Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

[Influence of Land Use Change on Ecosystem Service Value Based on GEE in the Beijing-Tianjin-Hebei Region from 1998 to 2018].

In sustainable development assessment of the Beijing-Tianjin-Hebei region, the ability to dynamically estimate the value of ecosystem services is of great significance. This study considers the Beijing-Tianjin-Hebei region as the research area, based on the google earth engine (GEE); the classification and decision tree (CART) classification algorithm was adopted to supervise and classify the Landsat Thematic Mapper/Operational Land Imager (TM/OLI) images in the study area in 1998, 2003, 2008, 2013, and 2018, and land use types in these five periods were obtained. Quantitative analysis of the dynamic changes of land use in the Beijing-Tianjin-Hebei region from 1998 to 2018 was carried out. Then, the ecosystem service value (ESV) equivalent estimation method was used to quantitatively estimate the ESV in the Beijing-Tianjin-Hebei region and combine it with a 15 km×15 km scale grid to detect its temporal and spatial dynamics. The main results were as follows. ① From 1998 to 2018, the area of construction land (increased by 16.67%) and grassland (reduced by 13.73%) in the six land use types in the Beijing-Tianjin-Hebei region was the largest, and the change in the proportion of water bodies (0.2%) was the smallest. ② The total value of ESV in the Beijing-Tianjin-Hebei region experienced a short-term increase from 1998 to 2003 (an increase of 91.97×108 yuan), and continued to decrease from 2003 to 2018 (a decrease of 239.07×108 yuan), mainly related to the expansion of construction land area in the other three time periods excluding 1998 and 2003. Among the six land use types, the forest provides the highest value of ecosystem services, and the construction land and unused land provide the lowest value of ecosystem services. ③ The ESV time-space analysis based on the 15 km×15 km scale grid showed that the ESV medium area in the Beijing-Tianjin-Hebei region gradually decreased from 1998 to 2018, the ESV lower area and the higher area gradually increased, and the ESV lower-area growth rate was higher than for the higher area. ④ The revised value of the Beijing-Tianjin-Hebei region (sensitivity coefficient range 0-0.83) has good significance and reliability. In future economic development, the Beijing-Tianjin-Hebei region should rationally optimize the land use pattern and strengthen the protection of forest land, grassland, water bodies and cultivated land.

A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects.

AIM: To investigate the pharmacokinetics, pharmacodynamics, and safety of higenamine, an active ingredient of Aconite root, in healthy Chinese volunteers. METHODS: Ten subjects received continuous, intravenous infusion of higenamine at gradually escalating doses from 0.5 to 4.0 µg·kg(-1)·min(-1), each dose was given for 3 min. Blood and urine samples were collected at designated time points to measure the concentrations of higenamine. Pharmacodynamics was assessed by measuring the subject's heart rate. A nonlinear mixed-effect modeling approach, using the software Phoenix NLME, was used to model the plasma concentration-time profiles and heart rate. RESULTS: Peak concentrations (C(max)) of higenamine ranged from 15.1 to 44.0 ng/mL. The half-life of higenamine was 0.133 h (range, 0.107-0.166 h), while the area under concentration-time curve (AUC), extrapolated to infinity, was 5.39 ng·h·mL(-1) (range, 3.2-6.8 ng·h·mL(-1)). The volume of distribution (V) was 48 L (range, 30.8-80.6 L). The total clearance (CL) was 249 L/h (range, 199-336 L/h). Within 8 h, 9.3% (range, 4.6%-12.4%) of higenamine was recovered in the urine. The pharmacokinetics of higenamine was successfully described using a two-compartment model with nonlinear clearance. In the pharmacodynamic model, heart rates were related to the plasma drug concentrations using a simple direct effect model with baseline. The E(0), E(max), and EC(50) were 68 bpm, 73 bpm and 8.1 µg/L, respectively. CONCLUSION: Higenamine has desirable pharmacokinetic and pharmacodynamic characteristics. The results provide important information for future clinical studies on higenamine.

[Influence on pubertal reproductive function in female rats by immune challenge in early life].

OBJECTIVE: To investigate the long-term programming effects on pubertal reproductive function by immunological challenge in early life. METHODS: Female Sprague-Dawley rats were administered by endotoxin (lipopolysaccharide, LPS) at a dosage of 50 µg/kg and saline intraperitoneally on postnatal day 3 and 5. Body weight was measured weekly. Puberty onset (vaginal opening) and oestrous cyclicity were monitored from postnatal day 30. At the age of 6 weeks, bilateral ovariectomy was performed. The histological and morphological change of the ovaries (the thickness of the theca interna and the number of different kinds of follicles) were observed and the immunoreactivity of the ovarian sympathetic nerve markers (low affinity receptor of nerve growth factor, p75NGFR) was evaluated by immune staining. RESULTS: Immunological challenge (exposed to LPS) in early life delayed vaginal opening significantly [LPS-treated (40.6 ± 0.7) days versus controls (38.6 ± 0.5) days, P < 0.05], decreased the percentage of normal oestrous cyclicity (LPS-treated 26.1% versus controls 66.8%, P < 0.05), decreased the total number of different types of follicles (primordial follicles: LPS-treated 610 ± 47 versus controls 1181 ± 57, P < 0.05; primary follicles: LPS-treated 624 ± 41 versus controls 960 ± 30, P < 0.05; preantral follicles: LPS-treated 183 ± 16 versus controls 260 ± 14, P < 0.05; antral follicles: LPS-treated 32 ± 4 versus controls 79 ± 7, P < 0.05) and increased the thickness of the theca interna [LPS-treated (15.8 ± 0.4) µm versus controls (11.4 ± 0.3) µm, P < 0.05]. The immunostaining of p75NGFR was obviously enhanced in the LPS-treated ovaries when compared with that of controls (P < 0.05). CONCLUSIONS: Immunological stress during early critical developmental windows could have long dysfunctional effects on the pubertal reproductive function. It delayed puberty onset, reduced the percentage of the normal oestrous cycles, decreased follicles reserve and increased the thickness of the theca interna which might involve the up-regulation of the local ovarian sympathetic nerve activity.

[Correlation between serum inhibin B level after treatment with gonadotropin releasing hormone agonist and outcome of in vitro fertilization-embryo transfer].

OBJECTIVE: To evaluate the decreased level of serum inhibin B (INHB) treated by gonadotropin releasing hormone agonist (GNRH-a) in predicting ovarian response and pregnancy in in vitro fertilization-embryo transfer (IVF-ET). METHODS: The prospective study enrolled 124 women given by GnRH-a+ recombine follicle stimulating hormone (rFSH) + human chorionic gonadotrophin (hCG) long term stimulation protocol undergone their first cycle of IVF-ET treatment. The following predictive factors were collected and analyzed, such as age, basal level of follicle stimulating hormone (FSH), the ratio of FSH/ luteinizing hormone (LH), the concentration of INHB after down-regulation, total number of antral follicle count (AFC) and mean ovarian volume. Ovarian response was evaluated by the number of oocytes obtained. A multiple regression analysis and logistic regression model were used for all possible prognostic variables to evaluate the value of different hormones in predicting ovarian response and pregnancy after IVF-ET. Receiver operating characteristic (ROC) analysis was used to evaluate the level of INHB in predicting the number of oocytes obtained. The sensitivity and specificity were calculated at the discriminating cut-off point. RESULTS: The concentration of INHB after down-regulation showed a highly significant positive correlations with the number of oocytes obtained (r = 0.435, P < 0.01). The multiple regression analyses showed INHB was the most significant predictor of the number of retrieved oocytes, but INHB was not associated with IVF-ET outcome significantly (P > 0.05). ROC analyses showed INHB after down-regulation had the largest area under curve (AUC) 0.933 (95%CI: 0.878 - 0.988). When a threshold of 15 ng/L of INHB was established, 95.5% sensitivity and 50.0% specificity in ovarian response were observed. CONCLUSIONS: The level of INHB was the best factor in predicting ovarian response in IVF-ET. Decreased level of INHB was the early sign of ovarian reserve function failure, however, useless in predicting IVF-ET outcome.

[Effect of desogestrel and ethinyl estradiol pretreatment in superovulation cycles with short protocol].

OBJECTIVE: To study the effect of desogestrel and ethinyl estradiol (DEE) pre-treatment combined with gonadotropin releasing hormone agonist (GnRH-a) stimulation in in vitro fertilization-embryo transplantation (IVF-ET). METHODS: A retrospective analysis was performed in 101 infertile women who received a short protocol of GnRH-a for IVF-ET treatment from June 2004 to June 2007 in the Reproductive Medicine Center of First Affiliated Hospital of Wenzhou Medical College. Patients had been pre-treated with oral contraceptive pill (OCP) for two months before GnRH-a combined with recombinant follicle stimulation hormone (r-FSH) treatment (study group, n = 42) or had not been pretreated with OCP (control group, n = 59). A statistical analysis of two groups was carried out for the assessment of ovulation stimulating effect of OCP and its influence on the IVF. RESULTS: Serum FSH was significantly decreased after OCP in the study group. Twelve pregnancies were obtained including 1 case of spontaneous abortion at 7 weeks in the study group, and 11 pregnancies were obtained including 2 cases of spontaneous abortion during 7 -9 weeks in control group. The clinical pregnancy rates in the study group (23%, 12/53) was higher than that in the control group (17%, 11/63), but the differences were not significant (P > 0.05). The miscarriage rate in the study group (8%, 1/12) was lower than that in the control group (18%, 2/11), however no significant differences were found between them (P > 0.05). The cycle cancellation rate in patients of the study group (5%, 3/56)was significantly lower than that in patients of the control group (17%, 13/76, P < 0.05). The differences between patients of the two groups with respect to age, basal level of FSH and luteinizing hormone (LH), antral follicle counts, the mean number of oocyte retrieval, the days of stimulation, total dose of r-FSH used, fertilization rate and embryo cleavage rate, however were insignificant. CONCLUSION: OCP pretreatment combined with short protocol of GnRH-a stimulation in IVF could significantly decrease the cycle cancellation rate, with a declining miscarriage rate and increasing pregnancy rate.

[HLA-A site genotyping on single blastomeres is studied by nest-PCR-SSP method].

OBJECTIVE: To assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos. METHODS: By nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits. RESULTS: The amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%). CONCLUSION: The above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.

[In vitro maturation and fertilization of unstimulated immature oocyte for the treatment of infertile women].

OBJECTIVE: To evaluate the efficacy of in vitro maturation (IVM) and fertilization of unstimulated immature oocytes for the treatment of infertile women. METHODS: Fifty-four cycles of IVM were carried out in 40 patients including 26 women with infertility due to polycystic ovary syndrome (PCOS), and 14 patients with history of assisted reproductive technology (ART) failure. Transvaginal ultrasound-guided oocyte collection was performed during menstrual cycle days 9 - 12 without pretreatment of gonadotropins. After 24 - 48 hours of culture, the metaphase II stage oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was performed 2 or 3 days after ICSI. Laser assisted hatching was done before embryo transfer. RESULTS: Seven cycles were cancelled and the cancel rate was 13% (7/54). A total of 857 immature oocytes were obtained with the mean numbers of 18.2 per cycle, and 632 oocytes developed at MII stage (73.7%, 632/857). A total of 476 oocytes were fertilized by ICSI (75.3%, 476/632), with a cleavage rate of 91.2% (434/476). Embryo transfer was performed in 47 cycles and the mean number of embryos transferred were 4.3 per cycle (range: 2 - 6). The mean endometrial thickness on the day of embryo transfer was 8.9 mm. Nineteen clinical pregnancies were obtained, giving a pregnancy rate of 35% (19/54) per start cycle and 40% (19/47) per transfer cycle. CONCLUSIONS: IVM of unstimulated immature oocytes for the treatment of women with various causes of infertility especially due to PCOS is an effective alternative method. The clinical pregnancy rate of 40% (19/47) is similar to that by conventional in vitro fertilization treatment in our unit.

[Analysis of pregnancy outcomes after in vitro fertilization-embryo transfer and intracytoplasmic sperm injection].

OBJECTIVE: To compare pregnancy and perinatal outcomes between in vitro fertilization-embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI). METHODS: A retrospective study was carried out to measure pre-clinical and clinical abortion, ectopic pregnancies, multiple gestations, birth weight, gestational age, congenital malformation and perinatal mortality in patients receiving either IVF-ET (n = 143, group 1) or ICSI (n = 173, group 2) from January 1999 to June 2001. The outcomes of singleton and twin were compared separately. RESULTS: The maternal age, infertility duration, parity and the number of transferred embryo were comparable between the two groups. There were no significant differences in abortion rate (16.1% vs 13.3%), birth rate (65.7% vs 74.6%) between IVF-ET and ICSI groups (P > 0.05). In singleton, the rates of low birth weight, small for gestational age and pre-term birth were 1.8%, 7.3%, 5.5% respectively in IVF-ET group and 6.8%, 8.1%, 14.9% respectively in ICSI group. In twin, the rates of low birth weight, small for gestational age and pre-term birth were 34.2%, 30.3%, 42.1% respectively in IVF-ET group and 42.6%, 38.0%, 46.3% respectively in ICSI group. There were no significant differences between the two groups (P > 0.05). But the rates of low birth weight, small for gestational age and pre-term birth were higher in twin than in singleton (P < 0.01). The incidence of congenital malformation was 2.2% and 1.6% in IVF-ET and ICSI group respectively (P > 0.05). CONCLUSIONS: The pregnancy and perinatal outcomes are similar between IVF-ET and ICSI groups. Twin is the main cause of low birth weight, small for gestational age and pre-term birth.

[Effects of metformin on gonadotropin-induced ovulation in patients with polycystic ovary syndrome].

OBJECTIVE: To evaluate the effects of metformin on gonadotropin-induced ovulation in patients with polycystic ovary syndrome (PCOS). METHODS: Forty patients with PCOS (study group) and 20 women with normal weight and menstrual cycle (control group) were enrolled. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), fasting glucose (FG), fasting insulin (FINS) and fasting leptin were measured before and after treatment. In the study group, 20 cases (group A) were assigned to take 500 mg of metformin three times daily for 12 weeks, if pregnancy did not occur, high purified FSH (FSH-HP) was added for one cycle; another 20 cases (group B) were induced ovulation with FSH-HP alone for one cycle. RESULTS: There were significant high FINS and leptin levels in the study group as compared with the control group [(20 +/- 16) vs (12 +/- 6) nmol/L, P < 0.05; (14 +/- 16) vs (8 +/- 4) mg/L, P < 0.05]. The obese PCOS group had markedly higher serum FINS and leptin than the non-obese PCOS group [(24 +/- 18) vs (14 +/- 8) nmol/L, P < 0.05; (20 +/- 22) vs (8 +/- 4) mg/L, P < 0.05], but serum FINS and FG were not significantly different between the non-obese PCOS and the control group (P > 0.05). After administration of metformin for 12 weeks, serum LH, T, leptin and FINS decreased significantly (P < 0.05 - 0.01), serum FSH levels and body mass index showed a slight decrease, whereas no change was found in FG. In the study group, 3 cases conceived during metformin therapy, the remaining 37 were induced ovulation with FSH-HP or FSH-HP and metformin, 7 cases obtained pregnancy. The rates of ovulation and pregnancy in group A were higher than those in group B (88% vs 70%, 24% vs 15%), but no significant difference was found. CONCLUSIONS: Metformin therapy in PCOS can decrease the FINS and leptin levels, normalize the endocrine abnormalities, resumes ovulation and pregnancy in some patients, and may improve the ovarian response to gonadotropin.

[Endocrine and metabolic effects of metformin in combination with compound cyproterone acetate in women with polycystic ovarian syndrome].

OBJECTIVE: To study the endocrinologic and metabolic effects of metformin in combination with compound cyproterone acetate (CPA) on patients with polycystic ovarian syndrome (PCOS). METHODS: A prospective study involved total 45 PCOS patients as group A and 20 non-PCOS infertility patients as control (group B). Complete baseline work-up including body mass index (BMI), waist/hip ratio (WHR), ferriman-Gallwey score (FGS), gonadotrophin, testosterone (T), sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (Ds), insulin (FI) and glucose tolerance test, were performed in all patients. Patients in group A were treated with CPA alone (group A1), metformin alone (group A2) or combination of CPA with metformin (group A3), respectively by randomization. At the end of 12-week therapy, subjects were re-evaluated and above parameters were measured. RESULTS: Women in group A had significant increases in BMI, WHR, FGS, luteinizing hormone (LH), T, FI, insulin resistance, and significantly decrease in high-density lipoprotein (HDL)-C comparing with the control group (P < 0.01). No significant difference among A1, A2 and A3 was found at baseline. LH, T, free testosterone (FT) were significant decreased from (13.9 +/- 5.9) IU/L, (2.1 +/- 0.8) nmol/L and (2.8 +/- 2.3) nmol/L respectively to (5.8 +/- 2.2) IU/L, (1.2 +/- 0.4) nmol/L and (0.8 +/- 0.5) nmol/L respectively and SHBG was significant increased from (99 +/- 42) nmol/L to (187 +/- 64) nmol/L in group A3, when compared with LH, T and FT from (13.8 +/- 7.6) IU/L, (2.2 +/- 1.1) nmol/L and (2.5 +/- 1.9) nmol/L respectively to (11.8 +/- 6.5) IU/L, (1.8 +/- 0.8) nmol/L and (1.7 +/- 1.0) nmol/L respectively and SHBG from (99 +/- 40) nmol/L to (120 +/- 51) nmol/L in group A2 (P < 0.05 approximately 0.001). HDL-C were significantly increased from (1.5 +/- 0.3) mmol/L to (1.8 +/- 0.3) mmol/L in group A3 comparing with HDL-C from (1.5 +/- 0.4) mmol/L to (1.6 +/- 0.4) mmol/L in group A1 (P < 0.001). CONCLUSIONS: The PCOS patients treated with metformin in combination with compound cyproterone acetate may be more effective in inhibiting hyperandrogen and hypersecretion of LH than metformin alone and more obvious in improving lipid profiles than CPA alone.