Search

Purpose: Developmental and epileptic encephalopathies (DEEs) are severe clinical conditions characterized by stagnation or decline of cognitive and behavioral abilities preceded, accompanied or followed by seizures. Because DEEs are clinically and genetically heterogeneous, next-generation sequencing, especially exome sequencing (ES), is becoming a first-tier strategy to identify the molecular etiologies of these disorders., Methods: We combined ES analysis and international data sharing., Results: We identified 11 unrelated individuals with DEE and de novo heterozygous truncating variants in the interferon regulatory factor 2-binding protein-like gene (IRF2BPL). The 11 individuals allowed for delineation of a consistent neurodevelopmental disorder characterized by mostly normal initial psychomotor development followed by severe global neurological regression and epilepsy with nonspecific electroencephalogram (EEG) abnormalities and variable central nervous system (CNS) anomalies. IRF2BPL, also known as enhanced at puberty protein 1 (EAP1), encodes a transcriptional regulator containing a C-terminal RING-finger domain common to E3 ubiquitin ligases. This domain is required for its repressive and transactivating transcriptional properties. The variants identified are expected to encode a protein lacking the C-terminal RING-finger domain., Conclusions: These data support the causative role of truncating IRF2BPL variants in pediatric neurodegeneration and expand the spectrum of transcriptional regulators identified as molecular factors implicated in genetic developmental and epileptic encephalopathies.

Retained fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNA-185. Stromal interaction molecule 1 (STIM1), a potential target gene of miRNA-185, could influence placenta release via regulating Ca 2+ concentration intracellular. The aim of this study was to explore the mechanism of RFM by investigating the regulatory relationship between miRNA-185 and STIM1 in primary uterine caruncel epithelial (UCE) cells. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect Ca 2+ concentration at prepartum 1-5 d and postpartum 6, 12 and 24 h. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (Q-PCR) and western blotting (WB) were performed to detect the mRNA and protein levels of STIM1, respectively. UCE cells were cultured by the explant culture method, further purified, and subsequently treated with PmirGLO-STIM1-Mut + miRNA-185 mimics and mirGLO-STIM1-Mut + miRNA-185 NC. Q-PCR and WB were performed to detect mRNA and protein levels of STIM1 with treated miRNA-185 mimics. Serum levels of Ca 2+ from RFM cows were abnormally decreased at prepartum 1 d and postpartum 6, 12 and 24 h. Expression level of STIM1 was down-regulated in the caruncle tissue of RFM cows. The luciferase activity was decresed about 30.9% by miRNA-185 mimics (p < 0.01), and the mRNA and protein levels of STIM1 were downregulated miRNA-185-mimics. It was suggesting that miRNA-185 might play an important role in RFM through regulating the expression of STIM1., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Retention of fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNAs. Vascular endothelial growth factor (VEGF)A, regulated by miRNA-185, can activate arachidonic acid (ARA) release via the VEGFA signaling pathway, which influences RFM. The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect VEGFA and ARA concentrations at 6, 12, and 24 h after calving. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (qPCR) and western blotting (WB) were performed to detect the mRNA and proteins levels, respectively, of genes involved in the VEGFA signaling pathway. Uterine caruncle epithelial (UCE) cells were cultured by the explant culture method, further purified, and subsequently treated with miRNA-185 mimics, miRNA-185 mimics + MEK inhibitor, or left untreated as a control for detection of the mRNA and protein levels of genes involved in the VEGFA signaling pathway. The cellular supernatant was collected for measurement of ARA levels at 12, 24 and 48 h after treatment. Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving, as compared to those in healthy dairy cows. Expression levels of most of the investigated genes (VEGFA, PLC, PRK, RAF, MEK, MAPK, and PLA) were down-regulated in the caruncle tissue of RFM cows. However, P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM (p < .01). In UCE cells treated with the miRNA-185 mimics, expression of VEGFA, PLC, RAF, MEK, MAPK and PLA was significantly down-regulated, while that of P-p44/42 MAPK was significantly up-regulated. Expression of genes involved in the VEGFA signaling pathway was similar to that in the in vivo assay. In UCE cells treated with the miRNA-185 mimics + MEK inhibitors, expression of VEGFA, PLC, RAF, MEK, MAPK and P-p44/42 MAPK was significantly down-regulated, while that of PLA was significantly up-regulated. Meanwhile, the release of ARA was increased (p < .01). These results demonstrate that miRNA-185 can regulate the VEGFA signaling pathway, especially via abnormal expression of P-p44/42 MAPK, which influences the release of the fetal placenta after calving., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Biocompatibility of dentin bonding agents (DBA) and composite resin may affect the treatment outcome (e.g., healthy pulp, pulpal inflammation, pulp necrosis) after operative restoration. Bisphenol-glycidyl methacrylate (BisGMA) is one of the major monomers present in DBA and resin. Prior studies focused on salivary esterase for metabolism and degradation of resin monomers clinically. This study found that human dental pulp cells expressed mainly carboxylesterase-2 (CES2) and smaller amounts of CES1A1 and CES3 isoforms. Exposure to BisGMA stimulated CES isoforms expression of pulp cells, and this event was inhibited by catalase. Exogenous addition of porcine esterase prevented BisGMA- and DBA-induced cytotoxicity. Interestingly, inhibition of CES by bis(p-nitrophenyl) phosphate (BNPP) and CES2 by loperamide enhanced the cytotoxicity of BisGMA and DBA. Addition of porcine esterase or N-acetyl-l-cysteine prevented BisGMA-induced prostaglandin E(2) (PGE(2)) and PGF(2α) production. In contrast, addition of BNPP and loperamide, but not mevastatin, enhanced BisGMA-induced PGE(2) and PGF(2α) production in dental pulp cells. These results suggest that BisGMA may induce the cytotoxicity and prostanoid production of pulp cells, leading to pulpal inflammation or necrosis via reactive oxygen species production. Expression of CES, especially CES2, in dental pulp cells can be an adaptive response to protect dental pulp against BisGMA-induced cytotoxicity and prostanoid release. Resin monomers are the main toxic components in DBA, and the ester group is crucial for monomer toxicity., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background and Aims: The predictive power of adiposity and body compositions measured from bioelectrical impedance analysis (BIA) for identifying the risk of metabolic syndrome is unknown among ethnic Chinese., Methods and Results: We designed a nested case-control study by recruiting 1000 cases of metabolic syndrome and 986 matched controls from a health checkup center. For identifying the metabolic syndrome status, the highest areas under receiver operating characteristic curve (AUCs) were waist-height ratio (WHtR) (0.967, 95% confidence interval [CI], 0.960-0.976). The body fat mass vs. lean body mass and body mass index (BMI) had a similar AUC (0.896 for fat mass vs. lean body mass, 0886 for BMI, P=0.07). WHtR and waist circumference had the highest correctly classified proportions (0.89-0.90) and the highest Youden's index (0.77-0.81). The optimal cut point for WHtR was 52.5, with a sensitivity of 0.92 and specificity of 0.89 for discriminating metabolic syndrome risk. The incremental values of AUC, net reclassification improvement and integrated discrimination improvement values were still highest among WHtR, waist circumference and the percent body fat in the multivariate logistic model., Conclusion: Waist circumference and BIA-derived body component measures are suitable for clinical application in identifying the metabolic syndrome status among ethnic Chinese in Taiwan., (Copyright © 2010. Published by Elsevier B.V.)

Background: The cuff pressures may be different in oesophageal and tracheal intubations. We conducted a study to evaluate if cuff pressures of endotracheal tubes (ETTs) could provide information to distinguish tracheal or oesophageal intubations in a pig trachea-oesophagus model., Methods: In each preparation of pig trachea-oesophagus model, the trachea and the oesophagus were intubated separately with a cuffed ETT, and the cuff pressures were measured after each 1 ml increment of air (1-10 ml) during inflation. The cuff pressures and the pressure-volume relationships in both intubations were compared., Results: The cuff pressures of oesophageal intubations were significantly higher than those of tracheal intubations in all comparisons from 1 to 10 ml of cuff volumes (P < 0.05). The cuff pressure-volume curve was steeper in the oesophageal intubation group, and the difference between the two curves was the largest when the cuff volume was 4-5 ml., Conclusions: We conclude that the cuff pressures may be useful in detecting oesophageal intubations. This method is faster than other confirmation measures as it can detect inadvertent oesophageal intubations at the time of inflating the cuffs.

Objective: To delineate the clinical features of traumatic adrenal haematoma, to help emergency physicians recognise the condition early and so to prevent adrenal crisis., Methods: Review of the records of all patients admitted to Chi-Mei Medical Center, Tainan, Taiwan, with traumatic torso injuries, between 1 August 2003 and 31 August 2005. Of the 305 patients reviewed, 8 had adrenal haematoma. Injury Severity Scores, hospital course, need for intensive care unit admission and surgery or vascular embolisation, and concomitant injuries, were compared between patients with and without adrenal haematoma., Results: No adrenal haematoma was recognised in the emergency department. No case of adrenal haematoma needed surgery or vascular embolisation, but each had associated injuries to other internal organs. All eight patients with, but only 106 patients without, adrenal haematoma sustained injuries to more than one internal organ (p<0.01). The mean Injury Severity Scores and lengths of hospital and intensive care unit stays among the two groups were not significantly different. There was no statistical significance in the differences in rates of admission to the intensive care unit, mortality, laparotomy or vascular embolisation between the two groups., Conclusions: Emergency physicians were liable in trauma cases to miss adrenal haematoma, which of itself was a benign condition but was associated with increased internal organ injuries and days in the intensive care unit. Emergency physicians should be aware of the possibility of adrenal haematoma in trauma cases in order not to miss the bilateral condition, which might lead to a deadly adrenal crisis.

Background: Treating patients of bleeding peptic ulcers with heater probe thermocoagulation and haemoclip is considered to be safe and very effective. Yet, there is no report comparing the haemostatic effects of endoscopic haemoclip versus heater probe thermocoagulation plus hypertonic saline-epinephrine injection in these patients., Aim: To compare the clinical outcomes of both therapeutic modalities in patients with peptic ulcer bleeding., Methods: A total of 93 patients with active bleeding or non-bleeding visible vessels were randomised to receive either endoscopic haemoclip (n = 46) or heater probe thermocoagulation plus hypertonic saline-epinephrine injection (n = 47). Five patients from the haemoclip group were excluded because of the inability to place the haemoclip., Results: Initial haemostasis was achieved in 39 patients (95.1%) of the haemoclip group and 47 patients (100%) of the heater probe group (P > 0.1). Rebleeding occurred in four patients (10.3%) of the haemoclip group and three patients (6.4%) of the heater probe group (P > 0.1). The volume of blood transfused after entry into the study, duration of hospital stay, number of patients requiring urgent surgery and the mortality rates were not statistically different between the two groups., Conclusions: If the haemoclip can be applied properly, the clinical outcomes of the haemoclip group would be similar to those of the heater probe group in patients with peptic ulcer bleeding. However, if the bleeders are located at the difficult-to-approach sites, heater probe plus hypertonic saline injection is the first choice therapy.

Securing molecular evidence for transmission of RNA viruses is critically dependent on the time elapsed after infection. To establish HCV transmission by blood transfusions from specific donors, we deduced the sequence of amino acids 329 to 410 of HCV in 30 transfusion recipients and 120 of their blood donors. Phylogenetic analyses of hypervariable region 1 (HVR1) and genotyping furnished proof of transmission. Linear regression analysis showed no differences between HVR1 sequences from the recipient and the implicated donor up to 7.9 weeks post-transfusion, but donor and recipient sequences diverged subsequently (r=0.690). The choice of viral genomic region is also important. We compared four genomic regions in HIV-1 encoding variable regions V1, V2 and V3 in the env gene and p2 in the gag gene in documented cases of mother-to-infant HIV-1 transmission. The less variable regions of V3 and p2 provided positive evidence for virus transmission, in contrast to results obtained on highly variable regions V1 and V2 (p<0.05). Intrafamilial transmission of hepatitis B virus, a DNA virus, could be demonstrated on the molecular level by sequencing of a 100-nucleotide region of the viral genomes from 96 Chinese children and their parents. In 28 of the 30 mothers and 6 of 8 fathers, sequences were identical to those of their offspring (p<0.00005).

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH., (Copyright 2001 Academic Press.)

The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB(2), Bcl-2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB) cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR).

Thrombomodulin is an important endothelial anticoagulant protein that decreases thrombin activity and activates protein C. Our recent study has shown that the G-33A promoter mutation of thrombomodulin gene is associated with coronary artery disease. This study was conducted to determine whether the G-33A mutation in the promoter region of thrombomodulin gene is a genetic risk factor for ischemic stroke or carotid atherosclerosis. The functional significance of this mutation was also evaluated. We recruited 333 patients (mean age 64 years, 59% male) with ischemic stroke and 257 age- and sex-matched controls. In all study participants, carotid atherosclerosis was assessed by Duplex scanning, and thrombomodulin G-33A promoter mutation was detected by single-strand conformation polymorphism. Luciferase reporter gene assay was used to assess the influence of this mutation on thrombomodulin promoter activity. There was no significant difference in the thrombomodulin G-33A mutation frequency (GA+AA genotypes) between the stroke and the control groups (18.3 vs. 24. 1%, P=0.105). The G-33A mutation frequency was also similar between the study participants with and without carotid atherosclerosis (22.2 vs. 19.8%, P=0.550). When only younger subjects (age

We characterized the pH-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-domain fluorometry, with the objective of identifying lifetime-based sensors for low pH values. These three probes were originally designed as dual excitation wavelength-ratiometric probes, with high photostability and high quantum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays analysis reveals that these probes have double exponential intensity decays at intermediate pH values and that the decay time amplitudes are greatly dependent on pH. The longer lifetime components originated from the unprotonated forms and the shorter components from the protonated forms. Both forms can emit fluorescence at intermediate pH values. The apparent pKa values were also determined from the titration curves of phase angles and modulations versus pH for the purpose of pH sensing. The apparent pKa values range from pH 3 to 5, a range where lifetime-based sensors are not presently reported. Since these probes show low pKa values and display substantial phase and modulation changes with pH, they are suitable as lifetime-based pH sensors to monitor the pH changes in acidic environments. One potential application of these probes is to trace the pH in different cellular compartments., (Copyright 1999 Academic Press.)

Background/aims: Portal hypertension and hyperdynamic circulation (i.e. generalized vasodilation and increased cardiac output and regional organ blood flows) may play an important role in the development of portal hypertensive gastropathy. This study investigated the effect of chronic administration of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to portal hypertensive rats on hemodynamics and the development of portal hypertensive gastropathy., Methods: Partial portal vein-ligated or sham-operated rats were randomly assigned to receive either placebo (distilled water) or aminoguanidine (approximately 100 mg/kg per day subcutaneously) for 2 days prior to and 14 days. Hemodynamic studies with a thermodilution technique and gastric morphometric analysis were performed at 14 days after the operation., Results: In rats given placebo, portal vein-ligated rats had a significantly lower mean arterial pressure and systemic vascular resistance associated with a significantly higher cardiac index and portal pressure than sham-operated rats (p<0.05). In portal vein-ligated rats aminoguanidine induced a significant increase in mean arterial pressure and systemic vascular resistance accompanied by a significant decrease in cardiac index (p<0.05) without changes in portal pressure (p>0.05). Despite persistence of portal hypertension, the aminoguanidine-treated portal vein-ligated rats had similar mean arterial pressure, cardiac index, and systemic vascular resistance as seen in placebo-treated sham-operated rats. The mean cross-sectional area of gastric mucosal vessels was significantly higher in placebo-treated portal vein-ligated than in placebo-treated sham-operated rats (p<0.05). Treatment with aminoguanidine did not induce changes in the mean cross-sectional area of gastric mucosal vessels in either portal vein-ligated or sham-operated rats (p>0.05)., Conclusions: The results show that in portal hypertensive rats long-term aminoguanidine therapy corrects the hyperdynamic circulation without inducing changes in portal pressure and ameliorating the development of portal hypertensive gastropathy. This study suggests that, instead of correcting hyperdynamic circulation, treatment of portal hypertensive gastropathy should be aimed at reducing portal pressure.

To study the heterogeneity of hepatitis B virus (HBV) DNAs in Hong Kong, where HBV infection is endemic, serum specimens from 90 HBsAg carrier children were systematically tested with nine oligonucleotide probes representing conserved sequences in the viral genome. In a pair of twins and their cousin (belonging to family H) and an unrelated child (family Y) serum HBV DNA annealed to all but one probe, a sequence located between the core and pre-S regions of the viral genome (positions 2723-2738; EcoRI site 1). Serum from the H twins' aunt and the father and paternal grandparents in the Y family were also HBV DNA-positive. The nucleotide sequences in positions 2701-2800 were analysed. The same point mutation, C to T in position 2735, was present in the HBV DNAs from 7 individuals in the two families. All 4 H family members had the same HBV DNA sequence. The HBV DNA sequences found in the Y daughter, father, and grandfather were identical and they were different from the H family mutant. These results provided evidence at the DNA level of intrafamilial transmission within these Chinese families.

We have shown that ether-linked glycerolipids accumulated in the adrenal, liver, and spleen of a male Chinese infant with Wolman's disease; the increases were mainly in the alkyl and alk-1-enyl glycerolipids that did not contain phosphorus. Alkyldiacylglycerol accounted for a portion of the rise in the neutral alkylglycerols. The spleen also contained increased amounts of ether-linked phosphoglycerides of the alkyl and alk-1-enyl types. Organs from a Niemann-Pick patient were also included in this study; they did not show comparable rises in the content of ether-linked glycerolipids, suggesting the possibility that storage of these compounds may be characteristic of Wolman's disease, or a variant form thereof.

24 Chinese children aged 1.5-5 years and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis B virus DNA polymerase (HBV DNAp), and HBV DNA on at least three occasions in the 6 months before the trial were randomised to receive either vitamin B complex or intramuscular recombinant alpha 2-interferon (r-IFN) ('Roferon') 10 X 10(6) IU/m2 thrice weekly for 12 weeks. In all 12 subjects receiving r-IFN, HBV DNAp and HBV DNA levels fell during the course of r-IFN injections. Within 4 weeks of cessation of r-IFN injection, the HBV DNAp and HBV DNA returned to pre-trial levels except in 2 subjects, in whom loss of HBV DNAp and HBV DNA was sustained for up to 18 months from onset of the trial. 1 child lost HBeAg at 18 months. 2 of the 12 children in the placebo group also had a sustained loss of HBV DNAp and HBV DNA during the 18 months, with 1 child losing HBeAg at 18 months. All 24 subjects remained positive for HBsAg. r-IFN produced very slight side-effects except for pyrexia and the "flu" syndrome, both of which showed rapid tachyphylaxis. In the dose given r-IFN was safe but had no long-term beneficial effects on HBsAg carriage in Chinese children.

The mutagenic activity of dimethylnitrosamine activated by rat-liver microsomes in the presence of NADH was compared with that obtained with NADPH. 3 histidine auxotrophic strains of Salmonella underwent reversions after activation with NADH as the sole coenzyme. All 3 tester strains showed a dose-response relationship with dimethylnitrosamine (10-125 mumoles per plate) after NADH-supported activation. With NADH as the sole coenzyme, the most sensitive strain, hisG46, showed a 105-fold increase in mutagenesis frequency as compared with the 230-fold increase obtained with NADPH. Activation of dimethylnitrosamine in the presence of NADH and NADPH, in combination, produced mutagenesis at frequencies above those seen with NADH alone, but less than or equal to those seen with NADPH as the only coenzyme during the activation step. Experiments in vitro showed that microsomal incorporation of carbon from [14C]dimethylnitrosamine was highest in the presence of NADPH, lowest with NADH and reached intermediate levels when both coenzymes were present. The source of the microsomes in all experiments was liver from rats pre-treated with Aroclor 1254.

For studies on the coenzyme B12-dependent enzyme, leucine-2,3-aminomutase, (3R)- and (3S)-beta-leucines were synthesized. The 10-camphorsulfonamide p-nitrobenzyl esters could be resolved by normal-phase HPLC. A much better separation was obtained by reversed-phase HPLC of the diastereomeric derivatives obtained by treatment of -leucine with Marfey's reagent (N2-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide).