Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation.

Many integral membrane proteins might act as indispensable coordinators in specific functional microdomains to maintain the normal operation of known receptors, such as Notch. Gm364 is a multi-pass transmembrane protein that has been screened as a potential female fertility factor. However, there have been no reports to date about its function in female fertility. Here, we found that global knockout of Gm364 decreased the numbers of primordial follicles and growing follicles, impaired oocyte quality as indicated by increased ROS and γ-H2AX, decreased mitochondrial membrane potential, decreased oocyte maturation, and increased aneuploidy. Mechanistically, Gm364 directly binds and anchors MIB2, a ubiquitin ligase, on the membrane. Subsequently, membrane MIB2 ubiquitinates and activates DLL3. Next, the activated DLL3 binds and activates Notch2, which is subsequently cleaved within the cytoplasm to produce NICD2, the intracellular active domain of Notch2. Finally, NICD2 can directly activate AKT within the cytoplasm to regulate oocyte meiosis and quality.

Chinese Herbal Medicine Adjusting Brain Microenvironment via Mediating Central Nervous System Lymphatic Drainage in Alzheimer's Disease.

Due to its complex pathogenesis and lack of effective therapeutic methods, Alzheimer's disease (AD) has become a severe public health problem worldwide. Recent studies have discovered the function of central nervous system lymphatic drainage, which provides a new strategy for the treatment of AD. Chinese herbal medicine (CHM) has been considered as a cure for AD for hundreds of years in China, and its effect on scavenging ß-amyloid protein in the brain of AD patients has been confirmed. In this review, the mechanism of central nervous system lymphatic drainage and the regulatory functions of CHM on correlation factors were briefly summarized. The advances in our understanding regarding the treatment of AD via regulating the central lymphatic system with CHM will promote the clinical application of CHM in AD patients and the discovery of new therapeutic drugs.

Prenatal features of 17q12 microdeletion and microduplication syndromes: A retrospective case series.

OBJECTIVE: To present the experience on prenatal features of 17q12 microdeletion and microduplication syndromes. MATERIALS AND METHODS: Prenatal chromosomal microarray analysis (CMA) were conducted between January 2015 and December 2018 at a single Chinese tertiary medical centre. Information of cases identified with 17q12 microdeletion or microduplication syndromes were retrospectively collected. Foetal ultrasonographic findings were reviewed, and other information about the gestation week at diagnosis, inheritance and pregnancy outcomes were also included. RESULTS: Ten pregnancies with 17q12 microdeletion and 4 with 17q12 microduplication were identified. The copy number variation (CNV) sizes were 1.39-1.94 Mb in the deleted cases and 1.42-1.48 Mb in the duplicated cases, respectively. All the duplicated and deleted regions included HNF1B and LHX1 genes. Most individuals with 17q12 deletion presented kidney anomalies (9/10), with renal hyperechogenicity being the most common finding (7/10). Fetuses with 17q12 duplication presented a wide phenotypic spectrum, including "double bubble" sign, structural anomalies of the heart and growth anomalies. CONCLUSIONS: Our experience further demonstrated the high correlation between 17q12 microdeletion and renal anomalies especially hyperechogenic kidneys. Structural anomalies of the heart were newly identified phenotypes of 17q12 duplication during prenatal period. Besides, growth anomalies and duodenal atresia might be associated with the duplication.

GTPases Arf5 and Arl2 function partially distinctly during oocyte meiosis.

Mammalian female meiosis must be tightly regulated to produce high-quality mature oocytes for subsequent regular fertilization and healthy live birth of the next generation. GTPases control many important signal pathways involved in diverse cellular activities. ADP-ribosylation factor family members (Arfs) in mice possess GTPase activities, and some members have been found to function in meiosis. However, whether other Arfs play a role in meiosis is unknown. In this study, we found that Arl2 and Arf5 are the richest among Arfs in mouse oocytes, and they are more abundant in oocytes than in granular cells. Furthermore, Arl2 and Arf5 depletion both impeded meiotic progression, but by affecting spindles and microfilaments, respectively. Moreover, Arl2 and Arf5 depletion both significantly increased regular reactive oxygen species levels and decreased mitochondrial membrane potential and autophagy, indicating that oocyte quality was damaged by Arl2 and Arf5 depletion. These results suggest that Arl2 and Arf5 are two novel essential GTPases required for oocyte meiosis and quality control.

Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes.

OBJECTIVES: Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely "female fertility factor" in Fox3a-knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question. MATERIALS AND METHODS: Co-immunoprecipitation, immunogold labelling-electron microscopy, co-localization and yeast two-hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level. RESULTS: Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active ß-catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p-ß-catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation. CONCLUSIONS: Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non-canonical Wnt5a signalling pathways.

[Experience from four dimensions application of classic formulas for treatment of dysmenorrhea].

The application of classic formulas from Shanghan Zabing Lun has a long history in treatment of dysmenorrhea. However, the understanding on how to use classic formula in treatment of dysmenorrhea is still insufficient in the traditional Chinese medicine (TCM), mainly limited to syndrome differentiation and the formula-syndrome correlation. The treatment method of syndrome differentiation is flexible and changeable, but the process is tedious and difficult to copy. In addition, its experience is not easy to summarize and pass on, and it is difficult for beginners to comprehend the essence. Formula-syndrome correlation method is accurate, quick acting, but subjective, too mechanical, and will have obvious side effects in case of prescription mistakes. For complex diseases or when suffering from a variety of diseases, the efficacy of formula-syndrome correlation method is not so obvious. By analyzing the advantages and limitations of these two methods, the author has created a four-dimensional therapy which combines the advantages of these two, from four dimensions including disease differentiation, syndromes differentiation, time differentiation, and constitution differentiation in clinical application for dysmenorrheal. Its clinical curative effect is satisfactory. Four-dimensional therapy is a way of thinking, to guide clinicians for accurate diagnosis and precisely choosing drugs; meanwhile, the personalized health guidance is given according to different constitutions and different seasons, which also has certain significance to treat the undiseased.

Placenta-specific 1 regulates oocyte meiosis and fertilization through furin.

Placenta-specific 1 (Plac1) has been found to be essential for placentation, and abnormal Plac1 expression and distribution is highly correlated with preeclampsia and implantation failure; however, its function in mammalian oocytes has not been elucidated. Here, we report that Plac1 was more prominent in mouse oocytes and enriched at the membrane region throughout meiosis. On the one hand, Plac1 knockdown severely disrupted microvillus organization; however, on the other hand, Plac1 significantly decreased oocyte maturation and increased aneuploidy, consequently disrupting normal fertilization. On the basis of immunoprecipitate matrix-assisted laser desorption/ionization, we established a working model, then verified and suggested that, at the germinal vesicle stage, Plac1 enriches the membrane to activate furin, and active furin subsequently activates IGF-1 receptor to maintain regular microvillus organization. Upon meiosis onset, active furin/IGF-1 receptor relocates into the cytoplasm to activate (phosphorylate) Akt to promote meiosis. In summary, our finding suggests that Plac1, a protein that is crucial for placentation, is also essential for oocyte meiosis and fertilization.-Shi, L.-Y., Ma, Y., Zhu, G.-Y., Liu, J.-W., Zhou, C.-X., Chen, L.-J., Wang, Y., Li, R.-C., Yang, Z.-X., Zhang, D. Placenta-specific 1 regulates oocyte meiosis and fertilization through furin.

Pnma5 is essential to the progression of meiosis in mouse oocytes through a chain of phosphorylation.

PNMA (paraneoplastic antigen MA) family includes Pnma1-6. Although other members have been found to be involved in paraneoplastic neurological disorders, death receptor-dependent apoptosis, and tumorigenesis, Pnma5 was thought to be a female fertility factor, as indicated by one genome-wide study. But until now there have not been any further functional studies about Pnma5 in female meiosis. Our preliminary study indicated that Pnma5 might play important roles in meiosis. To further address this, Pnma5 was knocked down in in-vitro maturated (IVM) mouse oocytes, which are common models for mammalian female meiosis, by specific siRNA, and results showed that the loss of Pnma5 significantly delayed the progression of meiosis I and increased chromosome segregation errors during anaphase I. In in-vitro fertilization (IVF), Pnma5 knockdown caused significantly lower fertilization. To assess how it affects meiosis, Pnma5 knockdown was found to significantly decrease the stability of spindle microtubules and altered F-actin organization within actin cap regions, cause significantly abnormal mitochondria aggregation and lower ATP concentration. Next we have found that phosphorylation at Thr533 re-located Pnma5 strongly to spindles & cortex and was required for the phosphorylation of Akt and Gsk3ß, while Src and Erk1/2 phosphorylation was required for the phosphorylation of Pnma5, indicating that phosphorylated Pnma5 is the active form and subsequently activates Akt and Gsk3ß. Collectively this study suggests that Pnma5 is important for meiosis and is the pivot of Src→Erk1/2→Pnma5→Akt→Gsk3ß pathway.

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes.

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.

ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis.

ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD.

[Guiding Significance of "Mistreatment by Warming Therapy" in Treatment of Non-small Cell Lung Cancer].

"Mistreatment by warming therapy" refers to various symptoms/syndromes caused by improper use of fire methods mentioned in Treatise on Febrile Diseases, which are similar to damaged yin induced fire-heat syndrome (injury of yin, consumption of qi, loss of yin, consumption of body fluid) manifested after radiotherapy and/or chemotherapy in modern clinics. Better efficacy was obtained in treating various symptoms/syndromes by using nourishing yin purging fire method. In this paper authors focuses on exploring guiding significance of using "Mistreatment by warming therapy" in treating non-small cell lung cancer (NSCLC).

[Effect of Zhixin Formula on Gliocyte Protein Expressions, TLRs and NF-κB in Bone Cancer Pain Model Rats].

OBJECTIVE: To explore the analgesic effect of Zhixin Formula (ZF) and its effects on spinal glial fibrillary acidic protein (glial fibillary acidic protein, GFAP, a marker of astrocyte) , CD11b (a maker of microglia), Toll like receptors (TLR2 and TLR4) and nuclear factor κB (NF-κB) in bone cancer pain model rats. METHODS: Totally 20 male SD rats were randomly divided into the blank control group and the bone cancer pain group, 10 in each group. The bone cancer pain model was induced by injecting ascites tumor fluid containing 3 x 10(3) Walker256 cell line from the left tibia. Ethological tests, X-ray test, and HE staining were performed to confirm a successful modeling. After model was successfully established, 70 male SD rats were randomly divided into seven groups, 10 in each group: the blank control group, the bone cancer pain group (as the model group), the Western medicine (WM) group (Tramadol Hydrochloride), the high dose ZF group, the middle dose ZF group, the low dose ZF group, and the Chinese medicine (CM) group (Wulin Zhitong Capsule). Fourteen days after modeling, rats in the high, middle, and low dose ZF groups were administrated by gastrogavage with 9, 4.5, and 2.25 g/kg ZF water condensed preparation respectively, once a day for seven consecutive days. On day 21 MS typical protein expressions including GFAP, CD11b, TLR (2,4) and NF-κB from cornu dorsal medullae spindis L4-L5 were detected by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Compare with the blank control group, increased weight in the model group was slow and showed a decreasing trend (P < 0.01), spontaneous ambulatory pain score (SAPS) obviously increased (P < 0.01), paw withdrawal threshold (PWT) obviously decreased in the model group (P < 0.05, P < 0.01). Results of lateral tibial X-ray and HE staining showed obvious changes and damage occurred in bone structures of rats in the model group. Immunohistochemistry showed that GFAP expression significantly increased in the model group (P < 0.05). Protein levels of NF-κB also significantly increased in the model group (P < 0.05). Compared with the model group, CD11b expressions obviously decreased in the middle and high dose ZF groups (P < 0.01). Meanwhile, protein expressions of TLR2 and TLR4, as well as NF-κB also obviously decreased (P < 0.05). CONCLUSION: ZF had analgesic effect, which might be probably related to inhibiting proliferation and activation of gliocytes, as well as activation of TLRs and NF-κB.

[Effect-of Angong Niuhuang Pill on Th1/Th2 of cerebral infarction patients of phlegm-heat obstructing orifices in China and Indonesia].

OBJECTIVE: To observe the effect of Angong Niuhuang Pill (ANP) on Thl/Th2 in cerebral infarction patients and to explore the mechanism of phlegm-heat obstructing orifices. METHODS: Re- cruited were 30 cerebral infarction patients of phlegm-heat obstructing orifices syndrome (PHOOS) both in China and Indonesia. They were assigned to 4 groups according to the use of ANP, the Chinese treatment group, the Indonesia treatment group, the Chinese control group, and the Indonesia control group. Patients in the two control groups received conventional treatment, while those in the two treatment group additionally took ANP for 30 successive days. Their adverse reactions were observe, and levels of INF-γ and IL-4 were detected. RESULTS: The INF-γ level and the INF-γ/IL-4 ratio significantly decreased, and the IL-4 level increased after treatment in the four groups with statistical difference (P < 0.05). Compared with the control group after treatment in the same country, the INF-γ level and the INF-γ/IL-4 ratio were lower, and the IL-4 level was higher in the two treatment groups (P < 0.05). Compared with the two Chinese groups, the INF-γ level and the INF-γ/IL-4 ratio were higher, and the IL-4 level was lower in the two Indonesian groups (P < 0.05). There was no statistical difference in the post-treatment indices between the two treatment groups. CONCLUSIONS: ANP had moderating effect on Th1/Th2 in cerebral infarction pa- tients. Cerebral infarction patients of PHOOS might exist certain relation with Th1/Th2.

[Research on the relationship between chinese medical syndrome types and Th1/Th2 in bronchioloalveolar carcinoma by thoracoscopic technique].

OBJECTIVE: To study the relationship between Chinese medical syndrome types of bronchioloalveolar carcinoma (BAC) and Th1/Th2. METHODS: Totally 60 BAC patients were syndrome typed as qi and yin deficiency syndrome (QYDS) and qi stagnation and phlegm-blood stasis syndrome (QSPSS), 30 cases in each group. Meanwhile, 30 subjects with benign pulmonary nodules were recruited as the control group. The contents of interferon-gamma (INF-gamma), interleukin 4 (IL-4), IL-2, and IL-5 were detected using thoracoscopic technique. RESULTS: As for Th1 (INF-gamma and IL-2), it was ranked from high to low as the control group > the QSPSS group > the QYDS group (P < 0.05). As for Th2 (IL-4 and IL-5), it was ranked from high to low as the QYDS group > the QSPSS group >the control group (P < 0.05). As for Th1/Th2 (INF-gamma/lL-4, IL-2/IL-5), it was ranked from high to low as the control group > the QSPSS group >the QYDS group (P < 0.05). CONCLUSIONS: Compared with the tissue of benign nodules, Th1 function in tumor tissue of BAC patients was weaker and Th2 function stronger. Chinese medical syndrome types of BAC had correlation with Th1/Th2. Patients of excess syndrome had stronger immunity with Th1/Th2 shifting left,while those of deficiency syndrome were predispose to humoral immunity with Thl/Th2 shifting right.

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and apoptosis in colorectal cancer cells via p53-dependent and -independent pathways.

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.

[Observation on therapeutic effect of acupuncture in the treatment of German hypertension patients].

OBJECTIVE: To observe the clinical efficacy of acupuncture in the treatment of hypertension patients. METHODS: A total of 80 out-patients and in-patients with primary hypertension were randomly and equally divided into acupuncture group and medication group. Patients of the acupuncture group were treated with acupuncture stimulation of Fengchi (GB20), Qu-chi (LI 11), Taichong (LR 3), etc. for 30 min, combined with Xingjian (LR 2), Xiaxi (GB43) for hyperactivity of the liver-yang, with Ganshu (BL 18) and Fuliu (KI 7) for severe phlegm-damp, and with Guanyuan (CV 4) and Shenshu (BL23) for deficiency of both yin and yang, once per day, continuously for one month except weekends. In addition, patients of the medication group were ordered to take Diovan (80 mg) once daily, continuously for 30 days. Both casual blood pressure (CBP) and ambulatory blood pressure (ABP) were detected by using an auscult-type baumanometer and a dynamic blood monitor, respectively. RESULTS: Of the two 40 cases in the medication and acupuncture groups, 18 (45.0%) and 27 (67.5%) experienced marked improvement in the reduction of blood pressure, 14 (35.0%) and 11 (27.5%) were improved, 8 (20.0%) and 2 (5.0%) failed, with the effective rates being 80.0% and 95.0%, respectively. The therapeutic effect of the acupuncture group was significantly superior to that of medication group in improving hypertension (P < 0.05). CONCLUSION: Acupuncture is superior to Diovan in relieving hypertension.

[Determination of isoguanosine in Semen Crotonis Tiglii by high-performance liquid chromatography].

OBJECTIVE: To develop a high-performance liquid chromatographic method for the determination of isoguanosine in Semen Crotonis Tiglii. METHODS: The determination was done by using reverse-phase high-performance liquid chromatography with a Hedera ODS-2 column (4.6 mm x 250 mm, 5 microm). Elution was employed with the mobile phase of acetonitrile-methanol-water (1:4:95) at flow rate of 1.0 mL/min. The detection wavelength was set at 292 nm and the column temperature was 25 degrees centigrade. Injection volume was 10 microL. RESULTS: The linear range of isoguanosine was from 0.161 to 0.967 mg. The correlation coefficient of the calibration curves was 0.999 8. The average recovery rate was 98.78% with relative standard deviation of 0.02%. CONCLUSION: The results show that the method is simple, accurate, and repeatable, and it is suitable for quality control of Semen Crotonis Tiglii.

[Therapeutic effect of whole body hyperthermia combined with chemotherapy in patients with advanced cancer].

OBJECTIVE: To determine the short-term efficacy and security of whole body hyperthermia (WBH) combined with chemotherapy for advanced cancer. METHODS: Different chemotherapy regimens were applied in 138 patients with advanced cancer. Among them, 68 patients (Group A) didn't receive any other therapies. The other 70 patients (Group B) received WBH together with chemotherapy. WBH was maintained at 40 degrees C approximately 42 degrees C for 50 approximately 60 min (once or twice every week and 4 times a cycle). RESULTS: In Group A, the rate of complete remission (CR) was 2.9%, partial remission (PR) was 36.8%, stable disease was 35.3%, progressive disease was 25.0%, the overall response rate (CR + PR) was 39.7%; while in Group B, the corresponding figures were 5.7%, 52.9%, 25.7%, 25.0%, and 58.6%, respectively. There was significant difference between the two groups (P < 0.05). The rates of III + IV gastrointestinal tract andmyelosuppression toxicities were 26.5% and 16.2% in Group A, while 27.1% and 18.6% in Group B. No significant difference was found. CONCLUSION: WBH combined with chemotherapy is efficient and safe for advanced cancer, and is worth generalizing extensively.