To provide supports for Ginkgo biloba cell engineering for production of Terpene lactones (Ginkgolides and bilobalide), the cell suspension were established from calli induced from zygote embryos and stems of 30-day-old seedlings respectively. The relationship between cell growth, differentiation and the terpene lactone accumulation in these suspension cultures were investigated. HPLC determination indicated that, the ginkgolide B was found in the embryo derived cell suspension cultures at 0.044% of cell dry weight, and this result was the first time reported in this study. The accumulation of terpene lactone in the suspension cultures derived from both the embryo and seedling stems are effected by the level of the cell differentiation. The ginkgolide B was only found in small cell aggregates in the size smaller than 2mm, and the highest level of ginkgolide B was accumulated in cell aggregates in the size smaller than 1mm; however, the cell aggregates in the size bigger than 3mm could only produced bilobalide and ginkgolide A. In the same size aggregates of the suspension cultures the terpene lactone accumulation is strongly effected by the source of the explant. When the size of cell aggregates was in less than 1mm, the concentration of bilobalide, ginkgolide A and B in the cell suspension cultures derived from the embryos was 2, 1.4 and 0.56-fold, respectively, higher than that of cell cultures derived from seedling stems.
Bilobalides ; analysis ; Cell Differentiation ; physiology ; Cell Proliferation ; Culture Techniques ; methods ; Ginkgo biloba ; growth & development ; metabolism ; Ginkgolides ; analysis ; Lactones ; analysis

OBJECTIVE: To develop the single-tube one-step methylation variable position (MVP) analysis technology-single-tube post-digestion PCR-melting curve analysis (PDP-MCA). METHODS: Based on differentially methylated region (DMR) reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme (MSRE), DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples (peripheral venous blood, semen, and vaginal fluid, 5 samples each type) were tested by single-tube one step MVP and traditional MSRE-PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. RESULTS: When the melting temperature of the fragments had a differential of 2 degrees C, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions (digestion, amplification and detection) into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. CONCLUSION Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.
DNA/isolation & purification* ; DNA Methylation/genetics* ; DNA Primers/genetics* ; Humans ; Multiplex Polymerase Chain Reaction/standards* ; Nucleic Acid Denaturation

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with a rare non-deletional hemoglobin H disease (Hb H disease). METHODS: Peripheral venous blood samples of the proband and his parents were collected and subjected to routine blood testing, hemoglobin electrophoresis and screening for common mutations associated with thalassemia. Next generation sequencing was carried out to detect potential pathogenic variant, and candidate variant was verified by Sanger sequencing. RESULTS: The proband was found to harbor a rare HbA2: c272_279delAGCTTCGG variant, which compounded with southeast Asian type deletion of α-thalassemia (--^SEA). The two mutations were respectively inherited from his mother and father. CONCLUSION The rare HbA2: c.272_279delAGCTTCGG variant compounded with southeast Asian type α-thalassemia (--^SEA) probably underlay the non-deletion Hb H in the proband. Above finding has enabled genetic counseling and prenatal diagnosis for this family.
Female ; Pregnancy ; Humans ; alpha-Thalassemia/genetics* ; Pedigree ; Genetic Testing ; Genetic Counseling ; Mothers

Stain identification has long been a task in forensic biology. The identification of semen stain, one of the most common human stains, can provide crucial information for crime scene reconstruction and forensic investigation. Traditional detection of semen stain depends largely on the microscopic identification of spermatozoa, enzyme activity-based methods or antigen-antibody reactions. These morphological, proteinological and zymological approaches, however, are apparently inadequate in identifying tiny, admixed, degraded or contaminated samples. With the development of transcriptomics and epigenetics, many semen-specific mRNA markers, such as protamine-1 (PRM1) and -2 (PRM2), have been applied to semen and semen stain identification. Messenger RNA profiling shows great promise in identifying tissues as demonstrated by the recognition of specific markers. Further more, studies on tis-sue-specific differential DNA methylation will provide a scrumptious way of identifying difficult samples.
DNA Methylation ; Forensic Medicine ; methods ; Genetic Markers ; Humans ; Male ; RNA, Messenger ; analysis ; Semen

【Objective】To explore clinical manifestations and features of renal uric acid excretion in gout patients with obesity.【Methods】Totally 228 primary gout patients were enrolled and divided into three groups according to body mass index（BMI）. Clinical and fasting blood biochemical analysis data were collected. Indices of renal uric acid excretion were calculated according to 24 h urinary uric acid and urinary creatinine.【Results】The obese group（n = 44）was younger than overweight group（n = 88）and non-overweight group（n = 96）［43（32，57）years vs 55（45，65）years，58（45，67）years］，with earlier onset age［37（26，48）years vs 48（38，59）years］，higher serum uric acid［594（522，697）μmol/L vs 511（372，653）μmol/L］and had more hypercholesterolemia（56.8% vs 31.3%）and low density lipoproteinemia（59.1% vs 47.9%）compared with non-overweight group. The ratio of hypertriglyceridemia（43.5% and 37.5% vs 17.7%）and metabolic syndrome（50.0% and 36.4% vs 12.5%）in the overweight and obese group were both higher than non- overweight group. Fraction excretion of uric acid（FEUA）in obese group［5.5（3.6，7.4）% vs 7.0（5.2，9.8）%］was lower than non-overweight group，and the glomerular filtration load of uric acid［5.3（4.2，7.5）mg·min- 1 ·1.73 m- 2 vs 3.5（2.2，5.2）mg·min-1·1.73 m-2］in obese group was higher than that in non-overweight group（All P < 0.0167）. Multivariate regression analysis showed that overweight or obesity were negatively correlated with FEUA（All P < 0.05）.【Conclusion】High uric acid load of serum and glomerular filtration in gout patients with obesity may be due to the relative insufficiency of renal uric acid excretion.

OBJECTIVETo study the effects of gonadotroph-releasing hormone (GnRH) agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced follicle apoptosis in female rats.

METHODSThirty-six female Sprague- Dawley rats were randomized into 6 groups, namely normal saline (NS), CTX, GnRH-a+NS, GnRH-a+CTX, GnRH-ant+NS, and GnRH-ant+CTX groups. The rats were sacrificed between the first and second week after the treatments., and the follicle apoptosis was investigated using TUNEL assay and transmission electron microscopy.

RESULTSThe apoptosis rate of the granulose cells in the follicles in late development was significantly higher than that in early follicles, and the apoptosis rate of the oocytes and granulose cells in rats with CTX treatment was significantly higher than that in rats without CTX treatment (P<0.05). The apoptosis rate of the granulose cells in GnRH-a groups (ranging from 33.40 - or + 4.59 to 73.25 - or + 5.35) was significantly higher than that in GnRH-ant groups (27.46 - or + 4.52 to 49.38 - or + 5.02, P<0.05), but there was no significant difference in the oocytes of early follicles between GnRH-a groups (23.48 - or + 4.25 to 36.15 - or + 4.23) and GnRH-ant groups (21.47 - or + 3.81 to 34.04 - or + 5.54, P>0.05). Electron microscopy revealed characteristic apoptotic changes of the oocytes in early follicles and granulose cells in early and late follicles. The apoptotic changes were especially typical in the granulose cells showing the formation of the apoptotic bodies, and the oocytes only showed chromatin condensation and aggregation.

CONCLUSIONIn the rat mode, GnRH-a promotes while GnRH-ant suppressed follicle apoptosis induced by CTX. GnRH analogues regulates mainly granulose cell apoptosis, but have little effect on oocyte apoptosis.

Animals ; Apoptosis ; drug effects ; Cyclophosphamide ; toxicity ; Female ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; antagonists & inhibitors ; Granulosa Cells ; pathology ; Oocytes ; pathology ; Ovarian Follicle ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective: To investigate the characteristics of body composition (BC) in gout patients and its clinical significance. Methods: Consecutive gout patients were recruited between August 2017 and December 2018. Demographic information, clinical characteristics and comorbidities were collected. BC was assessed by bioelectric impedance analysis including body fat percentage (BF%), trunk and limb BF%, appendicular skeletal muscle index. Overfat was defined by BF% ≥25% for male and ≥35% for female. The association between BC and serum uric acid (sUA) was evaluated by multiple linear regression. Results: A total of 362 gout patients were recruited with median age 38 (30, 52) years, 96.1% (348/362) were male. Mean sUA was (551±133) μmol/L. The mean BF% was (25.8±6.4)% with 53.6%(194/362) patients overfat. Male gout patients with overfat showed more affected joints [4(2, 6) vs. 2(2, 5)], higher sUA [(576±126)μmol/L vs. (523±134) μmol/L], higher prevalence of dyslipidemia [70.1%(131/187) vs. 54.0%(87/161)], metabolic syndrome [60.8%(118/187) vs. 28.0%(47/161)], fatty liver [58.2%(113/187) vs. 35.1%(59/161)] and hypertension [44.4%(83/187) vs. 25.5%(41/161)] than male patients with normal fat (all P<0.05). Their BF%, trunk BF% and limb BF% were positively correlated with the numbers of affected joints, sUA, metabolic syndrome, fatty liver, and hypertension, respectively (r=0.154-0.435, all P<0.05). Multivariable linear regression suggested that BF% (β=4.29, P=0.020) and trunk BF% (β=9.11, P=0.007), but not limb BF%, were positively correlated with sUA. Conclusion Overfat is very common in gout patients. The proportion of trunk fat in male patients is positively correlated with sUA. When assessing obesity in gout patients clinically, body composition analysis should be performed simultaneously.

Objective: To investigate the characteristics of non-alcoholic fatty liver disease (NAFLD) and its associated factors in rheumatoid arthritis (RA) patients. Methods: This cross-sectional study recruited 385 RA patients [including 72 (18.7%) male and 313 (81.3%) female] who received abdominal sonographic examination from August 2015 to May 2021 at Department of Rheumatology, Sun Yat-Sen Memorial Hospital. There were 28 RA patients at 16-29 years old and 32, 80, 121, 99, 25 at 30-39, 40-49, 50-59, 60-69, ≥ 70 years old, respectively. Demographic and clinical data were collected including age, gender, history of alcohol consumption, disease duration, body mass index (BMI), waist circumference, blood pressure, RA disease activity indicators and previous medications. Logistic regression analyses were used to identify the associated factors of NAFLD in RA patients. Results: The prevalence of NAFLD was 24.2% (93/385) in RA patients, 26.3% (21/80) in 40-49 age group and 33.1% (40/121) in 50-59 age group. There were 22.1% (85/385) and 3.6% (14/385) RA patients with overweight and obese, in which the prevalence of NAFLD was 45.9% (39/85) and 78.6% (11/14) respectively, which was 2.6 folds and 4.5 folds that of RA patients with normal BMI. Although there was no significant difference of age, gender and RA disease activity indicators between RA patients with or without NAFLD, those with NAFLD had higher proportions of metabolic diseases including obese (11.8% vs. 1.0%), central obesity (47.3% vs. 16.8%), hypertension (45.2% vs. 29.8%) and type 2 diabetes mellitus (24.7% vs. 12.0%), consistent with higher levels of total cholesterol [(5.33±1.31) mmol/L vs. (4.73±1.12) mmol/L], triglyceride [(1.51±1.08) mmol/L vs. (0.98±0.54) mmol/L] and low-density lipoprotein cholesterol [(3.37±0.97) mmol/L vs. (2.97±0.78) mmol/L, all P<0.05]. Multivariate logistic regression analysis showed that BMI (OR=1.314) and triglyceride (OR=1.809) were the independent factors positively associated with NAFLD in RA patients. Conclusion: NAFLD is a common comorbidity in RA patients, especially in those with middle-aged, overweight or obese, which is associated with high BMI or high triglyceride. Screening and management of NAFLD in RA patients especially those with overweight, obese or dyslipidemia should be emphasized.
Adolescent ; Adult ; Aged ; Arthritis, Rheumatoid/epidemiology* ; Cholesterol, LDL ; Cross-Sectional Studies ; Female ; Humans ; Male ; Middle Aged ; Non-alcoholic Fatty Liver Disease/epidemiology* ; Obesity/epidemiology* ; Overweight/epidemiology* ; Triglycerides ; Young Adult

OBJECTIVETo evaluate the efficacy and safety of cyclosporine A(CsA) in the treatment of refractory nephrotic syndrome (RNS) in children.

METHODSThe Cochrane library, PubMed, EMBASE, CBMdisk, CNKI and VIP were searched from the time when the databases were established to December 31, 2008. Reports on RCTs on treating RNS in children with CsA were collected. Data were extracted and assessed independently by three reviewers. The methodological quality of included RCTs was assessed by the revised Jadad-scale (including randomization, allocation concealment, blinding method and withdrawal). Meta-analysis of homogenous RCTs was managed by using RevMan4.2.3.

RESULTNine RCTs involving 293 participants were included. Six RCTs were assessed as high-quality studies with scores from 4 to 7 and 3 RCTs were assessed as low-quality studies with scores from 1 to 3. Sub-category meta-analysis was based on different clinical types and interventions of RNS in children. Meta-analysis based on included RCTs showed the following results. (1) In children with steroid-dependent or frequent relapse nephrotic syndrome: the short-term efficacy of CsA plus prednisone was better than that of prednisone alone [OR 0.14, 95% CI (0.03, 0.71)]; the short-term efficacy of CsA, cyclophosphamide (CTX) and mycophenolate mofetil had no significant differences, but compared with chlorambucil, CsA had a worse short-term efficacy [OR 6.93, 95% CI (1.53, 31.38)] and a higher relapse rate [OR 0.06, 95% CI (0.01, 0.58)]; maintaining a blood level of CsA between 60 and 80 microg/L during remission period could reduce the long term relapse rate [OR 6.43, 95% CI (1.21, 34.19)]; the incidence of end-stage renal disease (ESRD) or mortality was zero in both groups. (2) In children with steroid-resistant nephrotic syndrome, the short-term efficacy of CsA was better than that of placebo or supportive treatment and CTX, OR and 95% CI were 0.15 (0.02, 0.96) and 0.41 (0.03, 5.00), respectively, but no significant differences were found in the relapse rate and the incidence of ESRD or mortality. (3) Side effects of CsA: the incidence of nephrotoxicity, hypertrichosis and gum hypertrophy was higher in the CsA group than in that of control group, OR and 95% CI were 0.19 (0.05, 0.79), 0.06 (0.02, 0.19), 0.05 (0.02, 0.18), respectively, but no significant differences were found in the incidence of hypertension and liver toxicity.

CONCLUSIONSAvailable evidence showed that CsA could improve short term efficacy in RNS in children, but could not improve long term and endpoint efficacy, therefore CsA could be one of the ideal second-line drugs for RNS in children. There was a trend that the effect of CsA on steroid-dependent or frequent relapse nephrotic syndrome was superior to that on steroid-resistant nephrotic syndrome.

Child ; Cyclosporine ; adverse effects ; therapeutic use ; Humans ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Nephrotic Syndrome ; drug therapy ; Randomized Controlled Trials as Topic ; Recurrence ; Treatment Outcome

OBJECTIVETo study the clinical and pathological features of Denys-Drash syndrome (DDS).

METHODThree DDS cases who were treated in our department from December 2009 to June 2011 were subjected to this study by reviewing of literature.

RESULTBoth case 1 and case 2 were female, with karyotype 46, XX. Case 3 was male with bilateral cryptorchidism. The ages of nephropathy onset of the three cases were 1 year and 9 months, 2 years and 8 moths, and 3 months respectively. Proteinuria in case 2 and case 3 were evidenced to be resistant to steroid. Case 1 was partially responsive to tacrolimus, plasma albumin and cholesterol were improved, although proteinuria was persistent after Tacrolimus was administered. Remission was achieved in case 2 after administration of cyclosporine A and later tacrolimus, and her renal function remains normal till present (4 years and 9 months). Residue renal histology revealed diffused mesangial sclerosis (DMS) in all three patients. All of the three patients had developed right unilateral Wilms tumor. A novel WT1 missense mutation exon 9 c.1213C > G was detected in case 1. WT1 exon 9 c.1168C > T nonsense mutation and exon 8 c.1130A > T missense mutation were detected in case 2 and case 3, respectively.

CONCLUSIONThe clinical manifestation of nephropathy in DDS is variable. The majority present with early onset nephropathy and reach renal failure before the age of 4 years. But in a few patients, nephropathy can also be present much later and progress slowly. Proteinuria in DDS is resistant to steroid but is responsive to calcineurin inhibitors, including Cyclosporine A. The effectiveness of tacrolimus was also observed in this study. DDS is evidently caused by WT1 mutation. DMS is the characteristic renal pathological change in DDS.

Cyclosporine ; therapeutic use ; Denys-Drash Syndrome ; drug therapy ; genetics ; pathology ; Fatal Outcome ; Female ; Genes, Wilms Tumor ; Heterozygote ; Humans ; Infant ; Male ; Mutation ; Nephrotic Syndrome ; drug therapy ; genetics ; pathology ; Proteinuria ; drug therapy ; Sclerosis ; drug therapy ; genetics ; pathology ; Tacrolimus ; therapeutic use ; Treatment Outcome ; WT1 Proteins ; genetics ; Wilms Tumor ; drug therapy ; genetics ; pathology