Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution.

HYPOTHESIS: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. EXPERIMENTS: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FINDINGS: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

H4K20me3 upregulated by reactive oxygen species is associated with tumor progression and poor prognosis in patients with hepatocellular carcinoma.

Epigenetic alteration by oxidative stress is vitally involved in carcinogenesis and cancer progression. Previously, we demonstrated that oxidative stress was increased in hepatocellular carcinoma (HCC) patients and associated with tumor aggressiveness. Herein, we immunohistochemically investigated whether histone methylation, specifically H4K20me3, was upregulated in human hepatic tissues obtained from HCC patients (n = 100). Also, we experimentally explored if the H4K20me3 was upregulated by reactive oxygen species (ROS) and contributed to tumor progression in HCC cell lines. We found that H4K20me3 level was increased in HCC tissues compared with the adjacent noncancerous liver tissues. H3K9me3 and H3K4me3 levels were also increased in HCC tissues. Cox regression analysis revealed that the elevated H4K20me3 level was associated with tumor recurrence and short survival in HCC patients. Experimentally, H2O2 provoked oxidative stress and induced H4K20me3 formation in HepG2 and Huh7 cells. Transcript expression of histone methyltransferase Suv420h2 (for H4K20me3), Suv39h1 (for H3K9me3), and Smyd3 (for H3K4me3) were upregulated in H2O2-treated HCC cells. H2O2 also induced epithelial-mesenchymal transition (EMT) in HCC cells, indicated by decreased E-cadherin but increased α-SMA and MMP-9 mRNA expression. Migration, invasion, and colony formation in HCC cells were markedly increased following the H2O2 exposure. Inhibition of H4K20me3 formation by A196 (a selective inhibitor of Suv420h2) attenuated EMT and reduced tumor migration in H2O2-treated HCC cells. In conclusion, we demonstrated for the first time that H4K20me3 level was increased in human HCC tissues, and it was independently associated with poor prognosis in HCC patients. ROS upregulated H4K20me3 formation, induced mRNA expression of EMT markers, and promoted tumor progression in human HCC cells. Inhibition of H4K20me3 formation reduced EMT and tumor aggressive phenotypes in ROS-treated HCC cells. Possibly, ROS-induced EMT and tumor progression in HCC cells was epigenetically mediated through an increased formation of repressive chromatin H4K20me3.

Acute effect of various dosages of sugar ingestion on vascular function in offspring of hypertensive and normotensive parents.

OBJECTIVES: We aimed to study vascular function in healthy men with a parental history of hypertension compared to those without. Acute effect of various dosages of sugar ingestion on vascular function was also investigated in both groups. METHODS: Thirty-two healthy men were recruited and divided into two groups, offspring of hypertensive parents (OHT) and offspring of normotensive parents (ONT). Participants were orally given 15, 30, and 60 g of sucrose solution compared to water. Peak forearm blood flow (FBF), forearm vascular resistance (FVR), pulse wave velocity (PWV), and oxidative stress markers were measured at baseline and after sucrose intake at 30, 60, 90, and 120 min. RESULTS: At baseline, peak FBF was significantly lower (22.40 ±â€Š1.18 vs. 25.24 ±â€Š0.63 ml × dl -1  × min -1 , P  < 0.001), FVR was significantly higher (3.73 ±â€Š0.42 vs. 3.30 ±â€Š0.26 mmHg × ml -1  × dl × min, P  = 0.002), and PWV was significantly faster (6.31 ±â€Š0.59 vs. 5.78 ±â€Š0.61 m/s, P  = 0.017) in OHT than ONT. After each sucrose intake, peak FBF significantly declined and was lowest at 30 min in both groups. The reduction in peak FBF was seen in all doses of sucrose and the higher dose of sucrose intervened, the longer reduction in peak FBF observed. CONCLUSIONS: Vascular function was attenuated in healthy men with a family history of hypertension and became worse after sucrose ingestion even at the low dose. Our findings suggest that the ones, especially those with a parental history of hypertension, should reduce sugar consumption as low as possible.

A study of cyanidin/alginate complexation: Influence of pH in assembly and chiral properties.

Cyanidin 3-O-glucoside (CND) is a frequently-used anthocyanin that has excellent antioxidant properties but a limited bioavailability in bloodstream. Complexation of CND with alginate can improve its therapeutic outcome. Here we have studied the complexation of CND with alginate under a range of pH values from 2.5 to 5. CND is positively charged at low pH, and becomes neutral, and then negatively charged as pH increases. CND/alginate complexation was studied by dynamic light scattering, transmission electron microscopy, small angle X-ray scattering, STEM, UV-Vis spectroscopy and circular dichroism (CD). CND/alginate complexes at pH 4.0 and 5.0 form chiral fibres with a fractal structure. At these pH values, CD spectra show very intense bands, which are inverted compared with free CND. Complexation at lower pH results in disordered polymer structures and CD spectra show the same features as for CND in solution. Molecular dynamics simulations suggest the formation of parallel CND dimers through complexation with alginate at pH 3.0, while at pH 4.0 CND dimers form in a cross like arrangement.

HydroZitLa inhibits calcium oxalate stone formation in nephrolithic rats and promotes longevity in nematode Caenorhabditis elegans.

Low fluid intake, low urinary citrate excretion, and high oxidative stress are main causative factors of calcium oxalate (CaOx) nephrolithiasis. HydroZitLa contains citrate and natural antioxidants and is developed to correct these three factors simultaneously. Antioxidants theoretically can prolong the lifespan of organisms. In this study, we preclinically investigated the antilithogenic, lifespan-extending and anti-aging effects of HydroZitLa in HK-2 cells, male Wistar rats, and Caenorhabditis elegans. HydroZitLa significantly inhibited CaOx crystal aggregation in vitro and reduced oxidative stress in HK-2 cells challenged with lithogenic factors. For experimental nephrolithiasis, rats were divided into four groups: ethylene glycol (EG), EG + HydroZitLa, EG + Uralyt-U, and untreated control. CaOx deposits in kidneys of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. Intrarenal expression of 4-hydroxynonenal in EG + HydroZitLa rats was significantly lower than that of EG rats. The urinary oxalate levels of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. The urinary citrate levels of EG + HydroZitLa and EG + Uralyt-U rats were restored to the level in normal control rats. In C. elegans, HydroZitLa supplementation significantly extended the median lifespan of nematodes up to 34% without altering feeding ability. Lipofuscin accumulation in HydroZitLa-supplemented nematodes was significantly lower than that of non-supplemented control. Additionally, HydroZitLa inhibited telomere shortening, p16 upregulation, and premature senescence in HK-2 cells exposed to lithogenic stressors. Conclusions, HydroZitLa inhibited oxidative stress and CaOx formation both in vitro and in vivo. HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans and human kidney cells. This preclinical evidence suggests that HydroZitLa is beneficial for inhibiting CaOx stone formation, promoting longevity, and slowing down aging.

Umami and Other Taste Perceptions in Patients With Parkinson's Disease.

OBJECTIVE: Studies of taste perceptions in Parkinson's disease (PD) patients have been controversial, and none of these studies have assessed umami taste. This study aimed to assess umami, along with the other 4 taste functions in PD patients. METHODS: Participants were tested for gustation using the modified filter paper disc method and olfaction using the modified Sniffin' Stick-16 (mSS-16) test (only 14 culturally suitable items were used). A questionnaire evaluated patients' subjective olfactory and gustatory dysfunction, taste preference, appetite, and food habits. RESULTS: A total of 105 PD patients and 101 age- and sex-matched controls were included. The body mass index (BMI) of PD patients was lower than that of controls (PD = 22.62, controls = 23.86, p = 0.028). The mSS-16 score was 10.7 for controls and 6.4 for PD patients (p < 0.001) (normal ≥ 9). Taste recognition thresholds (RTs) for sweet, salty, sour, bitter and umami tastes were significantly higher in PD, indicating poorer gustation. All taste RTs correlated with each other, except for umami. Most patients were unaware of their dysfunction. Patients preferred sweet, salty and umami tastes more than the controls. Dysgeusia of different tastes in patients was differentially associated with poorer discrimination of tastes, an inability to identify the dish and adding extra seasoning to food. BMI and mSS-16 scores showed no correlation in either patients or controls. CONCLUSION: PD patients have dysgeusia for all five tastes, including umami, which affects their appetite and diet. Patients preferred sweet, salty and umami tastes. This information can help adjust patients' diets to improve their nutritional status.

Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis.

Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-ßgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-ßgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Contamination of antibiotics and sul and tet(M) genes in veterinary wastewater, river, and coastal sea in Thailand.

Water systems in Southeast Asia accumulate antibiotics and antibiotic resistance genes (ARGs) from multiple origins, notably including human clinics and animal farms. To ascertain the fate of antibiotics and ARGs in natural water environments, we monitored the concentrations of these items in Thailand. Here, we show high concentrations of tetracyclines (72,156.9 ng/L) and lincomycin (23,968.0 ng/L) in pig farms, followed by nalidixic acid in city canals. The city canals and rivers contained diverse distributions of antibiotics and ARGs. Assessments of targeted ARGs, including sul1, sul2, sul3, and tet(M), showed that freshwater (pig farm wastewater, rivers, and canals) consistently contained these ARGs, but these genes were less abundant in seawater. Although sulfonamides were low concentrations (<170 ng/mL), sul1 and sul2 genes were abundant in freshwater (minimum 4.4 × 10-3-maximum 1.0 × 100 copies/16S), suggesting that sul genes have disseminated over a long period, despite cessation of use of this class of antibiotics. Ubiquitous distribution of sul genes in freshwater appeared to be independent of selection pressure. In contrast, water of the coastal sea in the monitored area was not contaminated by these antibiotics or ARGs. The density of Enterobacteriales was lower in seawater than in freshwater, suggesting that the number of ARG-possessing Enterobacteriales falls after entering seawater. From the pig farms, through rivers/canals, to the coastal sea, the occurrence of tetracyclines and tet(M) exhibited some correlation, although not a strong one. However, no correlations were found between concentrations of total antibiotics and ARGs, nor between sulfonamides and sul genes. This is the first comprehensive study showing Thai features of antibiotics and ARGs contaminations. The pig farm is hot spot of antibiotics and ARGs, and sul genes ubiquitously distribute in freshwater environments, which become less abundant in seawater.

Genetic characterization of coliform bacterial isolates from environmental water in Thailand.

INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with ß-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Supplementing post-wash asthenozoospermic human spermatozoa with coenzyme Q10 for 1 hr in vitro improves sperm motility, but not oxidative stress.

We investigated the effect of supplementing post-wash asthenozoospermic spermatozoa with coenzyme Q10 (CoQ10) in vitro, which may reduce oxidative stress and improve sperm motility. Semen samples were collected from 39 men with asthenozoospermia, and their spermatozoa were isolated by two-layer Percoll density-gradient centrifugation. Kinetic parameters of the isolated spermatozoa (baseline before intervention) were determined immediately by computer-aided semen analysis. Total anti-oxidant capacity and protein carbonyl levels, as markers of oxidative stress, were also measured in the baseline spermatozoa. The baseline spermatozoa suspension was divided equally into two portions, one for CoQ10 supplementation (50 µg/ml for 1 hr) and the other as an un-supplemented vehicle control. The total motility of the CoQ10-supplemented spermatozoa was significantly higher than in the control (p = .009) and progressive motility tended to be higher (p = .053). Immotile sperm concentration in the CoQ10-supplemented spermatozoa was significantly lower than in both the baseline (p = .026) and control (p = .009). Total anti-oxidant capacity and protein carbonyl levels between the baseline, CoQ10-supplemented and control spermatozoa were not significantly different. Our data suggest that CoQ10 treatment reactivated sperm motility. We propose short-term supplementation of post-wash asthenozoospermic spermatozoa with CoQ10 before intrauterine insemination.

Clinical validation of urinary indole-reacted calcium oxalate crystallization index (iCOCI) test for diagnosing calcium oxalate urolithiasis.

An indole-reacted calcium oxalate crystallization index (iCOCI) test was developed to quantify the total competence of urine to precipitate calcium oxalate (CaOx) crystals. We conducted the prospective cohort study in accordance with the STARD guideline to evaluate the accuracy of urinary iCOCI test (index test) for diagnosing urolithiasis. A total of 281 participants were recruited for the study. Levels of urinary iCOCI were determined in the pre-diagnostic 24-h urine samples. Positive urinary iCOCI (≥ 0.6 COM eqv., g/L) was accounted for 51% (144/281), and the rest of 49% (137/281) were negative. Non-contrast CT imaging (reference standard) was subsequently performed for the definite diagnosis of urolithiasis to divide the participants into two groups, non-stone subjects (NSS, n = 122) and stone-forming subjects (SFS, n = 159). It should be noted that only subjects who currently had urinary stone at the time of study were classified as SFS. Urinary iCOCI levels in the SFS were significantly higher than the NSS. ROC analysis revealed an area under curve (AUC) of 0.893 (95% CI: 0.855-0.932) in separating NSS from all SFS. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive likelihood ratio (LH+) and negative likelihood ratio (LH-) of urinary iCOCI test for diagnosis of all urolithiasis were 87%, 80%, 84%, 84%, 83%, 4.44 and 0.16, respectively. Of 159 SFS, 38 were confirmed to have CaOx stones. Among these 38 CaOx SFS, only 2 had negative urinary iCOCI test. The AUC of urinary iCOCI test for separating CaOx SFS from NSS was markedly high (0.946, 95% CI: 0.914-0.978). Sensitivity, specificity, PPV, NPV, accuracy, LH+ and LH- of urinary iCOCI test for diagnosing CaOx urolithiasis were 95%, 86%, 68%, 98%, 88%, 6.80 and 0.06, respectively. Conclusion, we clinically validated that an innovative non-invasive urinary iCOCI test was highly accurate to diagnose urolithiasis, especially CaOx stone. With its high sensitivity and NPV, urinary iCOCI test is clinically intended to use as a screening test for CaOx urolithiasis. LH- of 0.06 indicates that negative result of urinary iCOCI test is highly accurate to rule out the CaOx stone formation. It is noted that urinary iCOCI level is expressed as arbitrary unit, and it is not directly related to the actual physiological level of urinary oxalate.

Detection of CD33 expression on monocyte surface is influenced by phagocytosis and temperature.

CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli.

Oxidative stress and LINE-1 reactivation in bladder cancer are epigenetically linked through active chromatin formation.

Oxidative stress and reactivation of long interspersed element-1 (LINE-1) are coincidently observed in bladder cancer (BlCa), but the mechanistic connection between these two oncogenic phenomena is unknown. Previously, we reported increases in oxidative stress and LINE-1 protein (ORF1p) expression in human BlCa tissues. In this study, we measured 5-methylcytosine (5mC), 8-hydroxydeoxyguanosine (8-OHdG), 8-oxoguanosine DNA glycosylase-1 (OGG1), H3K9me3 and HP1α in bladder tissues obtained from BlCa patients. Reactivation of LINE-1 by reactive oxygen species (ROS) through chromatin remodeling was investigated in seven BlCa cell lines. We found that 5mC was decreased, but 8-OHdG, H3K9me3 and HP1α levels were increased in BlCa tissues relative to the adjacent non-cancerous tissues. OGG1, H3K9me3 and HP1α expression in BlCa tissues were positively correlated with 8-OHdG levels. Following H2O2 treatment, LINE-1 transcript expression was increased in VM-CUB-1 and TCCSUP, whereas AluYa5 and AluYb8 transcripts were increased in BFTC905 cells. Basal expression of LINE-1 ORF1p varied among BlCa cell lines from none to very high. H2O2 treatment clearly increased expression of ORF1p in VM-CUB-1, TCCSUP and BFTC905. Chromatin immunoprecipitation experiments revealed that 5'-LINE-1 promoters became further enriched in H3K4me3 and H3K18ac in VM-CUB-1 and BFTC905 cells treated with H2O2. In contrast, 5'-LINE-1 promoters became more enriched in H3K9me3 and H3K27me3 in UM-UC-3 treated with H2O2. In summary, decreased 5mC, but increased 8-OHdG, H3K9me3 and HP1α expression were demonstrated in human BlCa tissues, indicating global DNA hypomethylation, increased oxidative stress and altered histone methylation in BlCa. Chromatin structures were profoundly changed in BlCa cells exposed to ROS, but expression of LINE-1 transcript and protein were at most modestly increased. ROS enhanced expression of full-length LINE-1 elements only in cell lines with pre-existing activation, which was paralleled by increased formation of active chromatin at LINE-1 promoter loci.

Lime powder regimen supplement alleviates urinary metabolic abnormalities in urolithiasis patients.

BACKGROUND: Urolithiasis has high recurrent rate after surgical removal within 3 years. Potassium citrate compound is used to prevent stone recurrence but it has intolerable gastrointestinal adverse effects. We conducted a phase 2 clinical study of lime power regimen (LPR), a limeade-based supplement containing potassium and citrate for 6 months period of treatment, to evaluate its effects on biochemical and clinical aspects of recurrent urolithiasis. METHODS: Seventy-four urolithiasis patients were randomly allocated to receive either LPR or placebo for 6 months in a double-blinded manner. Plasma and 24 h urine samples were collected to measure urinary pH, mineral excretion and urinary total antioxidant status , plasma for creatinine and plasma protein carbonyl, and stone for elemental analysis at the initiation and end-of-treatment (6 month). Adverse effects were recorded. RESULTS: Administration of LPR significantly increased urinary pH, citrate and potassium excretion. Urinary levels of calcium and oxalate, and plasma protein carbonyl content were reduced, while urinary total antioxidant status was elevated by LPR treatment. Urinary supersaturation was decreased and urinary protein excretion was ameliorated in LPR-treated patients. Gastrointestinal adverse effects were rarely observed. None of the participants developed stone recurrence for the duration of the trial. CONCLUSION: Lime power regimen is a potential drug to correct urinary metabolic disorders associated with urolithiasis in high risk stone recurrent patients. A phase 3 clinical trial is underway to validate anti-stone recurrence property of LPR in long-term treatment.

rs11567842 SNP in SLC13A2 gene associates with hypocitraturia in Thai patients with nephrolithiasis.

Hypocitraturia is a profound risk for kidney stone formation and recurrence. Sodium-dicarboxylate cotransporter-1 (NaDC-1) is a main transporter responsible for citrate reabsorption in renal proximal tubules. To investigate an association of sodium-dicarboxylate cotransporter-1 (NaDC-1) polymorphism with hypocitraturia in Thai patients with nephrolithiasis (NL). Exonic SNPs in NaDC-1 were screened in peripheral blood DNA of 13 NL patients. The rs11567842 (A/G) variant was found and further genotyped in 145 NL patients and 115 non-stone forming controls. NL patients had significantly lower level of urinary citrate than the controls. Based on logistic regression, hypocitraturia was significantly associated with urinary stone formation (adjusted OR 8.34, 95% CI 4.63-15.04). Significant association of urinary citrate level with rs11567842 genotype was found only in the NL group. NL patients with GG genotype had significantly higher urinary citrate than those with AA and AG genotypes. GG carrying patients had significantly reduced risk for hypocitraturia (adjusted OR 0.15; 95% CI 0.05-0.48, AA as reference). In selected 15 calcium oxalate stone patients, AA carriers had significantly higher intrarenal NaDC-1 mRNA than GG and AG carriers. Homozygous GG of rs11567842 SNP in NaDC-1 gene was a protective genotype for hypocitraturia in kidney stone patients. The findings suggested that patients with AA genotypes were more susceptible to hypocitraturia than those with GG, hence carrying a higher risk for kidney stone recurrence.

LINE-1 ORF1 Protein Is Up-regulated by Reactive Oxygen Species and Associated with Bladder Urothelial Carcinoma Progression.

BACKGROUND/AIM: Reactivation of long interspersed nuclear element-1 (LINE-1) and oxidative stress are suggested to have oncogenic potential to drive tumorigenesis and cancer progression. We previously demonstrated that reactive oxygen species (ROS) caused hypomethylation of LINE-1 elements in bladder cancer cells. In this study, we investigated the expression of LINE-1-encoded protein (ORF1p) and oxidative stress marker 4-hydroxynonenal (4-HNE) in human bladder cancer tissues, as well as induction of ORF1p expression by ROS in bladder cancer cell lines. MATERIALS AND METHODS: Thirty-six cancerous and 15 non-cancerous adjacent tissues were immunohistochemically stained for ORF1p and 4-HNE. ORF1p expression and cell migration were determined in bladder cancer cells exposed to H2O2 Results: ORF1p and 4-HNE expression was higher in cancerous than non-cancerous tissues. Elevated ORF1p expression was associated with increased 4-HNE expression and with advanced tumors. H2O2 provoked oxidative stress and up-regulated ORF1p expression in VM-CUB-1 compared to the untreated control, and to a lesser degree in TCCSUP. H2O2 exposure enhanced cell migration in UM-UC-3, TCCSUP and VM-CUB-1. CONCLUSION: Elevated ORF1p expression is associated with tumor progression. ROS experimentally induce ORF1p expression and promote migration in bladder cancer cells.

Urinary stone risk factors in the descendants of patients with kidney stone disease.

BACKGROUND: Evidence has indicated that immediate family members of nephrolithiasis patients had high opportunity to develop stones. However, they are usually not regarded to be at risk, since it is unclear if there are any lithogenic abnormalities found in non-stone-forming nephrolithiasis relatives. Our aim was to investigate urinary metabolic abnormalities in the children of nephrolithiasis patients, compared with the general population. METHODS: The 24-h urinary metabolic profile was studied for 28 calcium oxalate nephrolithiasis patients (NL) and 46 of their descendants (ND), as well as 40 non-stone-forming volunteers (V) and 34 of their descendants (VD). RESULTS: There was no difference between age, gender, and serum creatinine between NL vs. V (parental groups) and ND vs. VD (descendant groups). High urinary oxalate in nephrolithiasis and urinary calcium in their descendants was detected. In addition, an elevated urinary excretion rate of calcium, phosphate, protein, and albumin, along with low citrate excretion and high urinary supersaturation was observed in both the nephrolithiasis patients and their descendants. Approximate 17.8-24.4% of the nephrolithiasis descendants had a urinary supersaturation higher than the nephrolithiasis level, but none was found in VD group. The level of urinary supersaturation index was correlated with urinary protein and albumin excretion in nephrolithiasis family. CONCLUSION: It was demonstrated that nephrolithiasis offspring carried several urinary metabolic risks predisposing to stone formation which are similar to their parents, and about one in every five nephrolithiasis children had nephrolithiasis level urinary supersaturation.

Oxidative Stress in Urothelial Carcinogenesis: Measurements of Protein Carbonylation and Intracellular Production of Reactive Oxygen Species.

Oxidative stress contributes substantially to urothelial carcinogenesis. Its extent can be assessed by measurements of reactive species (mainly reactive oxygen species (ROS)), oxidatively modified damage products, and levels of various antioxidants. We presented herein the methods for the measurement of protein carbonyl content and intracellular production of ROS. Protein carbonyl is the most commonly used indicator of protein oxidation because it is early formed and relatively stable under oxidative stress. Determination of protein carbonyl relies on the derivatization of carbonyl groups (aldehydes: R-CHO and ketones: R-CO-R) with 2,4-dinitrophenylhydrazine (DNPH) under strongly acidic conditions to yield stable dinitrophenyl (DNP) hydrazones. Absorbance of the DNP hydrazones at 370-375 nm is proportional to the content of carbonyl groups. To report the protein carbonyl content, it is usually normalized by total proteins. Detection of intracellular ROS production is based on oxidation of 2',7'-dichlorofluorescein-diacetate (DCFH-DA) by ROS to produce the highly fluorescent 2',7'-dichlorofluorescein (DCF). Fluorescent intensity measured at 480 nm excitation and 535 nm emission is directly proportional to the amount of ROS generated.

Stenotrophomonas maltophilia is highly prevalent among houseflies (Musca domestica).

Stenotrophomonas maltophilia is capable of surviving in a wide variety of environments and is considered to be among the antimicrobial-resistant bacteria of greatest public health concern in hospital settings. To clarify the role of houseflies (Musca domestica) in disseminating this bacterium, we collected 99 individuals from 15 locations (9 farms and 6 urban areas) in Thailand. S. maltophilia was isolated from 39 % (39/99) of these houseflies, with the isolation rates being similar in farms and urban areas. Multiple-antimicrobial resistance was evident among the S. maltophilia isolates obtained. Of note, the rate of resistance to trimethoprim/sulfamethoxazole (TMP/SMX), the recommended first-line antimicrobial for S. maltophilia infection, was relatively high (30 %). Almost all of the isolates had a different PFGE pattern. These results suggest that houseflies ingest and host S. maltophilia from several different environmental sources. In conclusion, houseflies may facilitate the spread of antimicrobial-resistant (including TMP/SMX-resistant) S. maltophilia from environmental sources to humans.

The novel mef(C)-mph(G) macrolide resistance genes are conveyed in the environment on various vectors.

BACKGROUND: The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. METHODS: Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)-mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. RESULTS: The mef(C)-mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)-mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10-4-10-5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (<10-9). CONCLUSIONS: These results suggest that mef(C)-mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)-mph(G).