are needed for recalcitrant trachyonychia. OBJECTIVE: To evaluate the efficacy and safety of oral alitretinoin for idiopathic recalcitrant trachyonychia. METHODS: A total of 21 adult patients with 210 nails affected by idiopathic recalcitrant trachyonychia were evaluated in this open-label prospective study. All patients took 30 mg of alitretinoin daily for at least 3 months. Clinical outcomes were assessed using the Physician Global Assessment (PGA) scale proposed by Park et al. (degree of roughness: 0, clear; 1, mild; 2, moderate; 3, marked; 4, severe) at baseline and 1, 3 and 6 months after treatment. RESULTS: After 1, 3 and 6 months of treatment, 74.3% (123/210), 98.1% (206/210) and 99.2% (119/120) of nails showed clinical improvement, respectively; 0% (0/210), 22.9% (48/210) and 69.2% (83/120) were completely free from nail abnormalities. The mean PGA score at baseline was 3.4, decreasing significantly to 2.7, 1.3 and 0.7 at 1, 3 and 6 months following treatment, respectively. LIMITATIONS: A small number of participants and lack of a control group were limitations. CONCLUSIONS: For the first time, this study evaluated the efficacy and safety of oral alitretinoin for idiopathic recalcitrant trachyonychia in adults. The results suggest that oral alitretinoin can be a good treatment option for adult patients with recalcitrant trachyonychia.

Association between the extent of urinary albumin excretion and glycaemic variability indices measured by continuous glucose monitoring.

AIMS: The contribution of glycaemic variability to the microvascular complication of diabetes has not been established. We examined whether there is an independent association between indices of glycaemic variability in continuous glucose monitoring and extent of albuminuria. METHODS: A total of 173 patients with Type 2 diabetes (without insulin therapy, n = 96; with insulin therapy, n = 77) who had unexplained large fluctuations in blood glucose values underwent three-day continuous glucose monitoring. We used a multinomial logistic regression model to determine whether the indices of glycaemic variability independently affected the odds of having a spot urine albumin/creatinine ratio of 30-299 mg/g and ≥ 300 mg/g. RESULTS: Higher standard deviation (P = 0.002), mean of daily differences (P = 0.023) and mean amplitude of glycaemic excursion (P = 0.043) significantly increased the odds of having a urine albumin/creatinine ratio of ≥ 300 mg/g. In multivariable analysis, only higher standard deviation, but not mean amplitude of glycaemic excursion and mean of daily differences, independently increased the odds of having a urine albumin/creatinine ratio of ≥ 300 mg/g (P = 0.025). Coefficient of variation (sd/mean) was not associated with the odds of having a urine albumin/creatinine ratio of 30-299 or ≥ 300 mg/g. CONCLUSIONS: The independent association between standard deviation and the extent of albuminuria was lost when the measures were normalized by mean glucose level. At least in terms of relative measures of glycaemic variability, we failed to demonstrate an independent association between glycaemic variability and albuminuria extent in patients with inadequately controlled Type 2 diabetes.

Functional characterization of the TSC2 c.3598C>T (p.R1200W) missense mutation that co-segregates with tuberous sclerosis complex in mildly affected kindreds.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by a combination of neurological symptoms and hamartomatous growths, and caused by mutations in the TSC1 and TSC2 genes. Overall, TSC2 mutations are associated with a more severe disease phenotype. We identified the c.3598C>T (R1200W) change in the TSC2 gene in seven different families. The clinical phenotypes in the families were mild, characterized by mild skin lesions, remitting epilepsy and a lack of severe mental retardation or major organ involvement. Functional analysis of the TSC2 R1200W variant, and four other TSC2 missense variants associated with a mild TSC phenotype, confirmed that the changes disrupted the TSC1-TSC2 function. Interestingly however, in each case, the TSC1-TSC2 interaction was not affected by the amino acid substitution.

Genome-wide methylation profile of nasal polyps: relation to aspirin hypersensitivity in asthmatics.

BACKGROUND: In addition to the dysregulation of arachidonic acid metabolism in aspirin-intolerant asthma (AIA), aspirin acetylsalicylic acid (ASA) exerts effects on inflammation and immunity; however, many of these effects are unknown. OBJECTIVE: The aim of the study was to evaluate the methylation status of whole genome in blood and polyp tissues with and without aspirin hypersensitivity. METHODS: Genome-wide DNA methylation levels in nasal polyps and peripheral blood cells were examined by microarray analysis using five subjects with AIA and four subjects with aspirin-tolerant asthma (ATA). RESULTS: In the nasal polyps of the patients with AIA, hypermethylation was detected at 332 loci in 296 genes, while hypomethylation was detected at 158 loci in 141 genes. Gene ontologic and pathway enrichment analyses revealed that genes involved in lymphocyte proliferation, cell proliferation, leukocyte activation, cytokine biosynthesis, cytokine secretion, immune responses, inflammation, and immunoglobulin binding were hypomethylated, while genes involved in ectoderm development, hemostasis, wound healing, calcium ion binding, and oxidoreductase activity were hypermethylated. In the arachidonate pathway, PGDS, ALOX5AP, and LTB4R were hypomethylated, whereas PTGES was hypermethylated. CONCLUSION: The nasal polyps of patients with AIA have characteristic methylation patterns affecting 337 genes. The genes and pathways identified in this study may be associated with the presence of aspirin hypersensitivity in asthmatics and are therefore attractive targets for future research.

Further delineation of Pitt-Hopkins syndrome: phenotypic and genotypic description of 16 novel patients.

BACKGROUND: Haploinsufficiency of the gene encoding for transcription factor 4 (TCF4) was recently identified as the underlying cause of Pitt-Hopkins syndrome (PTHS), an underdiagnosed mental-retardation syndrome characterised by a distinct facial gestalt, breathing anomalies and severe mental retardation. METHODS: TCF4 mutational analysis was performed in 117 patients with PTHS-like features. RESULTS: In total, 16 novel mutations were identified. All of these proven patients were severely mentally retarded and showed a distinct facial gestalt. In addition, 56% had breathing anomalies, 56% had microcephaly, 38% had seizures and 44% had MRI anomalies. CONCLUSION: This study provides further evidence of the mutational and clinical spectrum of PTHS and confirms its important role in the differential diagnosis of severe mental retardation.

Association of angiotensin I-converting enzyme gene polymorphisms with aspirin intolerance in asthmatics.

BACKGROUND: Aspirin-intolerant asthma (AIA) refers to the development of bronchoconstriction in asthmatic individuals following the ingestion of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). Angiotensin I-converting enzyme (ACE), a membrane-bound peptidase present in the lung, plays a pivotal role in the metabolism of the endogenous peptides involved in the pathogenesis of asthma. METHODS: We screened a Korean asthma cohort (581 asthmatics including 81 aspirin-intolerant asthmatics and 231 aspirin-tolerant asthmatics, and 181 normal controls) for four single nucleotide polymorphisms (SNPs; -262 A>T and -115 T>C in the 5'-flanking region and +5467 T>C [Pro450Pro] and+11860 A>G [Thr776Thr] in the coding region) and one ins/del (+21288 CT) in the ACE gene. RESULTS: None of the SNPs or haplotypes showed any association with the development of asthma, but they were significantly associated with the risk of AIA. Logistic regression indicated that the frequency of the rare alleles of -262 A>T and -115 T>C was higher in subjects with AIA than in subjects with aspirin-tolerant asthma (ATA) (P=0.003-0.01, P( corr)=0.015-0.05). Subjects homozygous for the rare alleles of -262 A>T and -115 T>C showed a greater decline in forced expiratory volume in 1 s (FEV(1)) after aspirin provocation than those homozygous for the common alleles (P<0.05). A luciferase reporter assay indicated that ACE promoters containing the rare -262 A>T allele possessed lower activity than did those containing the common allele (P=0.009). In addition, ACE promoters bearing the rare -115 T>C allele had no luciferase activity. DNA-protein binding assays revealed a band containing the ACE promoter region (including -262 A) and a protein complex. CONCLUSION: The -262 A>T polymorphism in the promoter of the ACE gene is associated with AIA, and the rare allele of -262 A>T may confer aspirin hypersensitivity via the down-regulation of ACE expression.

An electrochemical impedance measurement technique employing Fourier transform

We describe a novel technique for measuring electrochemical impedance, in which the electrode potential is ramped to a desired bias potential and a small potential step is applied to the working electrode after a short time delay. Fourier transform of the first derivative of the current signal thus obtained provides ac currents in the frequency domain, which allows the computation of impedances of the electrode/electrolyte interface in the whole frequency range. A home-built data acquisition system for these measurements and the results obtained therefrom were used for the measurements. The advantage of the technique includes an extremely short time of less than 1 ms for impedance measurements in the whole frequency region while equilibrium conditions of the electrochemical system are being maintained before and after the measurements, among many others. This technique is expected to revolutionize electrochemical measurements and to find important applications such as in situ measurements during battery cycling, corrosion testing, and other electrochemical experiments.

Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory.

Proper analysis of label distribution in metabolic pathway intermediates is critical for correct interpretation of experimental data and strategic experimental design. While, for example, 13C nuclear magnetic resonance (NMR) spectroscopy is usually limited to the measurement of degrees of 13C enrichment, more information about metabolic fluxes can be extracted from the fine structure of NMR spectra, or molecular weight distributions of isotopomers of metabolic intermediates (measured by gas chromatography-mass spectrometry). For this purpose, rigorous accounting for the contribution of all pathways to label distribution is required, especially contributions resulting from multiple turns of metabolic cycles. In this paper we present a mathematical model developed to analyze isotopomer distributions of tricarboxylic acid cycle (TCA) intermediates following the administration of 13C (or 14C) labeled substrates. The theory presented provides the basis to analyze 13C NMR spectra and molecular weight distributions of metabolites. In a companion paper (Park et al., 1999), the theory is applied to the analysis of several cases of biological significance. Copyright 1999 John Wiley & Sons, Inc.

A Rational Approach to Selective Recognition of NH(4)(+) over K(+).

An ion-selective electrode (ISE) based on receptor 1 is highly selective for binding NH(4)(+) over K(+) (lg K(NH(4)(+)/K(+))=-2.6); the three imine nitrogen atoms in 1 are ideally positioned for hydrogen bonding with the tetrahedral NH(4)(+) ion. This selectivity is considerably greater than that found for commercial ISEs based on nonactin (lg K(NH(4)(+)/K(+))=-1.0).

Γ-glutamyl transferase as an early and sensitive marker in ethanol-induced liver injury of rats.

γ-Glutamyl transferase (GGT) has been regarded as a biological marker of heavy alcohol consumption or hepatobiliary disease such as fatty liver. However, the role of GGT is unknown in the molecular pathway during alcohol-induced liver injury. To determine the role of GGT in alcohol-induced liver injury, Sprague-Dawley rats were administered 22% and 38% ethanol for 3 days as acute and 5 weeks as subchronic model. In serologic analysis, the level of GGT was significantly increased and the level of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were not changed at 3 days and 5 weeks. In histologic analysis, ethanol exposure induced granular deposit formation and sinusoidal dilation in the acute model for 3 days. In the subchronic model for 5 weeks, ethanol exposure further increased the granular deposit formation, sinusoidal congestion, and mild fatty liver change. To determine whether ethanol-exposed liver is associated with changes of antioxidants levels, we performed reverse-transcriptase polymerase chain reaction (RT-PCR) analysis on ethanol-exposed livers of rats. In RT-PCR analysis, the mRNA levels of GPX1 and SOD1 were significantly increased as well as up-regulation of CYP2E1. In the glutathione assay, the level of glutathione was significantly reduced in response to ethanol in rats. Therefore, in this study, ethanol increased the level of serum GGT but depleted the level of glutathione. Moreover, the CYP2E1 was rapidly reflected to ethanol in rats. Taken together, our findings suggest that the elevated GGT is associated with cellular antioxidant defense system, and the CYP2E1 can be used for early diagnosis in alcohol-related diseases.

The clinical and molecular characterization of patients with dyshormonogenic congenital hypothyroidism reveals specific diagnostic clues for DUOX2 defects.

CONTEXT: Mutations in the DUOX2 gene have been associated with transient or permanent congenital hypothyroidism due to a dyshormonogenic defect. OBJECTIVE: This study aimed to verify the prevalence of DUOX2 mutations and the associated clinical features in children selected by criteria supporting a partial iodide organification defect (PIOD). PATIENTS AND METHODS: Thirty children with PIOD-like criteria were enrolled and genotyped. A detailed clinical characterization was undertaken together with the functional analysis of the DUOX2 variations and the revision of the clinical and molecular data of the literature. RESULTS: In this large selected series, the prevalence of the DUOX2 mutations was high (37%). We identified 12 missense variants, one splice site, and three frameshift DUOX2 mutations. Functional analyses showed significant impairment of H2O2 generation with five missense variants. Stop-codon mutants were shown to totally abolish DUOX2 activity by nonsense-mediated RNA decay, exon skipping, or protein truncation. DUOX2 mutations, either mono- or biallelic, were most frequently associated with permanent congenital hypothyroidism. Moreover, the present data suggested that, together with goiter and PIOD, the most significant features to select patients for the DUOX2 analysis are the low free T4 and the high TSH concentrations at the first postnatal serum sampling, despite borderline blood spot TSH. Interestingly, the analysis of previously described DUOX2 mutated cases confirmed the validity of these findings. CONCLUSIONS: The defects in the peroxide generation system are common among congenital hypothyroidism patients with PIOD. The most robust clinical parameters for selecting patients for DUOX2 analysis have been identified, and several DUOX2 variants have been functionally characterized.

Disparate hypervariable region-1 of mitochondrial DNA did not induce skin allograft rejection in cloned porcine models.

INTRODUCTION: Alloantigen recognition in skin transplantation is the bane for surgeons. Several studies have mainly focused on the immunogenicity of major histocompatibility (MHC) antigens and H-Y minor histocompatibility antigens. However, the roles of the mitochondrial DNA (mtDNA) encorded miHA have not been identified. Therefore, we sought to address the antigenicity of the hypervariable region 1 (HV-1) of mtDNA in skin transplantation using cloned pig models. METHODS: Swine leukocyte antigen and HV-1 of mtDNA were analyzed using sequencing methods. Skin transplantation was performed between MHC-matched, mtDNA-mismatched cloned miniature pigs. Full-thickness skin was grafted between cloned pigs without any immunosuppressants. The grafted tissues were observed for 3 months and evaluated histologically. RESULTS: The cloned pigs shared identical MHC but mtDNA mismatched at 9 positions. Skin grafts between the cloned pigs were accepted and hair growth maintained, whereas MHC-mismatched grafts showed acute rejection within 7 days after transplantation and were replaced by hairless scar tissue. CONCLUSIONS: HV-1 disparate skin grafts were not recognized as alloantigenic by MHC-matched cloned pigs.

Fabrication of a biodegradable xenoantigen-free rat liver scaffold for potential drug screening applications.

BACKGROUND: The increasing market in biological pharmaceuticals raises the demand for human test systems. Although 2-dimensional (2D) models are mostly used for these purposes, these models not mimic responses of 3-dimensional (3D) native tissue. METHODS: After generation of a rat liver scaffold using 0.1% sodium dodecyl sulfate, we characterized the histology, blood vessel integrity, and residual DNA as well as retained amounts of collagen and glycosaminoglycan (GAG). Then, we examined the susceptibility of extracellular matrix (ECM) to enzymatic remodeling. Finally, a mixed lymphocyte reaction (MLR) was performed to evaluate the in vitro immunogenicity of the ECM against human peripheral blood mononuclear cells (PBMCs). RESULTS: Histologic examination of decellularized liver revealed the removal of nuclear and cytoplasmic materials with preservation of architecture. The vascular network was intact after decellularization. Biochemical analysis of ECM components revealed that only a negligible amount of DNA was retained compared with the native liver with preservation of large amounts of GAG and collagen. Scaffolds were degraded in response to collagenase treatment. MLR demonstrated that decellularized matrices did not exert any xenostimulatory response against human PBMCs. CONCLUSION: Our findings suggested that naturally derived rat liver scaffolds show natural biocompatibility besides the ability to preserve the intact 3D structure and components. Because of these characteristics, the whole decellularized rat liver can retain many aspects of native tissue structure and function upon recellularization enabling it to be used for drug screening.

Mouse adipose tissue-derived adult stem cells expressed osteogenic specific transcripts of osteocalcin and parathyroid hormone receptor during osteogenesis.

INTRODUCTION: Adult mesenchymal stem cells (MSCs) have potential to differentiate into various lineages, replacing cells during normal turnover and tissue regeneration to replace damaged or lost adult tissues during osteoporosis and arthritis, or traumatic injuries. We investigated the osteogenic signature in mouse adipose tissue (AD)- and bone marrow (BM)-derived MSCs. MATERIALS AND METHODS: MSCs from adipose tissue and bone marrow were compared for osteogenic endogenous mRNA markers by reverse-transcription polymerase chain reaction (RT-PCR). Cellular proliferation and immunophenotype analyzed by flow cytometry revealed that mouse AD-MSCs and BM-MSCs shared similar characteristics. RESULT: Isolated AD-MSC and BM-MSC showed high proliferation rates and fibroblast morphology. Flow cytometry revealed positive markers for mesenchyme, but negative for primitive hematopoietic and endothelial cells. At day 21, Alizarin red S and Von-kossa staining of differentiated cells showed high calcium deposits compared with undifferentiated cells. After 21 days of osteogenic differentiation, AD-MSCs expressed osteocalcin and parathyroid hormone (PTH) compared with undifferentiated cells. Osteogenic-specific transcript of osteocalcin (OC), bone gamma carboxyglutamate protein, and PTH receptor (PTHr) were detected only in differentiated not undifferentiated cells. Undifferentiated BM-MSCs, expressed all markers at low intensity, which amplified during differentiation. CONCLUSION: Our findings suggest that the OC and PTHr can be used as differentiation markers for osteogenesis of mouse AD-MSC.

Acute rejection after swine leukocyte antigen-matched kidney allo-transplantation in cloned miniature pigs with different mitochondrial DNA-encoded minor histocompatibility antigen.

INTRODUCTION: Graft rejection remains a major cause of morbidity and mortality following renal transplantation. One of the main determinants of success after renal transplantation is histocompatibility between donor and recipient. Most of the research on this topic has addressed human leukocyte antigen (HLA), but the roles played by minor histocompatibility antigens (mHAgs), such as mitochondrially transmitted antigens, are poorly understood. In this study, we evaluated immune responses induced by minor antigens originating from mitochondrial DNA (mtDNA) in a large animal model. METHODS: To characterize whole swine leukocyte antigen (SLA) allele in 8 cloned pigs, we performed SLA genotyping for SLA-1, SLA-2, SLA-3, SLA-DQB1, and SLA-DRB1 as well as the hypervariable region 1 (HV1) of mtDNA. Renal transplantation was performed using SLA-matched pigs with different mtDNA as well as SLA-mismatched cloned animals. Cytokine profiling was performed by incubating peripheral leukocytes with cellular components from SLA-matched different mtDNA and SLA-mismatched cells to evaluate mtDNA-mediated immune response. RESULTS: SLA types were confirmed to be identical, but mtDNA sequences of HV1 varied among cloned pigs. Rejection episodes in the SLA-matched group with different mtDNA were similar to those in the SLA-mismatched group; that is, plasma creatinine and BUN levels were increased and mononuclear cell infiltration was observed in perivascular regions in the matched and SLA-mismatched groups. Furthermore, in vitro studies showed interleukin (IL)-1ß expression to be elevated in SLA-matched and SLA-mismatched groups. CONCLUSION: Cloned pigs are a useful preclinical model to evaluate the immunogenicity of mtDNA encoding minor antigens. The mtDNA originating from nongenomic DNA induced cell-mediated immune rejection after kidney transplantation.

Ternary mixed monolayers for simultaneous DNA orientation control and surface passivation for label free DNA hybridization electrochemical sensing.

Target recognizing ternary mixed monolayer is constructed by simple sequential adsorption of thiol caped single stranded DNA (HS-ssDNA), 6-mercapto-1-hexanol (MCH) and 3-mercaptopropionic acid (MPA) diluents (abbreviated as HS-ssDNA/MPA/MCH) and applied for detection of DNA hybridization sequence by electrochemical method. The method is more reliable and reproducible on both conventional wire electrode and 100 nm scale gold-coated silicon chips. Detection limit of 10 pM is observed constantly on all types of electrodes. This ternary layer approach provides 60-80% discrimination effect between the hybridized and un-hybridized surfaces compared to the binary mixed monolayers HS-ssDNA/MCH and HS-ssDNA/MPA that showed 20-30% only. Characterization by impedance spectroscopy, X-ray photoelectron spectroscopy and surface Fourier Transform Infrared (FT-IR) techniques reveals 'head-to-head' anisotropic hydrogen bonding between MPA and MCH diluents that controls the HS-ssDNA orientation and enhancing the electrostatic blockade for K3[Fe(CN)6]. This is the first report characterizing the ternary layer for DNA molecular affinity sensing. Results provided unprecedented insight onto the label free electrochemical sensing and understanding of the complex sensing mechanism to develop sensors that is more reliable.

Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice.

Aflatoxin B(1) (AFB(1))-mediated hepatic damage is involved in production of AFB(1)-8,9-epoxide-bound DNA adducts and this is also affected by a pro-oxidant potential of the toxin. In this study we investigated the effects of quercetin on AFB(1)-treated HepG2 cells. We also examined the biochemical mechanisms associated with the effects of quercetin on AFB(1)-mediated liver damage in mice. Our results revealed that quercetin and isorhamnetin inhibit production of reactive oxygen species and cytotoxicity, and block the decrease of reduced glutathione (GSH) levels in AFB(1)-treated HepG2 cells. Isorhamnetin have inhibitory ability on lipid peroxidation stronger than quercetin in the cells. Oral supplementation with quercetin decreased serum lactate dehydrogenase levels, increased hepatic GSH levels and superoxide dismutase activity, and reduced lipid peroxidation in both the liver and kidney in AFB(1)-treated mice. However, quercetin did not show a significant reduction on serum levels of alkaline phosphate, alanine aminotransferase and aspartate aminotransferase that were increased in AFB(1)-treated mice. HPLC analysis revealed that quercetin in plasma is mainly present as glucoronides and/or sulfates of quercetin. Collectively, it is suggested that quercetin does not directly protect against AFB(1)-mediated liver damage in vivo, but exerts a partial role in promoting antioxidative defense systems and inhibiting lipid peroxidation.