[Phenotype and genotype of twelve Chinese children with mitochondrial DNA depletion syndromes].

Objective: To explore the phenotype and genotype of mitochondrial DNA depletion syndromes (MDS) in Chinese children. Methods: The clinical and genetic data of 12 MDS patients (8 were boys and 4 were girls) diagnosed in the Department of Neurology in Beijing Children's Hospital, Capital Medical University from October 2010 to April 2018 were retrospectively collected and analyzed. Results: The developmental milestones were normal or mildly retardated before disease onset. The age of onset ranged from 0 to 2.9-year-old. Most cases developed postnatal or after infection. The most common initial symptoms were feeding difficulty, seizure, muscle weakness, psychomotor regression and hepatic dysfunction. At the last evaluation, all the patients had developmental retardation, failure to thrive, muscle weakness, and dysphagia. Other clinical features were weight loss (9 cases), hearing impairment (7 cases), ptosis (6 cases), seizure (5 cases), dyspnea (4 cases), visual impairment (1 case), hirsutism (1 case), lactic acidosis (7 cases), elevated hepatic enzymes (4 cases) and creatine kinase (2 cases), elevated protein in cerebrospinal fluid (3 cases), abnormalities on screening for inborn error of metabolism (10 cases) and brain magnetic resonance imaging (MRI) (10 cases), abnormal electromyogram (including neurogenic or myogenic injury) (5 cases). Five patients died of infection or multiple organ failure. A total of 18 novel mutations presented below were detected in these patients. Among the 6 cases of encephalomyopathy, there were 3 with SUCLG1 mutation (c. 916G>T, c. 619T>C, c. 980dupT were novel), 2 with SUCLA2 mutation (c. 851G>A, c.971G>A were novel), and one with RRM2B mutation (c.456-2A>G, c.212T>C were novel). All the cases of hepatic encephalopathy all had POLG mutations (c. 3151G>A, c. 2294C>T, c. 2858G>C, c. 680G>A and c. 150_158delGCAGCAGCA were novel). Two cases of infantile-onset spinocerebellar ataxia had TWNK mutations (c. 1163C>T, c. 1319T>C, c. 1388G>A and c. 257_258delAG were novel). One case of myopathy had TK2 mutations (c.557C>G and c.341A>T were novel). Conclusions: The clinical and genetic features of MDS were heterogeneous. Eighteen novel mutations in six MDS related genes were reported, which expanded the genetic spectrum of MDS in Chinese children.

[Signaling pathway of M2-type polarization induced by Mycobacterium tuberculosis-specific peptide E7 in monocyte-macrophages].

Objective: To explore the signal pathway of M2-type polarization induced by Mycobacterium tuberculosis (MTB)-specific peptide E7. Methods: Monocyte-macrophages were divided into blank control group, M1 positive stimulus group [co-stimulated with lipopolysaccharide and gamma interferon (IFN-γ)], M2 positive group(co-stimulated with IL-4 and IL-13), and E7 experimental group (with MTB-specificity polypeptide E7 stimulated). The expression of M1 type markers CD(16), IL-6, TNF-α and M2 type markers CD(163), CD(206), IL-10 were detected at 12, 18, 24 and 36 h. Furthermore, peroxisome proliferators-activated receptors-γ (PPAR-γ) blocker was used in the blank control group, M2-positive stimulus group and E7 experimental stimulus group. T test was used to compare the expression of PPAR-γ and CD(163) before and after the addition of blockers. Results: Compared with the positive control group and the blank control group, the expression of TNF-α in the E7 experimental group gradually reached the peak when macrophages were stimulated for 18 h(the relative expression was 20.02), and then the expression of TNF-α gradually decreased and the expression of CD(163) increased. The expression of CD(163) peaked at 24 h (the relative expression was 2.44). After adding the inhibitor, the expression of PPAR-γ in E7 stimulation group was lower than before blocking (before blocking 0.94±0.06, after blocking 0.69±0.09, P=0.028). CD(163) expression level was significantly lower than that before blocking (before blocking 3.95±0.61, after blocking 2.87±0.20, P=0.047). Conclusion: The MTB-specific peptide E7 induced differentiation of macrophages into M2 type, a process that may be involving PPAR-γ in just another kinase-signal transducer and activator of transcription pathway.

[Application of purse string suture with Harmonious Clips and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect].

Objective: To evaluate the feasibility and safety of purse string suture with Harmonious Clips and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect. Methods: A total of 33 patients who underwent ESD of gastric antrum in single channel endoscopy at the First people's Hospital of Wujiang District from January 2015 to April 2018 were retrospectively analyzed. Everyone had one lesion, and the diameters were all more than 3 cm. After the resection and hemostasis, purse string suture with Harmonious Clips and Olympus endoloop or no suture in study group (n=16) and the control group (n=17). The degree of abdominal pain, postoperative gastrointestinal decompression time, incidence of delayed hemorrhage, postoperative hospital stay and the healing rate were observed and compared. Results: All patients successfully completed resection, no perforation occurred, and all the lesions were resected completely in one time. All patients in the study group were sutured successfully. The abdominal pain score on the first day after operation was (2.7±0.7) in the study group, and (3.6±0.8) in the control group(t=3.686, P=0.001). The mean time of postoperative gastrointestinal decompression was (1.6±0.5) days in the study group, while (2.4±0.7) days in the control group(t=3.675, P=0.001). No delayed bleeding occurred in the study group, while 5 cases in the control group had delayed bleeding. The rate of delayed hemorrhage was 29.4% in control group, 4 cases successfully achieved hemostasis through endoscopy therapy, 1 case was given surgical intervention after ineffective endoscopic hemostasis(P=0.044). The average postoperative hospital stay were (6.2±1.1) days and (5.9±2.0) days respectively (t=0.423, P=0.675). Two months after the operation, the two groups of patients reviewed the gastroscopy, the results showed that, all wounds in the study group were healed completely, the healing rate was 100%. There were 6 cases of incomplete wound healing in the control group, the healing rate was 64.7%(P=0.018). No recurrence was found after 6 months of operation through reviewing gastroscopy. Conclution: It indicates good feasibility and efficacy about the purse string suture with Harmonious Clip and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect. It is safe and reliable, worth of being generalized.

[Progressive cavitating leukoencephalopathy: four cases and literatures review].

Objective: To analyze the clinical and genetic features of progressive cavitating leukoencephalopathy (PCL). Method: The data of clinical and genetic features of 4 PCL patients diagnosed by Beijing Children's Hospital between January 2015 and January 2016 were analyzed. The cases with complete clinical data retrieved on literature search at China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform and PubMed (up to August 2016) by using search terms of"NDUFV1" ,"NDUFS1" , or"leukoencephalopathy" , were summarized. Result: There were three females and one male, two of which were compatriots. The age of onset ranged from 6 months to 15 months. All four children's first symptoms were motor development regression, and the developmental milestones were almost normal before the onset. Of the 4 patients, 3 had cognitive impairment, 1 had seizures, 4 had dystonia and pyramidal impairment, 2 had emaciation, and 1 had nystagmus. The lactate concentrations of 4 patients were normal in blood. One patient had lactaciduria in the urinary organic acid analysis. Cranial magnetic resonance imaging (MRI) of all patients showed leukoencephalopathy, involved in the corpus callosum, and three patients accompanied by cystic lesions. Follow up for 2-13 years showed that the physical and language development were improved. Genetic analysis revealed that mutations in NDUFS1 were found in three patients and NDUFV1 mutation was found in one patient. All six mutations (p.Arg377Cys and p. Arg377His in NDUFV1; p. Arg482Glyfs(*)5, p.Thr368Pro, p.Tyr454X and p. Asp565Gly in NDUFS1) are novel. Five English case reports including 10 PCL patients were collected. Together with this group of 4 cases, a total of 14 cases were involved. All 14 children patients had motor development regression, 11 cases had cognitive impairment and dystonia, 6 cases had pyramidal impairment, 5 cases had irritability, 4 cases had epilepsy and nystagmus, 3 cases had strabismus and swallowing difficulty. Cranial MRI showed patchy leukoencephalopathy with cavities, involved in the corpus callosum. Follow up for 19 months-15 years that the neurology development were improved slowly in all patients. Conclusion: NDUFS1 and NDUFV1 gene mutation screening should be performed firstly in patients with PCL clinical and imaging feature.

[Clinical and genetic characteristics of children with Leigh syndrome].

Objective: To investigate the clinically and genetic characteristics of children with Leigh syndrome. Method: Patients with clinically diagnosed Leigh syndrome(LS)in the department of Neurology, Beijing Children's Hospital from January 2013 to February 2016 underwent the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) detecting with next generation sequencing (NGS) technology. The clinical data of gene confirmed cases were retrospectively collected and analyzed. The differences in the onset age, clinical manifestations, lactic acid level and MRI results between the mtDNA variation and nDNA variation were compared and analyzed.t test, Chi-square test and Fisher's exact test were used for statistical analysis. Result: Thirty-five cases were diagnosed by gene detection, including 20 males and 15 females. The median onset age was 1 year (ranging from the neonatal period to 4.4 years old). The age of onset within 2 years accounted for 74%(26 cases). The onset age of initial symptoms, including developmental delay, developmental regression, and seizures, were 6 (4, 12) months, 12 (8, 14) months, and 6 (1, 23) months respectively. The onset age of ptosis, extrapyramidal symptoms and ataxia were 26 (18, 44) months, 28 (23, 40) months and 28 (19, 35) months, respectively. There were significant differences in the onset age between the three groups (H=21.919, P=0.01). Within the 35 cases, 29 were manifested with developmental delay (83%), 26 with dystonia (74%), 18 with growth retardation, 15 with myasthenia, 13 with developmental regression, 11 with dysphagia, 10 with feeding difficulties, 4 with skeletal dysplasia, and 2 with digestive tract symptoms; nystagmus and respiratory abnormalities were observed in 9 cases respectively; extrapyramidal symptoms, peripheral nerve injury, ptosis, seizures were observed in 8 cases respectively; and ataxia, ophthalmoplegia and hypertrichiasis were found in 5 cases respectively.The blood lactic acid was measured in 32 LS patients, within which 23 cases (72%) had increased results; 8 out of 11 cases who underwent were cerebrospinal fluid lactic acid test had increased results. The results of neuroimaging revealed that all the patients were involved in the brainstem and (or) basal ganglia, of whom 27 (77%) had brainstem involvement, 24 (69%) had basal ganglia involvement. Thirteen out of 14 patients who had medulla oblongata involvement had nDNA variation; while 7 out of 8 patients with cerebellar involvement had nDNA variation. Genetic etiology was confirmed in all patients, among whom there were 17 cases (49%) with mtDNA mutation, including 8993T>C/G (n=5), 14487T>C (n=4), 13513G>A (n=2), 9176T>C, 10158T>C, 3697G>A, 10191T>C, 14459A>G and 11777C>A (n=1) respectively. Remaining 18 cases(51%) had nDNA mutation, including SURF1 gene(n=10), PDHA1 gene(n=3) and one case each of NDUFV1, NDUFAF6, NDUFAF5, NDUFS1 and COQ7 genes. In this study, 27 types of mutations were founded, 15 of which had not been previously reported. Respiratory chain gene mutations have been found in 31 cases(89%); 3 cases had PDHc gene mutations, and 1 case had other mutation. Conclusion: LS usually occurs in infants. The most common primary symptoms are age-dependent abnormal movements, ocular symptoms, and seizures. Respiratory chain defects is the most common causes of LS.SURF1 is the most common variation, followed by 8993T>C/G, 14487 T>C and 13513G>A mutation.

Association between angiotensin-converting enzyme insertion/deletion polymorphisms and the risk of heart disease: an updated meta-analysis.

Insertion/deletion (I/D) polymorphisms of the gene encoding angiotensin converting enzyme (ACE) are a controversial risk factor for heart diseases (HDs). ACE I/D polymorphism has been reported to be associated with various cardiovascular diseases. However, some studies have presented conflicting results. In this study, we aim to explore the association between ACE I/D polymorphisms and the risk of coronary HD (CHD), coronary artery disease (CAD), and myocardial infarction (MI). A meta-analysis was conducted, which included 12,533 cases and 20,726 controls from 75 case-control studies. We performed overall analysis on the entire dataset and found that the D allele of ACE was significantly associated with increased risk of HDs in three different comparison models (dominant, recessive, and homozygote). We also performed analyses on subgroups based on ethnicity as well as disease type. Our results showed that the D allele of ACE was significantly associated with an increased risk of HDs in the Asian and European groups but not in the American group. In addition, in all three subgroups (CHD, CAD, and MI), the D allele of ACE was found to be significantly associated with increased risk of disease. Begg's funnel plots were generated to evaluate publication biases, but no obvious publication bias was found in the studies included in our meta-analysis. In conclusion, our meta-analysis demonstrated that the D allele of ACE was significantly associated with an increased risk of HDs.

Chronic treatment with a glucagon receptor antagonist lowers glucose and moderately raises circulating glucagon and glucagon-like peptide 1 without severe alpha cell hypertrophy in diet-induced obese mice.

AIMS/HYPOTHESIS: Antagonism of the glucagon receptor (GCGR) represents a potential approach for treating diabetes. Cpd-A, a potent and selective GCGR antagonist (GRA) was studied in preclinical models to assess its effects on alpha cells. METHODS: Studies were conducted with Cpd-A to examine the effects on glucose-lowering efficacy, its effects in combination with a dipeptidyl peptidase-4 (DPP-4) inhibitor, and the extent and reversibility of alpha cell hypertrophy associated with GCGR antagonism in mouse models. RESULTS: Chronic treatment with Cpd-A resulted in effective and sustained glucose lowering in mouse models in which endogenous murine Gcgr was replaced with human GCGR (hGCGR). Treatment with Cpd-A also led to stable, moderate elevations in both glucagon and glucagon-like peptide 1 (GLP-1) levels, which were completely reversible and not associated with a hyperglycaemic overshoot following termination of treatment. When combined with a DPP-4 inhibitor, Cpd-A led to additional improvement of glycaemic control correlated with elevated active GLP-1 levels after glucose challenge. In contrast to Gcgr-knockout mice in which alpha cell hypertrophy was detected, chronic treatment with Cpd-A in obese hGCGR mice did not result in gross morphological changes in pancreatic tissue. CONCLUSIONS/INTERPRETATION: A GRA lowered glucose effectively in diabetic models without significant alpha cell hypertrophy during or following chronic treatment. Treatment with a GRA may represent an effective approach for glycaemic control in patients with type 2 diabetes, which could be further enhanced when combined with DPP-4 inhibitors.

Somatostatin receptor subtypes 2 and 5 mediate inhibition of egg yolk-induced gall bladder emptying in mice.

BACKGROUND: Somatostatin inhibits gall bladder contraction. Impaired gall bladder emptying is associated with gall bladder stone formation. The incidence of cholecystolithiasis is high in patients treated with a somatostatin agonist octreotide, which predominantly interacts with somatostatin receptor subtype 2 (SSTR2). Therefore, it is believed that SSTR2 regulates gall bladder contraction; however, evidence has not been provided. Here, we evaluate the effects of SSTR1-SSTR5-selective agonists on egg yolk-induced gall bladder contraction in mice. METHODS: Homozygous deletion of SSTR2 and SSTR5 was generated by cross-mating of SSTR2(-/-) with SSTR5(-/-) mice. Mice of different genotypes were injected with SSTR1-5-selective agonists or octreotide 15 min before induction of gall bladder emptying by egg yolk. One hour later, gall bladders were removed and weighed. KEY RESULTS: Egg yolk-reduced gall bladder weights in all mice, irrespective of their genotype. Octreotide was the most potent inhibitor of gall bladder emptying in wild-type mice. In contrast, agonists with high selectivity for SSTR2 or SSTR5 inhibited gall bladder emptying by approximately 50-60%, whereas SSTR1-, SSTR3- and SSTR4-selective agonists failed to influence gall bladder contraction. In SSTR2(-/-) mice, octreotide and an SSTR5-selective agonist inhibited gall bladder emptying by approximately 50%, whereas SSTR2-selective agonists were inactive. Octreotide inhibited gall bladder emptying in SSTR5(-/-) mice by approximately 50%, without any effect in SSTR2(-/-)/SSTR5(-/-) mice. CONCLUSIONS & INFERENCES: Our study provides evidence for the role of SSTR2 and SSTR5 in regulating gall bladder emptying in mice.

Mass spectrometry screening of combinatorial mixtures, correlation of measured and predicted electrospray ionization spectra.

Methodology was developed to afford rapid characterization of multicomponent mixtures of small organic molecules prepared by split-and-mix combinatorial synthesis. This methodology involved the use of liquid chromatography mass spectrometry (LC/MS) combined with correlation analysis of measured versus predicted electrospray ionization mass spectra. Low-resolution mass spectra of complex mixtures revealed predictable patterns that confirm library products, assisted in identifying chemical synthesis errors, and assessed overall library integrity. In general, equal signal intensities were observed for most combinatorial mixture components, indicating that differences in electrospray ionization efficiency was not a major limitation to this approach. High-throughput data processing programs and informatics tools were used to speed data analysis and to simplify the presentation of the library characterization results. This approach has been used to characterize combinatorial libraries that were synthesized for a variety of drug-discovery programs. Examples are shown for library formats of 1, 40, 66, 280, and 400 component(s)/well. The applicability of this approach to large combinatorial mixtures should allow direct characterization of massive combinatorial libraries.

Versatile and efficient solid-phase syntheses of pyrazoles and isoxazoles.

[reaction: see text] Condensation of aromatic or aliphatic esters with resin-supported acetyl carboxylic acids 2, followed by cyclization with hydrazines or hydroxylamine, activation of the linker, and cleavage using amines provides highly substituted, isomeric pyrazoles or isoxazoles 5. This general method gives products in excellent yields and purities in which the ratio of the two isomers can be easily controlled. A variation of this scheme generates 1,4,5- and 1,3, 4-trisubstituted pyrazoles and related isoxazoles. Post-cleavage reduction with borane converts pyrazole amides to amines such as 11.

Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry.

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

Kinetics of methyl mercury in blood and brain during chronic exposure in the monkey Macaca fascicularis.

The disposition parameters derived from a compartmental model kinetic analysis of blood Hg levels in nonpregnant, adult female Macaca fascicularis given daily doses of MeHg did not vary with either dosage level (50, 70 or 90 micrograms MeHg/kg b.wt.day) or duration of exposure (up to 507 day). In contrast, blood clearance of Hg in pregnant females was dose-dependent; it being higher at the 90 micrograms MeHg/kg b.wt.day than at the lower dosage levels. Hg levels in the brain of adult fascicularis relative to blood Hg also increased at the highest level of exposure. Blood Hg half-life in neonate fascicularis was similar to half-life in their mothers (adults). Finally, the regional distribution of mercury in the brains of adult and neonate fascicularis exposed to low and intermediate levels of MeHg resembles the reported distribution of mercury in the brains of adult and neonate humans environmentally exposed to MeHg. Consequently, M. fascicularis may be an especially appropriate animal model for studying the neurotoxic mechanisms of chronic methyl mercury exposure.

[A preliminary study on serum alpha-L-fucosidase assay in the diagnosis of hepatocellular carcinoma].

Serum alpha-L-fucosidase (AFU) was determined in 33 patients with hepatocellular carcinoma (HCC), 4 with secondary metastatic liver cancer, 61 with various liver diseases, 12 with gastrointestinal tumor and 50 healthy controls. The results showed that AFU level was significantly higher in HCC (14.48 +/- 5.77) than that in the controls (3.33 +/- 0.72) and in patient with other diseases (P less than 0.01). Serum AFU level was also increased in fulminant hepatitis (8.96 +/- 3.99), acute hepatitis (8.94 +/- 4.94) and chronic hepatitis (7.27 +/- 2.58), P less than 0.01 or 0.05. There was no significant difference in AFU level between the controls and patients with secondary metastatic liver cancer (6.25 +/- 0.84), cirrhosis (6.30 +/- 3.17), gastrointestinal tumor (4.43 +/- 1.64), liver hemangioma and liver abscess (4.86 +/- 2.22). A level exceeding 10.5u was a useful marker for the diagnosis of HCC with 78.8% sensitivity and 90.0% specificity. The diagnostic positivity was 81.8% in low AFP producing HCC, whereas 93.9% in those with elevated AFP. Our data indicate that serum AFU is a useful tumor marker for HCC, particularly in detection of AFP-low or negative HCC patients.