Objective: To clarify the molecular basis of patients with Bartter syndrome type I and explore the therapeutic effect of trafficking-defective variations by chemical chaperone 4-Phenylbutyric acid(4-PBA). Methods: The clinical characteristics, laboratory findings and genetic data of 3 patients diagnosed with Bartter syndrome type I who were admitted to Department of Nephrology, Children's Hospital of Nanjing Medical University from 2017 to 2018 were retrospectively analyzed. Wild type and variant SLC12A1 gene constructs were transiently overexpressed in HEK293 cells. Western blotting was used to detect the expression levels of Na⁺-K⁺-2Cl^-cotransporter(NKCC2) protein. Immunofluorescent staining was applied to investigate the subcellular localization of NKCC2 protein. In addition, the effect of the chemical chaperone 4-PBA on the expression and localization of the SLC12A1 gene variants was investigated. Unpaired t test was used for statistical analysis of 4-PBA treatment. Results: All the 3 patients (2 males and 1 female), aged 3.0, 4.0 and 1.2 years, respectively. All patients had antenatal onset with polyhydramnios and were born prematurely. After birth, all patients presented with hypochlorine alkalosis accompanied by hypokalemia and hyponatremia. Sequencing analysis revealed that the 3 patients were homozygotes or compound heterozygotes for variants in the SLC12A1 gene. In HEK293 cells, the surface expression of NKCC2 in 3 variants (p.L463S, p.L479V, p.507-510del) are all lower than in wild type (0.718±0.039, 0.287±0.081, 0.025±0.156 vs. 1.001±0.028, t=5.92, 8.35, 30.49, all P<0.01). Moreover, the total protein expression of p.L479V and p.507-510del group were all lower than that in wild type group (0.630±0.032, 0.043±0.003 vs. 1.000±0.111, t=3.21, 8.65, all P<0.05). 4-PBA treatment increased the mature protein expression level of the p.L463S and p. L479V group in 4-PBA treatment group are all higher than the untreated group (0.459±0.018 vs. 1.123±0.024, 0.053±0.012 vs. 1.256±0.037, t=2.75, 18.35, all P<0.05). Cytoplasmic retention of the L479V and 507-510del variants were observed by immunofluorescent staining. 4-PBA treatment could rescue a number of NKCC2 L479V variants to the membrane. Conclusions: The 3 SLC12A1 variants cause expression or subcellular localization defects of the protein. The findings that plasma membrane expression and activity can be rescued by 4PBA might help to develop novel therapeutic strategy for Bartter syndrome type Ⅰ.
Bartter Syndrome/genetics* ; Child, Preschool ; Female ; HEK293 Cells ; Homozygote ; Humans ; Infant ; Male ; Pregnancy ; Retrospective Studies ; Solute Carrier Family 12, Member 1/genetics*

OBJECTIVES: To study the effect of glucose metabolism disorders on the short-term prognosis in neonates with asphyxia. METHODS: A retrospective analysis was performed on the medical data of the neonates with asphyxia who were admitted to 52 hospitals in Hubei Province of China from January to December, 2018 and had blood glucose data within 12 hours after birth. Their blood glucose data at 1, 2, 6, and 12 hours after birth (with an allowable time error of 0.5 hour) were recorded. According to the presence or absence of brain injury and/or death during hospitalization, the neonates were divided into a poor prognosis group with 693 neonates and a good prognosis group with 779 neonates. The two groups were compared in the incidence of glucose metabolism disorders within 12 hours after birth and short-term prognosis. RESULTS: Compared with the good prognosis group, the poor prognosis group had a significantly higher proportion of neonates from secondary hospitals (48.5% vs 42.6%, CONCLUSIONS Recurrent hyperglycemia in neonates with asphyxia may suggest poor short-term prognosis, and it is necessary to strengthen the early monitoring and management of the nervous system in such neonates.
Asphyxia ; Asphyxia Neonatorum/epidemiology* ; Humans ; Hyperglycemia ; Infant, Newborn ; Prognosis ; Retrospective Studies

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
China ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; RUNX1 Translocation Partner 1 Protein ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; WT1 Proteins

OBJECTIVE: To develop a method for simultaneous detection of 4 kinds of bisphenol A analogs in human urine by high performance liquid chromatography and tandem quadrupole mass-spectrometry. METHODS: The urine samples were extracted by solid phase extraction( Bond Elut Plexa) after hydrolyzed using β-glucuronidase and sulfatase. The gradient elution was carried out with acetonitrile-water. The bisphenol A-d16 and tetrabromobisphenol A-d10 were used as internal standard substances to conduct the quantitative analysis using the combined mode of liquid chromatography-mass spectrometry and mass spectrometry with the negative electrospray ionization,and the multiple reaction monitoring scan mode. RESULTS: The linear range of all the 4 kinds of bisphenol A analogs( Bisphenol F, Bishpenol A,Tetrachlorobisphenol A and Tetrabromobisphenol A) in urine was found to be 0. 50-100. 00 μg / L, and the linear correlation coefficients were 0. 998 6,0. 999 1,0. 999 6 and 0. 999 7,respectively. The limits of detection were 0. 21,0. 25,0. 01 and 0. 14 μg / L. The recovery rate was 69. 2%-99. 9%. The relative standard deviations of within-run and between-run precisions were 5. 5%-22. 8% and 2. 4%-14. 4%,respectively. The samples could be kept for at least 7 days at room temperature. The method was applied to detect urine samples of 38 children and 20 medical workers and the results were statistically analyzed. The detection rates of bisphenol A, bisphenol F, tetrachlorobisphenol A and tetrabromobisphenol A for children group were 94. 7%,94. 7%,0. 0% and 34. 2%,respectively,and those of the medical workers group were 100. 0%,100. 0%,5. 0% and 75. 0%,respectively. CONCLUSION: The method is effective for the detection of 4 kinds of bisphenol A analogs in human urine with simple operation,high sensitivity and good selectivity.

OBJECTIVEWe aimed to establish a sensitive quantified method for the simultaneous determination of melamine and cyanuric acid residues in water and urine by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry (HILIC-ESI-MS/MS) with the pretreatment of hydrophilic functional silica gel and cation exchange resin mixed solid phase extraction column(MCT), and to investigate the melamine and cyanuric acid residues in 501 water and 216 urine from several province and city.

METHODSAbout 100 ml water (or 10 ml urine) was adjusted to pH 3.0 with concentrated hydrochloric acid, and then mixed with the internal standard solution((15)N3-melamine and (15)N3-(13)C3 -cyanuric acid) and 100 ml acetonitrile (10 ml for urine). The solution was cleaned with MCT solid-phase extraction column, and eluted once by 3 ml methanol and twice by 2.5 ml methanol (containing 5% ammonia water). The effluent was collected and dried by N2 flow at 40 °C, and then diluted to 2 mmol/L ammonium acetate containing 90% volume fraction acetonitrile. The completely dissolved solution was then filtered with 0.22 µm organic membrane; and the filtrate was detected by high performance liquid chromatography-tandem mass spectrometry and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. Then we detected the melamine and cyanuric acid residues in 501 water and 216 urine samples collected from several province and city.

RESULTSBy the quantification of internal standard (15)N3-melamine and (15)N3-(13)C3-cyanuric acid, the melamine and cyanuric acid were linear in the range of 2.0-1000.0 µg/L with correlation coefficient of 0.9998 and 0.9997. The detection limits of the method were separately 0.4 ng/L (melamine) and 0.3 ng/L (cyanuric acid) for water, and 4.0 ng/L (melamine) and 3.0 ng/L (cyanuric acid) for urine. The average recovery rate was around 95.3%-100.1% with the relative standard deviation (RSD) was <4.02%. Out of the 501 water samples, melamine was detected out in 19.9% (100/501) and cyanuric acid was detected out in 5.2% (26/501). The content was around 0.03-5.00 g/L. Melamine or cyanuric acid was detected out in 24.5% of the urine samples (53/216), with the content around 0.01-1.00 g/L.

CONCLUSIONThe established method of solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry can satisfy the requirement for detection of melamine and cyanuric acid residues in all sorts of water and urine. Meanwhile, the two substances widely existed in water and Chinese population.

Chromatography, Liquid ; methods ; Environmental Monitoring ; methods ; Humans ; Mass Spectrometry ; Solid Phase Extraction ; methods ; Triazines ; analysis ; urine ; Urinalysis ; methods

There are two types of decoction of Smilax glabra due to its reddish brown or off-white colored cross section. These two kinds of decoction were found that they have large difference in anti-inflammatory effects and chemical constituents in the preliminary experiments. Comparing and analyzing the content of total tannin in these two kinds of decoction of S. glabra from 28 areas by UV-Vis spectrophotometry were first used to provide some experimental and theoretical development and utilization of this medicinal resource and quality control. Also, the sample recovery test required in Chinese Pharmacopoeia was improved by adding tannic acid instead of gallic acid to samples.
Geography ; Pigmentation ; Reproducibility of Results ; Smilax ; chemistry ; Tannins ; analysis ; isolation & purification

BACKGROUNDCurrently, all the diagnostic indicators for endometriosis lack perfect sensitivity and specificity. According to the characteristic of endometriosis, we analyzed the new biomarker neutrophil-to-lymphocyte ratio (NLR) and the combination of NLR and serum CA-125 to investigate their diagnostic value for identifying stages III and IV endometriosis.

METHODSThe values of serum CA-125 and routine blood tests were collected from 197 patients with endometriosis, 102 with benign tumors and 112 healthy individuals. We investigated the sensitivity and specificity of NLR and its combination with serum-CA-125 for diagnosing stages III and IV endometriosis by using receiver operating characteristic (ROC).

RESULTSThe mean values of NLR, the combination of serum CA-125 and NLR (combination) of the groups with stages III and IV endometriosis were significantly higher than the other two groups. Serum CA-125, NLR, and the combined biomarkers could significantly discriminate the stages III and IV endometriosis group from the other two groups (P < 0.05). NLR shows a lower sensitivity of 57.9% and specificity of 65.2% with a cutoff value at 1.82. And the combination of biomarkers has the highest AUC of 0.949 with a sensitivity of 86.8% and specificity of 92.0% at the cutoff value of 44.40. In addition, for patients with negative CA-125, 55.36% and 53.57% of the patients were able to be diagnosed with endometriosis by using NLR alone and the combination of biomarkers.

CONCLUSIONFor diagnosing stages III and IV endometriosis, the neutrophil-to-lymphocyte ratio is a better adjuvant to serum CA-125, and the neutrophil-to-lymphocyte ratio is valuable in diagnosing stages III and IV endometriosis for patients with negative serum CA-125.

Adult ; Biomarkers ; blood ; CA-125 Antigen ; blood ; Endometriosis ; blood ; diagnosis ; Female ; Humans ; Lymphocytes ; Neutrophils ; Retrospective Studies

OBJECTIVETo develop the national neglect norms for urban primary school students in China.

METHODSAccording to multi-stage stratified cluster sampling principle, 24 cities of 13 provinces (municipalities) in China were selected during December 1 to 31, 2008. A total of 1491 students in grade 1 - 3 and 2236 students in grade 4 - 6 were selected. Questionnaire was designed by authors and the final norms were determined through several statistical analysis methods, such as item analysis method, factor analysis method, reliability analysis method. The reliability analysis and validity analysis were used to test the stability and reliability of the norms. The evaluation criteria of the scale was determined by the percentile method, then the initial development of the norm was completed.

RESULTSThe two questionnaires of grade 1 - 3 and grade 4 - 6 students consisted of 55 and 57 items, respectively, whose item loadings were ranged from 0.301 to 0.687 and 0.321 to 0.730, which met the statistical requirements. For grade 1 - 3 students, the scale's total Cronbach α coefficients was 0.914, the total split-half reliability coefficients was 0.896, the Cronbach α coefficients of four level was above 0.737 except medical and social neglect, split-half reliability was ranged from 0.461 to 0.757; for grade 4-6 students, the scale's total Cronbach α coefficients was 0.916, split-half reliability was 0.883, except social neglect, the Cronbach α coefficients of other level was ranged 0.457 to 0.856, split-half reliability was ranged from 0.500 to 0.798. The total neglect cut-off score of the two scales grade 1-3 and 4-6 were 125 and 155, respectively.

CONCLUSIONThe structure of two norms was reasonable. The scales have good stability and reliability.

Child ; Child Abuse ; prevention & control ; statistics & numerical data ; China ; epidemiology ; Female ; Humans ; Male ; Reproducibility of Results ; Schools ; Students ; Surveys and Questionnaires

OBJECTIVETo develop the national neglect norms for rural children aged 3 to 6 years, which are suitable for Chinese situations.

METHODSAccording to the multi-stage stratified cluster sampling principle, 84 towns of 10 provinces or municipalities were selected in China. Children aged 3 to 6 years were surveyed in November 2010, the sample of analysis were 3240 (of whom males were 49.6% (1608/3240) and the Han nationality were 93.3% (3023/3240)). Questionnaire was designed by authors and deleted items that did not meet the requirements through several statistical analysis methods, such as item analysis method, factor analysis method, reliability analysis method. The reliability analysis and validity analysis were used to test the stability and reliability of the norm. The evaluation criteria of the scale was determined by the percentile method, then the initial development of the norm completed.

RESULTSAfter deleting inappropriate items by statistical processing, finally, the scale consisted of 57 items, and included 6 neglected dimensions (physical neglect, emotional neglect, educational neglect, safe neglect, medical neglect and social neglect). Its item loadings ranged from 0.359 to 0.789, which met the statistical requirements. The scale's total Cronbach α coefficients 0.904, the total split-half reliability coefficients were 0.820, the 6 neglect dimensions' Cronbach α coefficients ranged from 0.620 to 0.815, the 6 neglect dimensions' split-half reliability coefficients ranged from -0.034 to 0.789, the scale's parallel reliability were 0.785 and it's re-test reliability were 0.613. After construct validity, external validity and content validity testing, the result showed that this scale could effectively reflect the real neglected status of children investigated. The total neglect cut-off score of this scale were 121.

CONCLUSIONThe scale has good stability and reliability. And it adapts Chinese conditions and it's convenient to operate.

Child Abuse ; diagnosis ; prevention & control ; Child, Preschool ; China ; Female ; Humans ; Male ; Psychological Tests ; Reproducibility of Results ; Rural Population ; Sampling Studies ; Surveys and Questionnaires ; standards

Objective To develop the National Norms of Negligence (NNN) for rural children aged 0 to 35 months.Methods According to multi-stage stratified cluster sampling principle,10 provinces or municipalities (Jilin,Shaanxi,Shanxi,Beijing,Anhui,Jiangsu,Hunan,Hubei,Yunnan,Chongqing) in China were selected.A national research group was formed collaboratively.A questionnaire was designed by ourselves.According to several statistical analysis methods,such as item,factor and reliability analysis etc.we determined the norm.The evaluation criteria of the scale were determined by percentile method.Finally,the reliability and validity of the norm were evaluated.Results In total,2310 children were surveyed,in which the effective sample were 2227,with an effective rate as 96.4％.The scale consisted of 6 neglected dimensions and 65 items in total.The total Cronbach's α coefficient of the scale was 0.903,with the split-half reliability coefficient as 0.829,the parallel reliability as 0.720 and the re-test reliability as 0.678,respectively.The total neglect cut-off score of this scale was 139.Conclusion The scale seemed to have perfect stability and reliability and all the statistical indicators met the psychometric demands.