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

OBJECTIVEIdiopathic collapsing glomerulopathy (ICG) is a clinically and pathologically distinct variant of focal segmental glomerulosclerosis, which is characterized by proteinuria (often nephrotic range) and rapid progression to end-stage renal failure. The typical pathological changes are global/segmental glomerular collapse, hypertrophy and hyperplasia of podocytes and severe tubulointerstitial lesions. Most ICG patients who have been reported in previous published papers are adults. ICG in children is rare. The study aimed to analyze and investigate clinical manifestations, renal histopathological findings, treatment and outcomes of ICG in children.

METHODSData of two cases of ICG, a 7-year-old boy and a 12-year-old girl, were analyzed. Both of them were Chinese and Han. Clinical characteristics, results of laboratory tests, renal histopathological findings, treatment, outcomes and prognosis of the two children with ICG were retrospectively analyzed. Results were compared with published data.

RESULTSThese two children presented typical clinical features of nephrotic syndrome. The quantity of 24 hr urine protein was 7.6 g/d (0.47 g/kg x d for boy) and 10.67 g/d (0.35 g/kg x d for girl). Both of them had hypertension (blood pressure ranged from 130/90 to 150/110 mmHg) and hypercholesterolemia (15.4 mmol/L for the boy and 11.3 mmol/L for the girl). The serum albumin was 12 g/L for girl and 23 g/L for boy. The creatinine clearance rate gradually decreased from normal range to 30 ml/min for the girl. The histopathological changes in renal biopsy of them were focal segmental or global glomerular collapse, hypertrophy and hyperplasia of podocytes and severe tubulointerstitial lesions. These two cases were steroid-resistant and were treated with pulse intravenous methylprednisolone and pulse intravenous cyclphosphamade in one case, who rapidly progressed to end-stage renal failure and died half a year later. Another one was treated with cyclosporine. He showed continuous hypertention and heavy proteinuria for eight months.

CONCLUSIONICG in the 2 children was a severe disease which presented steroid-resistant nephrotic syndrome and rapidly progressive renal failure. The pathological characteristics was global/segmental glomerular collapse, hypertrophy and hyperplasia of podocytes and severe tubulointerstitial lesions. In children with ICG treatment was difficult and the prognosis was poor.

Child ; Disease Progression ; Female ; Glomerulosclerosis, Focal Segmental ; complications ; diagnosis ; pathology ; therapy ; Glucosinolates ; Humans ; Kidney ; pathology ; Kidney Failure, Chronic ; etiology ; Kidney Glomerulus ; pathology ; Male ; Nephrotic Syndrome ; etiology ; Proteinuria ; etiology ; Treatment Outcome

The megakaryocyte and platelet lineage may be one of the major sites of human cytomegalovirus (HCMV) infection. However, whether HCMV aggravates apoptosis in normal megakaryocytes was not well investigated. Megakaryocytic cell line CHRF-288-11 and HCMV AD 169 strain were co-cultured in this study. PCR was used to detect the direct infection of the cells by HCMV IEA expression. The apoptotic cells were analyzed by morphologic observation, DNA ladder formation, annexin V/PI and PI assay with flow cytometry. The results showed that HCMV significantly inhibited the growth of CHRF cells in three different concentrations of viral infection groups (10(-3), 10(-2), 10(-1)). The viability levels in each infection groups were 77%, 73% and 68% respectively after incubation for 7 days, compared with 98% in the control group. Using annexin V/PI with flow cytometry, it was shown that the percentages of apoptotic cells viral infection in groups (10(-3), 10(-2), 10(-1)) were (21.3 +/- 2.49)%, (25.8 +/- 3.65)% and (31.4 +/- 3.91)% at 7 days after infection, while the control was (3.68 +/- 1.47)%. The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, PCR detection also showed the direct infection by identification of HCMV IEA expression in CHRF cells. This study suggested that HCMV could directly infect megakaryocytes and aggravated apoptosis in HCMV-infected megakaryocytes.
Apoptosis ; Cell Survival ; Cells, Cultured ; Cytomegalovirus ; pathogenicity ; DNA, Viral ; analysis ; Humans ; Megakaryocytes ; cytology ; virology ; Polymerase Chain Reaction