Immunogenicity and safety of SARS-CoV-2 recombinant protein subunit vaccine (IndoVac) adjuvanted with alum and CpG 1018 in Indonesian adults: A phase 3, randomized, active-controlled, multicenter trial.

BACKGROUND: Bio Farma has developed a recombinant protein subunit vaccine (IndoVac) that is indicated for active immunization in population of all ages. This article reported the results of the phase 3 immunogenicity and safety study in Indonesian adults aged 18 years and above. METHODS: We conducted a randomized, active-controlled, multicenter, prospective intervention study to evaluate the immunogenicity and safety of IndoVac in adults aged 18 years and above. Participants who were SARS-CoV-2 vaccine-naïve received two doses of either IndoVac or control (Covovax) with 28 days interval between doses and were followed up until 12 months after complete vaccination. RESULTS: A total of 4050 participants were enrolled from June to August 2022 and received at least one dose of vaccine. The geometric mean ratio (GMR) of neutralizing antibody at 14 days after the second dose was 1.01 (95 % confidence interval (CI) 0.89-1.16), which met the WHO non-inferiority criteria for immunobridging (95 % CI lower bound > 0.67). The antibody levels were maintained through 12 months after the second dose. The incidence rate of adverse events (AEs) were 27.95 % in IndoVac group and 32.15 % in Covovax group with mostly mild intensity (27.70 %). The most reported solicited AEs were pain (14.69 %) followed by myalgia (7.48 %) and fatigue (6.77 %). Unsolicited AEs varied, with each of the incidence rate under 5 %. There were no serious AEs assessed as possibly, probably, or likely related to vaccine. CONCLUSIONS: IndoVac in adults showed favourable safety profile and elicited non-inferior immune response to Covovax. (ClinicalTrials.gov: NCT05433285, Indonesian Clinical Research Registry: INA-R5752S9).

Impact of GSTT1 AND GSTM1 variants and hair mercury concentration in maternal blood pressure among coastal pregnant women in Central Java, Indonesia.

The GSTT1 and GSTM1 genes have a role in mercury metabolism and excretion, as well as blood pressure response, impacting birth outcomes. The present study assesses whether GSTT1 and GSTM1 deletion variants and maternal hair Hg concentration are associated with blood pressure and birth outcomes among the Indonesian coastal pregnant mother population. A cross-sectional study was conducted on 139 pregnant women in the Jepara coastal area of Central Java, Indonesia. Maternal characteristics during pregnancy, including blood pressure and birth outcomes, were collected. GSTT1 and GSTM1 gene variants were detected using polymerase chain reaction (PCR). Hair Hg levels were measured using the reducing-vaporization mercury analyzer. The mean maternal hair Hg concentration was 0.727±0.558 µg/g. GSTT1 genotype homozygous deletion was found in 41.7% of subjects, while no GSTM1 deletion was found. No statistically significant difference was found between deletion and non-deletion groups for hair Hg. GSTT1 deletion genotype shows protection but is inconclusive toward diastolic hypertension (p=0.048, OR 0.285, CI 0.077-1.052) and insignificant with birth outcomes (all p>0.05). High hair Hg concentration and positive history of cardiovascular diseases increase the risk of systolic and diastolic hypertension during pregnancy with OR 6.871 (CI 95% 1.445-32.660) and 8.518 (CI 95% 2.126-34.125), respectively, while not in birth outcomes. Maternal Hg exposure and history of cardiovascular diseases are independent risk factors for pregnant hypertension, whereas the GSTT1 homozygous deletion genotype has no role in diastolic hypertension and birth outcomes among the Indonesian coastal pregnant mother population.

Characteristics, attitudes, and the odds for positive attitude toward clinical trial: A study on Indonesian COVID-19 vaccine trial participants.

Aims: This study was performed to understand the Indonesian population's characteristics and the factors that contribute to a more positive attitude toward participation in a clinical trial. Methods: A cross-sectional survey was conducted involving 402 COVID-19 vaccine trial participants in Semarang, Indonesia, utilizing self-reporting questionnaires consisting of questions related to socio-demographic characteristics and statements in a 5-scaled Likert Scale to assess the attitude toward vaccine trial. The odds for positive attitude were analyzed using Ordinal Logistic Regression to obtain the odd-ratio and 95% confidence interval. The P < 0.05 was considered statistically significant. Results: Most of the respondents were adults aged 22-64-year-old (89.30%), males (63.68%), married (77.61%), worked as an employee (59.70%), obtained information about the clinical trial from the Public Health Service (41.29%), had a low education level (40.80%), a low monthly income level (68.41%), with no previous participation in a clinical trial (90.80%). All respondents showed a good attitude toward the trial, with low education level, nonemployment status, fewer or no previous participation in clinical trials, and getting the information from the public health centers were the main predictors for better attitude toward vaccine trials. Conclusion: There was a positive attitude toward vaccine trials in the Indonesian population. The positive attitude could be driven by having a low education level, nonemployment status, fewer or no previous participation in the clinical trial, and getting information from public health centers.

The efficiency of selective pooling strategy in a COVID-19 diagnostic laboratory.

INTRODUCTION: Mass testing is essential in the surveillance strategy for fighting the COVID-19 pandemic. It allows early detection of suspected cases and subsequently early isolation to mitigate spread. However, the high cost and limited consumables and reagents hinder the mass testing strategy in developing countries such as Indonesia. The specimen pooling strategy is an option to perform mass screening with limited resources. This study aims to determine the positivity rate cut-off and to evaluate the efficiency of pooling strategy for the laboratory diagnosis of COVID-19. METHODOLOGY: Between August 4th, 2020, and November 11th, 2020, a four-sample pooling strategy testing to detect SARS-CoV-2 was carried out at the Microbiology Diagnostic Laboratory of Diponegoro National Hospital, Semarang, Indonesia. Pools with positive results were subjected to individual specimen retesting. Spearman's correlation and linear regression analysis were used to determine the best positivity rate cut-off to apply pooling strategy. RESULTS: A total of 15,216 individual specimens were pooled into 3,804 four-sample pools. Among these pools, 1,007 (26.47%) were positive. Five hundred and ten (50.64%) were 1/4 positive. A maximum positivity rate of 22% is needed to save at least 50% extraction and qRT-PCR reactions in a four-sample pooling strategy. CT values between individual specimens and pools showed a good interval agreement. CONCLUSIONS: Pooling strategy could reduce personnel workload and reagent cost, and increase laboratory capacity by up to 50% when the positivity rate is less than 22%.

Degradation of ciprofloxacin in aqueous solution using ozone microbubbles: spectroscopic, kinetics, and antibacterial analysis.

Ciprofloxacin (CIP) has been listed in the last version of the surface water due to its ability to kill human cells by inhibiting the activity of DNA topoisomerase IV. Thus, CIP, along with other antibiotic pollution has become a serious threat to the environment and public health. Ozonation has been used as an advanced technique that is applied in wastewater treatment to remove CIP, but the primary limitation of this method is the low solubility of ozone in water. This study is the first report of CIP removal in a scale-up of its aqueous solution using a self-developed aerator pump-enhanced ozonation (APO) system, which only employs a propeller and a zigzag arrangement of meshes. This aerator pump decreased the size of ozone bubbles by 90% and increased the effective ozone solubility to 0.47 ppm. The mechanism of degradation of CIP is attributed to an oxidation reaction of the antibiotic with reactive oxygen species, such as hydroxyl, oxygen, and hydroperoxyl radicals, generated on the surface of the ozone microbubbles. It was found that the rate and efficiency of degradation of CIP using the APO system were 3.64 × 10-3/min and 83.5%, respectively, which is higher compared with those of conventional flow ozonation (FO) systems (1.47 × 10-3/min and 60.9%). The higher degradation efficiency of CIP by the APO system was also revealed by its higher electrical energy efficiency (0.146 g/kWh), compared to that of the FO system (0.106 g/kWh). The degradation of CIP was also monitored by the resulting antibacterial activity against Escherichia coli and Staphylococcus aureus.

Trimetazidine improves left ventricular global longitudinal strain value in patients with heart failure with reduced ejection fraction due to ischemic heart disease.

Heart failure with reduced ejection fraction (HFrEF) due to ischemic heart disease (IHD) showed a progressive decline in left ventricular contractile function. However, no previous study has examined the left ventricular global longitudinal strain (LV GLS) parameter that represents LV contractile function. We investigated whether trimetazidine could improve the LV GLS value in patients with HFrEF due to IHD. We performed a double-blind, randomized controlled trial (RCT) including 26 patients with HFrEF due to stable IHD who were given modified-release trimetazidine 35 mg twice per day (n = 13) or placebo (n = 13) for 3 months in addition to standard medication. Left ventricular systolic function including GLS values was assessed at baseline and after 3 months using echocardiography. A total of 25 participants (13 control and 12 trimetazidine groups) were recruited with a baseline average age of 57.1 ± 10 years, and LV ejection fraction (LVEF) value of 34.6% ± 4.4%, and a GLS value of 7.4% ± 2.1%. Baseline clinical characteristics and echocardiogram were similar between the two groups. There was significant GLS improvement in the trimetazidine group (-6.9% ± 2.4% to -8.4% ± 2.6%, p = 0.000), but no significant differences were noted in the control group. The GLS improvement was significantly higher in the trimetazidine group than the control (1.5% + 0.9% vs. -0.7% + 1.7%, p = 0.001). No adverse drug reactions from the administration of trimetazidine in this study. Trimetazidine may improve GLS values in patients with HFrEF due to IHD.

Cytochrome b-245 Alpha Chain Gene Variants and Arterial Function in Indonesian Short Stature Children.

BACKGROUND: The association between short stature, undernutrition and the risk to cardiovascular disease has been clinically established. Genetic factor, particularly the variants in cytochrome b-245 alpha chain (CYBA) gene, which alter the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase level, might affect arterial function. This study aimed to observe the association between single nucleotide variants (SNVs) of the CYBA gene and the arterial function of short stature children to understand the reason why some people with short stature develop cardiovascular disease. METHODS: A total of 142 genomic deoxyribonucleic acid (DNA) samples have been collected from short stature children in Brebes, Central Java, Indonesia. Four common single-nucleotide polymorphisms (SNPs): C242T (rs4673), A640G (rs1049255), -930A>G (rs9932581) and *49A>G (rs7195830) in the CYBA gene were examined using TaqMan allelic discrimination assay. The arterial function was measured using transthoracic echocardiography and described as aortic stiffness and distensibility index. Statistical analysis was done to find a significant difference in arterial function between genotypes of each SNV. RESULTS: A P-value of < 0.05 was considered significant. In rs9932581 (-930A>G) of CYBA gene, the subjects with GG genotype were found to have significantly lower arterial stiffness and higher distensibility compared to AA and AG genotypes. No significant difference was found in the other SNVs. CONCLUSION: The GG genotype in rs9932581 of the CYBA gene might have a protective effect on cardiovascular disease in short stature children.

Effects of Cooking Method on the Antioxidant Activity and Inhibition of Lipid Peroxidation of the Javanese Salad "Pecel" Vegetables and Its Peanut Sauce Dressing.

Vegetables are essential in our diet to maintain health, partly due to their antioxidant properties. A well-known Javanese salad called "Pecel" is prepared by boiling the vegetables and dressed with seasoned peanut sauce. Cooking can reduce or improve the antioxidant properties of foods; therefore, the purpose of this study was to evaluate the effects of brief water boiling (1 min), steaming (1 min), and water blanching (20 s) of the Javanese Pecel vegetables, with or without the peanut sauce. We assessed the in vitro antioxidant capacity and lipid peroxidation inhibition of the salad samples prepared using each cooking method. Six vegetables, i.e., Sesbania grandiflora (turi) flower, Amaranthus hybridus L. (spinach), Carica papaya (papaya) leaves, Cosmos caudatus L. (kenikir) leaves, Vigna unguiculata ssp. Sesquipedalis (yard-long beans), and Vigna radiata (mung-bean) sprouts were cooked by boiling or steaming for 1 min or blanching for 20 s. Peanut (Arachis hypogaea), the raw material for peanut sauce, was fried in either fresh palm oil or repeatedly used palm oil. Our results revealed that the highest antioxidant capacity (percent inhibition of DPPH radicals) was observed following boiling for 1 min in case of spinach (41.94 ± 9.8%), papaya (59.04 ± 5.35%), kenikir (54.93% ± 6.32%), and yard-long beans (70.21 ± 8.91%); steaming for 1 min in case of turi flower (60.25 ± 3.63%); and blanching for 20 s in case of mung-bean sprouts (49.27 ± 3.69%). Peanut sauce prepared by frying peanuts in fresh or repeatedly used palm oil reduces the natural antioxidant and lipid peroxidation inhibition properties. However, seasoning the peanut sauce with fresh garlic and lime leaves can restore the lost antioxidant properties. Our study provides the first and clear evidence of the optimal cooking method for Pecel vegetables and sheds light on the wisdom behind the existing traditional cooking method.

Uric Acid as a Risk Factor for Chronic Kidney Disease and Cardiovascular Disease　- Japanese Guideline on the Management of Asymptomatic Hyperuricemia.

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies.

Activation of MIP-2 and MCP-5 Expression in Methylmercury-Exposed Mice and Their Suppression by N-Acetyl-L-Cysteine.

Methylmercury (MeHg) is a well-known neurotoxin of the central nervous system (CNS). Neuroinflammation is one of the main pathways of MeHg-induced CNS impairment. This study aims to investigate the expressions of IL-6, MIP-2, and MCP-5, as biomarkers in relation with MeHg-induced CNS impairment and N-acetyl-L-cysteine (NAC) treatment in mice, as well as histopathological changes of brain tissue and clinical symptom such as ataxia. Twenty male Balb/c mice, aged 8-9 weeks, were divided into 4 groups and treated with saline (control), NAC [150 mg/kg body weight (BW) day], MeHg (4 mg Hg/kg BW), or a combination of MeHg and NAC for 17 days. MeHg induced the expression of IL-6, MIP-2, and MCP-5 in the serum, with median values (those in controls) of 55.06 (9.44), 15.94 (9.30), and 458.91 (239.91) mg/dl, respectively, and a statistical significance was observed only in IL-6 expression (p < 0.05). MIP-2 and MCP-5 expressions tended to increase in the cerebrum of MeHg-treated group compared with controls; however, the difference was not statistically significant. MeHg treatment also increased IL-6 expression in the cerebellum (7.73 and 4.81 mg/dl in MeHg-treated group and controls, respectively), with a marginal significance. NAC significantly suppressed MeHg-induced IL-6 and MIP-2 expressions in the serum (p < 0.05 for both), and slightly reduced MCP-5 expression in the cerebrum. Ataxia was observed in all MeHg-treated mice after 9-day exposure as well as the decrease of intact Purkinje cells in brain tissue (p < 0.05). These findings suggest that MeHg induced neurotoxicity by elevating the expression of IL-6, MIP-2, and MCP-5 and causing ataxia symptoms, and NAC reduced MeHg-mediated effects on the CNS.

Evidence for Urate Uptake Through Monocarboxylate Transporter 9 Expressed in Mammalian Cells and Its Enhancement by Heat Shock.

Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions. Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132. Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

Uric Acid-Induced Enhancements of Kv1.5 Protein Expression and Channel Activity via the Akt-HSF1-Hsp70 Pathway in HL-1 Atrial Myocytes.

BACKGROUND: Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents. CONCLUSIONS: Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

Sociocultural aspects of disorders of sex development.

Disorders of sex development (DSD) is a congenital condition in which the development of chromosomes, gonads, hormones, and reproductive structures are atypical. DSD brings with it a psychological impact on the affected individual and their families. The consensus statement on management of DSD strongly advised an integrated and multidisciplinary approach in providing care to the affected individuals. Studies have been conducted focusing on medical intervention, and more recently, there is increasing attention paid to psychological aspects of DSD. However, studies reporting cultural aspects of DSD are lacking. This review provides an overview on how culture impacts the affected individuals in coping with DSD and making decisions with regard to gender assignment or reassignment, help-seeking behavior for medical treatments, attitudes toward medical treatment, religious beliefs, and values concerning marriage and fertility. The involvement of social scientists is needed to study sociocultural aspects of DSD from more diverse cultures, to help affected individuals and their families in gaining better social acceptance. Birth Defects Research (Part C) 108:380-383, 2016. © 2016 Wiley Periodicals, Inc.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

Hyperuricemia and Atrial Fibrillation.

The importance of atrial fibrillation (AF) as a cause of mortality and morbidity has prompted research on its pathogenesis and treatment. Recognition of AF risk factors is essential to prevent it and reduce the risk of death. Hyperuricemia has been widely accepted to be associated with the incidence of paroxysmal or persistent AF, as well as to the risk of AF in post cardiovascular surgery patients. The possible explanations for this association have been based on their relation with either oxidative stress or inflammation. To investigate the link between hyperuricemia and AF, it is necessary to refer to hyperuricemia-induced atrial remodeling. So far, both ionic channel and structural remodeling caused by hyperuricemia might be plausible explanations for the occurrence of AF. Inhibition of xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, or the use of antioxidants, along with serum uric acid (SUA) level reduction to prevent inflammation, might be useful. Uric acid transporters (UATs) play a key role in the regulation of intracellular uric acid concentration. Intracellular rather than serum uric acid level is considered more important for the pathogenesis of AF. Identification of UATs expressed in cells is thus important, and targeting UATs might become a potential strategy to reduce the risk of hyperuricemia-induced atrial fibrillation.

Molecular Mechanisms Underlying Urate-Induced Enhancement of Kv1.5 Channel Expression in HL-1 Atrial Myocytes.

BACKGROUND: Hyperuricemia induces endothelial dysfunction, oxidative stress and inflammation, increasing cardiovascular morbidities. It also raises the incidence of atrial fibrillation; however, underlying mechanisms are unknown. METHODS AND RESULTS: The effects of urate on expression of Kv1.5 in cultured mouse atrial myocytes (HL-1 cells) using reverse transcriptase-PCR, immunoblots, flow cytometry and patch-clamp experiments were studied. Treatment with urate at 7 mg/dl for 24 h increased the Kv1.5 protein level, enhanced ultra-rapid delayed-rectifier K(+)channel currents and shortened action potential duration in HL-1 cells. HL-1 cells expressed the influx uric acid transporter (UAT), URATv1, and the efflux UATs, ABCG2 and MRP4. An inhibitor against URATv1, benzbromarone, abolished the urate effects, whereas an inhibitor against ABCG2, KO143, augmented them. Flow cytometry showed that urate induced an increase in reactive oxygen species, which was abolished by the antioxidant, N-acetylcysteine (NAC), and the NADPH-oxidase inhibitor, apocynin. Both NAC and apocynin abolished the enhancing effects of urate on Kv1.5 expression. A urate-induced increase in the Kv1.5 proteins was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and was abolished by an ERK inhibitor, PD98059. NAC abolished phosphorylation of ERK by urate. CONCLUSIONS: Intracellular urate taken up by UATs enhanced Kv1.5 protein expression and function in HL-1 atrial myocytes, which could be attributable to ERK phosphorylation and oxidative stress derived from nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Depletion of Uric Acid Due to SLC22A12 (URAT1) Loss-of-Function Mutation Causes Endothelial Dysfunction in Hypouricemia.

BACKGROUND: Uric acid (UA) serves as an antioxidant in vascular endothelial cells. UA transporter 1 (URAT1) encoded by SLC22A12 is expressed in the kidney and vessels and its loss of function causes hypouricemia. The purpose of this study was to examine whether there is any endothelial dysfunction in patients with hypouricemia. METHODS AND RESULTS: Twenty-six patients with hypouricemia (<2.5 mg/dl) and 13 healthy control subjects were enrolled. Endothelial function was evaluated using flow-mediated dilation (FMD). mRNA of UA transporters expressed in cultured human umbilical endothelial cells (HUVEC) was detected on RT-PCR. There was a positive correlation between FMD and serum UA in the hypouricemia group. URAT1 loss-of-function mutations were found in the genome of 21 of 26 patients with hypouricemia, and not in the other 5. In the hypouricemia groups, serum UA in homozygous and compound heterozygous patients was significantly lower than in other groups, suggesting that severity of URAT1 dysfunction may influence the severity of hypouricemia. Thirteen of 16 hypouricemia subjects with homozygous and compound heterozygote mutations had SUA <0.8 mg/dl and their FMD was lower than in other groups. HUVEC do not express mRNA of URAT1, suggesting the null role of URAT1 in endothelial function. CONCLUSIONS: Depletion of UA due to SLC22A12/URAT1 loss-of-function mutations causes endothelial dysfunction in hypouricemia patients.

Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria. METHODS: Genomic DNA of a patient was extracted from peripheral white blood. Exons and flanking sequences of UMOD were amplified by PCR with primers. Mutation analysis was performed by direct sequencing of the PCR products. The wild-type and mutant uromodulin were expressed in HEK293 cells and analyzed by western blotting, immunoprecipitation, immunofluorescence, and flow cytometry. RESULTS: We identified an FJHN patient who carried a novel UMOD mutation G335A (C112Y). The levels of both cytosolic and secreted C112Y protein were significantly decreased compared with the wild-type, whereas the level of ubiquitination was higher in C112Y than that in the wild type. The half-life of C112Y was shortened and it was restored by a proteasome inhibitor MG132. Immunofluorescence revealed decreased levels of C112Y in the Golgi apparatus and on the plasma membrane. Expression of C112Y induced cellular apoptosis as revealed by flow cytometry. Apoptosis induced by C112Y was suppressed by topiroxostat. CONCLUSION: C112Y causes its protein instability resulting cellular apoptosis which could be suppressed with topiroxostat.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.