CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo.

Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

A distinct serum metabolic signature of distant metastatic papillary thyroid carcinoma.

BACKGROUND: Although the incidence rate for thyroid cancer seems to have begun stabilizing in recent years, an increased rate of advanced stage of this disease has been reported. Additionally, distant metastasis is one of the most important prognostic factors of patients with papillary thyroid carcinoma (PTC). Unfortunately, the underlying mechanisms of distant metastasis, as well as cell status like metabolism changes in distant metastatic tumours have not been clearly elucidated. OBJECTIVE: To identify serum metabolic signature of distant metastatic PTC. DESIGN, PATIENTS AND MEASUREMENTS: In this study, gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) was used to analyse the serum from 77 patients diagnosed with PTC (37 in distant metastasis group and 40 in ablation group). Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) scores plots were used to analyse the data. RESULTS: Principal component analysis and OPLS-DA analyses demonstrated an evident trend of separation between 40 serum samples from the ablation group and 37 samples from distant metastasis group. A total of 31 metabolites were identified, which are related to amino acid, lipid, glucose, vitamin metabolism and diet/gut microbiota interaction. Pathway analysis showed "alanine, aspartate and glutamate metabolism" and "inositol phosphate metabolism" were the most relevant pathways. CONCLUSION: Serum metabolomics profiling could significantly discriminate papillary thyroid cancer patients according to distant metastasis. Potential metabolic aberration in distant metastatic PTC could be involved in different biological behaviours of tumour cells including proliferation, invasion/migration and immune escape. Diet/gut microbiota-produced metabolites could play an important role in these effects. This work may provide new clues to find the underlying mechanisms regarding the distant metastasis of PTC as well as potential adjuvant therapy targets.

Development of a Time-Resolved Fluorescence Immunoassay for the Diagnosis of Hepatocellular Carcinoma Based on the Detection of Glypican-3.

Glypican-3(GPC3), an oncofetal protein, is a potential novel marker for hepatocellular carcinoma (HCC). In this study, we attempted to establish a new method to detect serum GPC3 using the antibodies identified in our previous research, and then evaluated its clinical application for the diagnosis of HCC. Herein, a sandwich time-resolved fluorescence immunoassay (TRFIA) for detecting serum GPC3 was developed. The detection limit, analytical recovery, specificity and precision of the proposed TRFIA assay were satisfactory. A total of 415 patients were collected and divided into seven groups: hepatocellular carcinoma (101), colorectal cancer (67), gastric cancer (44), esophageal cancer (15), cirrhosis (55), hepatitis (61), normal liver (72). Using this proposed method, the concentration of serum GPC3 in these clinical samples was detected. The results demonstrated that the levels of GPC3 in serum from HCC patients were significantly higher than that in others. Compared with the results of chemiluminescence immunoassay (CLIA), a high consistency (Kappa =0.84) was observed. Thus, an effective, sensitive and reliable TRFIA-GPC3 kit for diagnosing HCC was successfully developed. It offers a suitable alternative to existed methods of determining GPC3 and is expected to be used in clinic in the future.

Rapid and Sensitive Lateral Flow Immunoassay Method for Procalcitonin (PCT) Based on Time-Resolved Immunochromatography.

Procalcitonin (PCT) is a current, frequently-used marker for severe bacterial infection. The aim of this study was to develop a cost-effective detection kit for rapid quantitative and on-site detection of PCT. To develop the new PCT quantitative detecting kit, a double-antibody sandwich immunofluorescent assay was employed based on time-resolved immunofluorescent assay (TRFIA) combined with lateral flow immunoassay (LFIA). The performance of the new developed kit was evaluated in the aspects of linearity, precision, accuracy, and specificity. Two-hundred thirty-four serum samples were enrolled to carry out the comparison test. The new PCT quantitative detecting kit exhibited a higher sensitivity (0.08 ng/mL). The inter-assay coefficient of variation (CV) and the intra-assay CV were 5.4%-7.7% and 5.7%-13.4%, respectively. The recovery rates ranged from 93% to 105%. Furthermore, a high correlation (n = 234, r = 0.977, p < 0.0001) and consistency (Kappa = 0.875) were obtained when compared with the PCT kit from Roche Elecsys BRAHMS. Thus, the new quantitative method for detecting PCT has been successfully established. The results indicated that the newly-developed system based on TRFIA combined with LFIA was suitable for rapid and on-site detection for PCT, which might be a useful platform for other biomarkers in point-of-care tests.

The Establishment of an HE4-CLIA Method and the Combined Analysis of HE4 and CA125 in Ovarian Cancer.

BACKGROUND: The human epididymal secretory protein 4 (HE4) is a novel, verified biomarker for the early diagnosis of ovarian cancer. METHODS: Magnetic beads were coated with capture antibodies and were used with acridinium ester labeled detection antibodies in a sandwich-type immunoassay. The patient's HE4 serum levels were measured simultaneously with the chemiluminescence immunoassay (CLIA) kit we developed and electrochemiluminescence immunoassay (ECLIA) kit from Roche (Mannheim, Germany). CA125 was also detected by time-resolved fluoroimmunoassay. The diagnostic value was analyzed. RESULTS: The assay demonstrated a linear range from 2.5 to 2,000 pmol/l, with an analytical sensitivity of 2.5 pmol/l. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. Compared with the ECLIA kit from Roche in 124 serum samples (40 patients with ovarian cancer, 35 patients with benign gynecological diseases, and 49 health controls), there is a satisfied correlation coefficient of 0.875. The area under the receiver-operating curve (ROC-AUC) was 0.903 (95% CI was 0.839-0.966, P < 0.001) for HE4, 0.787 (95% CI was 0.694-0.879, P < 0.001) for CA125, and 0.914 (95% CI was 0.866-0.962, P < 0.001) for combined analysis of HE4 and CA125. CONCLUSIONS: A quantitative method (HE4-CLIA) for detecting HE4 in serum was successfully established. Preliminary clinical sample analysis showed HE4-CLIA has a certain clinical value in the screening and diagnosis of ovarian cancer. Moreover, in distinguishing benign from malignant ovarian lesions, HE4 has higher demonstrated accuracy than CA125.

Development of a clinical chemiluminescent immunoassay for serum GPC3 and simultaneous measurements alone with AFP and CK19 in diagnosis of hepatocellular carcinoma.

BACKGROUND: Glypican-3 (GPC3) is an oncofetal antigen that shows great promise as a biomarker for diagnosis of hepatocellular carcinoma (HCC), but there is no reliable kit that can be used to detect it in clinics. The aim of this study is to develop a stable performance kit for GPC3 detection in clinics. DESIGN AND METHODS: The paired antibodies were identified through cycle-screening methods based on our previous research. Then, a double antibodies sandwich chemiluminescent immunoassay for detecting serum GPC3 was developed. The performance of the developed GPC3 diagnostic kit was evaluated by detecting the concentration of serum GPC3 and assessing its single or combined use with alpha fetoprotein (AFP) and cytokeratin 19 fragment (CK19) for HCC diagnosis. RESULTS: The assay demonstrated a linear range of 10-800 ng/ml, the cross-reactivity rate at 0.018% (AFP), 0.020% (carcino-embryonic antigen), and 0.021% (CK19), respectively. The minimum detectable concentration was 0.05 ng/ml; the intraassay coefficient of variation (CV) and interassay CV were both less than 10%, with good stability and reproducibility. GPC3 has a high sensitivity (54.2%) and specificity (99.4%) in diagnosing HCC. The level of GPC3 in HCC was robust higher than that in healthy or other liver diseases' sera (108.67 ± 230.04 ng/ml vs. 3.99 ± 7.68 ng/ml). The diagnostic sensitivity of GPC3 single or combined with CK19 and AFP for HCC was evaluated, and the rates were 54.2 and 90.6%, respectively. CONCLUSIONS: An applicable chemiluminescent immunoassay with stable performance against GPC3 in diagnosing HCC has been established and the combination of GPC3 with CK19 and AFP could improve the diagnostic sensitivity for HCC.

Autosomal dominant polycystic kidney disease with ectopic unilateral multicystic dysplastic kidney.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder. In most cases, ADPKD similarly affects bilateral kidneys. CASE PRESENTATION: Among the 605 ADPKD patients that were followed up by our center, we identified two male patients with unilateral ADPKD. The cases were remarkable because the patients also had ectopia and multicystic dysplasia in the contralateral kidney, which are generally sporadic disease conditions. Both patients tested positive for polycystic kidney disease 1 mutation, but negative for hepatocyte nuclear factor 1 beta mutation. Moreover, the deterioration of their kidney function seemed to be quicker than their age- and sex-matched controls and siblings. Both patients had started a long-term hemodialysis in their 40s. CONCLUSION: Anatomical and genetic abnormality in patients with ADPKD may be more frequent and complex than previously believed. The compensatory capacity in patients with ADPKD is fragile, and missing one kidney could accelerate the deterioration of renal function.

Association of a SLC30A8 genetic variant with monotherapy of repaglinide and rosiglitazone effect in newly diagnosed type 2 diabetes patients in China.

OBJECTIVE: To investigate a potential relationship between Solute carrier family 30 (zinc transporter) member 8 (SLC30A8) rs13266634 variant and efficacy of rosiglitazone or repaglinide in treating newly diagnosed Chinese type 2 diabetes patients. METHODS: A total of 209 diabetic patients without any antihyperglycemic history were recruited and treated with repaglinide or rosiglitazone randomly for 48 weeks (104 and 105 patients, respectively). Anthropometric measurements and clinical laboratory tests were carried out before and after the treatment. An non-synonymous variant rs13266634 was genotyped by matrix-assisted laser desorption ionization-time of flight mass spectroscopy. RESULTS: Ninety-one patients in repaglinide group and ninety-three patients in rosiglitazone group completed the study. Δ value of homeostasis model assessment of beta cell function (HOMA-B) and Δ value of fasting proinsulin levels were statistically significant between three genotype groups (P=0.0149 and 0.0246, respectively) after rosiglitazone treatment. However, no genotype association was observed in the repaglinide or rosiglitazone group with other parameters. CONCLUSION: The SLC30A8 variant was associated with the efficacy of insulin sensitizer monotherapy on insulin secretion in patients with newly diagnosed type 2 diabetes mellitus in Shanghai, China.

Pseudorabies Virus Inhibits Expression of Liver X Receptors to Assist Viral Infection.

Pseudorabies virus (PRV) is a contagious herpesvirus that causes Aujeszky's disease and economic losses worldwide. Liver X receptors (LXRs) belong to the nuclear receptor superfamily and are critical for the control of lipid homeostasis. However, the role of LXR in PRV infection has not been fully established. In this study, we found that PRV infection downregulated the mRNA and protein levels of LXRα and LXRß in vitro and in vivo. Furthermore, we discovered that LXR activation suppressed PRV proliferation, while LXR inhibition promoted PRV proliferation. We demonstrated that LXR activation-mediated reduction of cellular cholesterol was critical for the dynamics of PRV entry-dependent clathrin-coated pits. Replenishment of cholesterol restored the dynamics of clathrin-coated pits and PRV entry under LXR activation conditions. Interestingly, T0901317, an LXR agonist, prevented PRV infection in mice. Our results support a model that PRV modulates LXR-regulated cholesterol metabolism to facilitate viral proliferation.

A variation in NOS1AP gene is associated with repaglinide efficacy on insulin resistance in type 2 diabetes of Chinese.

AIM: To investigate a potential association between SNP rs10494366 in the neural nitric oxide synthase adaptor protein (NOS1AP) and efficacy of repaglinide (an insulin secretagogue) in newly diagnosed Shanghai Chinese type 2 diabetes patients. METHODS: A total of 104 newly diagnosed type 2 diabetes patients (69 men, 35 women) were recruited and treated with repaglinide for 24 weeks. Anthropometric measurements, clinical laboratory tests were obtained at baseline and after 24-week treatment. Genotyping was performed by sequencing. RESULTS: The baseline value of BMI, HOMA-IR, HOMA-B, and fasting insulin level were significantly different between GG, GT, and TT genotypes (P=0.024, 0.030, 0.005, and 0.007, respectively). Carriers of TT genotype were in significant insulin resistance at baseline. After 24-week repaglinide monotherapy, the Delta value of fasting insulin (P=0.019) and HOMA-IR (P=0.011) were significantly different. TT carriers had the least insulin resistance after treatment. The mixed model analysis showed that the variation had an interaction effect with repaglinide treatment only on HOMA-IR (P=0.013). CONCLUSION: A common variant in rs10494366 is associated with repaglinide monotherapy efficacy on insulin resistance in newly diagnosed Shanghai Chinese type 2 diabetes patients.

Precise microdeletion detection of Prader-Willi Syndrome with array comparative genome hybridization.

OBJECTIVE: Prader-Willi Sydrome (PWS) is a human disorder related to genomic imprinting defect on 15q11-13. It is characterized by a series of classic features such as hypotonia, hyperphagia, obesity, osteoporosis, typical facial and body dysmorphosis, hypogonadism, mental and behaviour disorders. Our study was designed to precisely detect the microdeletions, which accounts for 65%-70% of the PWS. METHODS: Physical and laboratory examinations were firstly performed to diagnose PWS clinically, and to discover novel clinical features. Then the patient was screened with bisulfite-specific sequencing and precisely delineated through high-density array CGH. RESULTS: With the bisulfite-specific sequencing, the detected CpG island in the PWS critical region was found homozygously hypermethylated. Then with array CGH, a 2.22 Mb type II microdeletion was detected, covering a region from MKRN3, MAGEL2, NDN, PWRN2, PWRN1, C12orf2, SNURF-SNRPN, C/D snoRNAs, to distal of UBE3A. CONCLUSIONS: Array CGH, after the fast screening of Bisulfite-specific sequencing, is a feasible and precise method to detect microdeletions in PWS patients. A novel feature of metacarpophalangeal joint rigidity was also presented, which is the first time reported in PWS.

[No association of vascular endothelial growth factor A gene rs9369425 polymorphism with glucose metabolism in Chinese Han population].

OBJECTIVE: To investigate the relationship between the vascular endothelial growth factor A gene (VEGFA) rs9369425 single nucleotide polymorphism (SNP) and type 2 diabetes in Chinese Han population. METHODS: One thousand eight hundred and ninety two type 2 diabetes patients and 1808 controls with normal glucose were recruited in this study. Phenotypes including body mass index, waist, waist hip ratio, plasma glucose and serum insulin levels of blood obtained both at 0 and 120 minute during standard 75-gram glucose oral glucose tolerance tests, were analyzed. Insulin resistance and beta cell function were assessed by homeostasis model assessment (HOMA-IR and HOMA-B). Genotyping was performed by time-of-light mass spectrum using a Sequenom platform. RESULTS: The frequencies of minor allele G in the diabetic patients and controls were 10.8% and 11.3% respectively. No significant difference of allele distribution was detected between the cases and controls (P=0.5086). No significant difference (P>0.05) was detected on the association between rs9369425 SNP and clinical phenotypes. CONCLUSION: VEGFA rs9369425 was not associated with type 2 diabetes in Chinese Han population. Whether there is association in any other loci in this gene remained to be investigated.

[Effects of long-term fertilization on soil organic phosphorus fractions and wheat yield in farmland of Loess Plateau]. / 长期施肥对黄土旱塬农田土壤有机磷组分及小麦产量的影响.

Understanding the effects of long-term fertilization on soil organic phosphorus fractions and wheat yield in the Loess Plateau can provide theoretical support for improving phosphorus conversion, utilization, and rational use of fertilizer. We examined the effects of different fertilizer treatments on soil organic phosphorus fractions, wheat yield and soil properties of a farmland in the long-term (1984-2016) positioning test station of Changwu loess soil. There were eight treatments, including no fertilizer (CK), single application of nitrogen fertilizer (N), single application of phosphorus fertilizer (P), application of nitrogen and phosphorus fertilizer (NP), single application of organic fertilizer (M), nitrogen combined with organic fertilizer (MN), phosphorus combined with organic fertilizer (MP), nitrogen and phosphorus combined with organic fertilizer (MNP). The results showed that the range of soil organic phosphorus content was 244.7-429.1 mg·kg-1 after long-term fertilization. Except for the N treatment, organic phosphorus content was significantly increased by 15.4%-47.9% compared to CK. Long-term application of phosphorus fertilizer changed the content of organic phosphorus fractions in the surface soil (0-20 cm). The treatments of MP and MNP significantly increased the contents of labile organic phosphorus and moderately labile organic phosphorus. The treatments of N, P and NP significantly reduced the content of moderately stable organic phosphorus. The treatments of N, P, NP, MN, MP, MNP all significantly increased the highly stable organic phosphorus. The ratio of soil organic phosphorus fractions to total organic phosphorus content was in order of moderately labile organic phosphorus > highly stable organic phosphorus > labile organic phosphorus > moderately stable organic phosphorus. After long-term fertilizer application, the combination of nitrogen and phosphorus fertilizers, especially with organic fertilizers, significantly increased wheat biomass yield and grain yield. Among all the examined soil properties, organic matter, Olsen-P and total inorganic phosphorus were significantly positively correlated with wheat yield. MP and M could significantly increase the content of Olsen-P, total phosphorus, total inorganic phosphorus, labile organic phosphorus and moderately labile organic phosphorus in the loess soil of Loess Plateau. Our results indicated that the organic and phosphorus fertilizers could improve soil phosphorus components that could be more easily absorbed by crops. In summary, the combination of nitrogen and phosphorus fertilizers, especially with organic fertilizers, could increase soil phosphorus supply in the region and promote the wheat yield, which is important for improving soil quality in the Loess Plateau.

[Comparison of the application of three diagnostic criteria of metabolic syndrome in familial type 2 diabetic pedigrees].

OBJECTIVE: To compare the significance of the application of three diagnostic criteria of metabolic syndrome (MS), issued by the National Cholesterol Education Program Adult Treatment Panel II (ATPIII) in 2005, International Diabetes Federation (IDF) in 2005 and Chinese Diabetes Society (CDS) in 2004, in type 2 diabetes mellitus pedigrees. METHODS: Totally,4468 subjects (including spouses) from 715 type 2 diabetic pedigrees were selected in this study. Complete laboratory data, including blood pressure, lipid profile and plasma glucose, were collected. The prevalence rates of MS and the unity of three criteria were analyzed. RESULTS: The prevalence rates of MS were 44.94% (2008/4468), 37.87% (1692/4468) and 23.86% (1066/4468) according to the ATPIII, IDF and CDS criteria respectively. It subsequently increased in second-degree relatives, spouses, first-degree relatives and probands (ATP III: 23.78% (117/492), 35.77% (318/889), 45.40% (1077/2372) and 69.37% (496/715); IDF: 20.53% (101/492), 31.61% (281/889), 38.74% (919/2372) and 54.69% (391/715); CDS: 8.94% (44/492), 16.99% (151/889), 25.08% (595/2372) and 38.60% (276/715); ATPIII: chi2 = 266.359, IDF: chi2 = 155.950, CDS: chi2 = 165.087, respectively, P < 0.01). The prevalence rates of MS, as defined by the ATP III and IDF criteria, were higher in females than in males (ATP III: 47.47% (1156/2435) and 41.91% (852/2033); IDF: 43.00% (1047/2435) and 31.73% (645/2033); chi2 = 13.871 and 60.169, respectively, P < 0.01), and was lower in females than in males as defined by the CDS criterion (22.38% and 25.63%, respectively, chi2 = 6.423, P = 0.011). The agreement in the diagnosis of MS using ATPIII and IDF, ATPIII and CDS, IDF and CDS was 92.93%, 75.56% and 77.21% respectively. Kappa index were 0.855, 0.484 and 0.478 respectively (P < 0.01). CONCLUSION: ATP III criterion showed the highest prevalence of MS and the percent of risk factor aggregation which best reflected the characteristics of MS in familial type 2 diabetic pedigrees.

[Effects of long-term fertilization on soil microbial abundance in farmland of the Loess Plateau, China]. / 长期施肥对黄土旱塬农田土壤微生物丰度的影响.

Based on a long-term experiment in the Changwu Agro-ecological Experimental Station, the real-time PCR analysis was used to examine the soil microbial abundance and to reveal the effects on soil microbial community under different long-term fertilization systems. The results showed that compared to the CK (barren field), the population of bacteria increased by 21% and archaea by 32% in treatment with inorganic fertilizer application. The abundance of bacteria in the treatment of chemical fertilizer combined with organic fertilizer increased by 37% and archaea by 36%. The treatment with chemical fertilizer mixed with organic fertilizer significantly increased the abundance of bacteria and archaea. The soil AOB increased by 7.13 times while the soil AOA only by 0.2 folds after 30-year application of chemical nitrogen fertilizer. AOB was highly responsive to fertilizer application, especially to nitrogen fertilizer. Compared with the single nitrogen application and the application of nitrogen fertilizer mixed with organic fertilizer, phosphorus fertilizer significantly increased the abundance of nifH and pmoA. The content of nifH, nirS cd and pmoA in the abandoned land was significantly higher than that in the cultivated soil. Results from the correlation analysis on soil basic physical and chemical properties indicated that soil pH, total nitrogen and organic carbon were key factors affecting soil microbial community abundance. In conclusion, long-term fertilization significantly changed soil microbial abundance, and fertilization patterns and cultivating methods had significant effect on microbial community abundance.

Association of KCNJ11 and ABCC8 genetic polymorphisms with response to repaglinide in Chinese diabetic patients.

AIM: The aim of this study was to investigate the association of KCNJ11 E23K and ABCC8 exon16-3T/C with the therapeutic effect of repaglinide in patients with type 2 diabetes. METHODS: A total of 100 Chinese patients with newly diagnosed type 2 diabetes were treated with repaglinide for 24 weeks. Arginine stimulation tests were performed to evaluate beta cell function. Gene variations were detected with PCR-restriction fragment length polymorphism. Responders were defined by a greater than 25% decrease in fasting plasma glucose or a greater than 20% decrease in hemoglobin A1c (HbA1c) values (or both) after the 24 week repaglinide treatment. RESULTS: Both baseline HbA1c and the decrease of HbA1c were significantly higher in patients with E/K and K/K genotypes of the KCNJ11 E23K variant when compared with E/E homozygotes (P=0.0103 and 0.0221, respectively). The decrease in 2 h postprandial plasma glucose (2hPG) was significantly greater in E/K heterozygotes than E/E homozygotes (P=0.0367). There was a significant difference in the response rate to repaglinide treatment between the E and K alleles (68% vs 82%, P=0.0324). The changes in fasting insulin and the homeostasis model assessment of insulin resistance were significantly greater in patients with ABCC8 exon16-3 C/C versus the T/C and T/T genotypes (P=0.0372 and 0.0274, respectively). CONCLUSION: The KCNJ11 E23K variant was associated with the therapeutic effect of repaglinide. In addition, The C/C homozygotes of the ABCC8 exon16-3T/C variant responded better to repaglinide in insulin sensitivity than the T/C and T/T genotypes.

Intracellular retention of human melanocortin-4 receptor: a molecular mechanism underlying early-onset obesity in F261S pedigree of Chinese.

OBJECTIVE: To investigate how F261S mutation identified from Chinese obese patients affects the function of melanocortin 4 receptor (MC4R) and to analyze the obesity-related phenotypes in subjects carrying the F261S mutation. METHODS: F261S mutant of MC4R was generated by site-directed mutagenesis. Plasmids encoding wild-type or F261S mutant of MC4R were transfected into HEK293 and COS-7 cells to examine their functional characteristics. Signaling properties of F261S MC4R were assessed by measuring intracellular cAMP levels in response to alpha-MSH stimulation. Cell surface expression of F261S MC4R was compared with that of wild-type MC4R. Clinical examinations were performed in subjects carrying F261S mutation and in non-mutated controls. RESULTS: The alpha-MSH-stimulated reporter gene activity was significantly reduced in cells expressing F261S MC4R, with a maximal response equal to 57% of wild-type MC4R. The F261S mutation also led to a significant change in the Es50 value compared with the wild-type receptor (P<0.01). Immunofluorescent assay revealed a marked reduction in plasma membrane localization of the MC4R in cells expressing the F261S mutant receptor. The resting metabolic rate and fat composition of the mutant carriers were not significantly different from those of the non-mutated obese controls. CONCLUSIONS: The decreased response to alpha-MSH due to the intracellular retention of MC4R may cause early-onset obesity in the F261S pedigree of Chinese.

[Comparison between genotyping and serological phenotyping in RhCE blood group].

OBJECTIVE: To genotype the RHCE gene of Hans, Xinjiang's Uigurs and Kazakstans in China, and to compare the results of RHCE genotyping with that of RhCc/Ee phenotyping. METHODS: RHCE genes of 98 Hans with RhD positive and 230 Hans, 72 Uigurs and 18 Kazakstans with RhD/RHD negative were genotyped with PCR-sequence specific primer (SSP) technique. RESULTS: The results of RHE/RHe genotyping from samples with RhD positive and negative were in accord with that of phenotyping. It would result in 4.44% error using C-->G polymorphism at nt48 of RHCE gene to genotype RHCE, and 4.05% failure of detection using the 109 bp insertion to detectRHCE gene in Chinese Hans. The results of RHE/RHe genotyping in unrelated 72 Uigurs and 18 Kazakstans with RhD phenotype were consistent with that of phenotyping, and false positive and false negative were not found in genotyping in Uigurs and Kazakstans tested. CONCLUSION: The results of RHE/RHe and RHc genotyping were correct with PCR-SSP and accordant with that of phenotyping. Using the C48G polymorphism in exon 1 of RHCE to genotype RHC gene would result in false positive resulting from RHc mutation at this locus, and using the 109 bp insertion to genotype RHC gene would result in false negative because of the absence of the 109 bp. Therefore it is necessary to genotype RHC gene using more than two polymorphic loci.

[Genetic characteristics of familial type 2 diabetes pedigrees: a preliminary analysis of 4468 persons from 715 pedigrees].

OBJECTIVE: To analyze the inheritance character of type 2 diabetes mellitus (T2DM) pedigrees. METHODS: 4468 persons from 715 T2DM pedigrees (including the spouses) undergo peripheral blood sample collection to examine blood sugar and physical examination. Questionnaire survey was conducted to explore the family history. Type 1 DM and maturity-onset DM of young people were to be ruled out. Pedigree chart were made. RESULTS: The prevalence rates of T2DM and impaired glucose regulation (IGR) was 47.62%, including 218 T2DM and 422 IGR newly discovered. The prevalence rates of T2DM and IGR were 38.33% and 14.25% in the siblings, and 56.81% and 12.58% in the parents, all significantly higher than those in the second-degree relatives (9.55% and 6.10%) and spouses (10.57% and 9.55% respectively, all P < 0.01). The prevalence and newly discovered rates of IGR in the offspring were 12.46% and 11.73% respectively, both significantly higher than those in the spouses (9.55% and 9.55% respectively, all P < 0.01). CONCLUSION: There is significant familial aggregation in T2DM. The first-degree relatives of T2DM patients are high risk populations, so long term monitoring and early screening should be performed.

[Association of -6735T-->C variant of glucokinase-associated dual-specificity phosphatase 12 gene with type 2 diabetes in Chinese].

OBJECTIVE: To investigate the impact of glucokinase-associated dual-specificity phosphatase 12 gene (DUSP12) single nucleotide polymorphism (SNP) on type 2 diabetes mellitus in Chinese. METHODS: The genotypes of -6735T-->C of DUSP12 were determined by PCR-RFLP in 577 Chinese in Shanghai, 359 with normal glucose tolerance (NGT) and 218 being newly diagnosed DM patients without taking any drug. Oral glucose tolerance test was conducted. Height, weight, glucose and insulin concentrations, serum lipid profiles, and fat distribution parameters were determined. RESULTS: The genotype frequency distributions of -6735T-->C variant in DUSP12 gene of the DM group and NGT group were not significantly different (all P > 0.05). The fasting plasma glucose concentration of the NGT subjects with C allele was 5.00 (4.69 - 5.28) mmol/L, significantly higher than that of the subjects with TT genotype [4.90 (4.50 - 5.26) mmol/L, P = 0.035]. The serum HDL-c concentration of the NGT subjects with C allele was (1.25 +/- 0.30) mmol/L, significantly lower than that of those with TT genotype [(1.34 +/- 0.30) mmol/L, P = 0.010]. These two results still showed significant statistical differences after adjusted with sex, age, and BMI (P = 0.035). CONCLUSION: -6735T-->C variant in DUSP12 doesn't play a major role in diabetes, but it may have some effects on glucose and lipid metabolism.