ObjectiveTo investigate the mechanism of Ershiwuwei Guijiu pill in preventing and treating postmenopausal osteoporosis （PMOP） by activating the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway and inhibiting excessive autophagy. MethodFemale SD rats were ovariectomized and randomly divided into the sham operation group （Sham）， the operation group （OVX）， the Ershiwuwei Guijiu pill （GJ） group， and the raloxifene hydrochloride （RLX） group， with 10 rats in each group. Enzyme-linked immunosorbent assay （ELISA） and colorimetric methods were used to detect the levels of estrogen， bone metabolism markers in serum， and total superoxide dismutase （T-SOD）， malondialdehyde （MDA）， and glutathione peroxidase （GSH-Px） in tibial tissue. Flow cytometry was used to detect reactive oxygen species （ROS） levels in bone marrow mesenchymal stem cells. Masson staining was used to observe pathological changes in the proximal tibia， and micro-computed tomography （Micro-CT） was used to observe changes in tibial microstructural parameters. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the expression of autophagy-related proteins Beclin1， microtubule-associated protein 1A/1B-light chain 3 （LC3）， autophagy-related 5 （Atg5）， as well as PI3K， Akt， and mTOR in tibial tissue. ResultCompared with the Sham group， the OVX group showed a significant decrease in serum levels of estradiol （E₂） and calcium ion （Ca²⁺）， and T-SOD， GSH-Px， PI3K， Akt， and mTOR mRNA levels in bone tissue （P<0.05， P<0.01）， significantly reduced bone mineral density （BMD）， bone surface/bone volume （BS/BV）， trabecular thickness （Tb.Th）， trabecular number （Tb.N）， and trabecular connectivity （Con） in the tibia （P<0.05， P<0.01）， thinner epiphyseal growth plate， and the bone marrow cavity filled with fat vacuoles. Moreover， the levels of phosphorus （P）， MDA， ROS， and mRNA and protein expression of Beclin1， LC3， and Atg5， as well as trabecular separation （Tb.Sp） were significantly elevated （P<0.05， P<0.01）. Compared with the OVX group， the GJ and RLX groups showed significant increases in serum E₂ and Ca²⁺， and bone tissue levels of SOD， GSH-Px， and the mRNA levels of PI3K， Akt， and mTOR （P<0.05， P<0.01）， significantly increased BMD， BS/BV， Tb.Th， Tb.N， and Con in the tibia， thickened epiphyseal growth plate， and significantly reduced fat vacuoles in the bone marrow cavity （P<0.05， P<0.01）. Additionally， the levels of P， MDA， ROS， Beclin1， LC3， Atg5 mRNA and proteins， and Tb.Sp were significantly decreased （P<0.05， P<0.01）. The ratios of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR， which were significantly reduced in the OVX group （P<0.01）， were significantly increased in the GJ and RLX groups （P<0.01）. ConclusionThe Ershiwuwei Guijiu pill reduces oxidative stress and inhibits autophagy， thereby preventing and treating postmenopausal osteoporosis. Its mechanism may be related to the activation of the PI3K/Akt/mTOR signaling pathway， which inhibits autophagy.

A 74-year-old male patient was hospitalized for cerebral infarction, hypertension, diabetes mellitus, and coronary atherosclerotic heart disease. During the hospitalization, a thyroid ultrasound examination revealed suspicious nodules. In order to make a definite diagnosis, thyroid ultrasonography was performed, during which 2.4 ml of sulphur hexafluoride microbubbles for injection and 5 ml of 0.9% sodium chloride injection were given intravenously. Ten minutes later, the patient developed fever, confusion, profuse sweating, red skin rash, and blood pressure 80/60 mmHg, and anaphylactic shock caused by sulphur hexafluoride microbubbles for injection was considered. The patient was given intravenous injection of dexamethasone 10 mg, intravenous infusion of adrenaline 1 mg dissolved in 0.9% sodium chloride injection 250 ml, intramuscular injection of promethazine 25 mg, and mask oxygen. About 3 minutes later, the patient′s consciousness gradually turned clear, and the blood pressure rose (103/72 mmHg); 1.5 hours later, the patient′s consciousness was completely recovered, the blood pressure was 143/86 mmHg, the heart rate was 98 beats/min, and the blood oxygen saturation was 0.98. Twenty-four hours later, the rash was basically disappeared, and the patient′s condition was stable.

A 74-year-old male patient was hospitalized for cerebral infarction, hypertension, diabetes mellitus, and coronary atherosclerotic heart disease. During the hospitalization, a thyroid ultrasound examination revealed suspicious nodules. In order to make a definite diagnosis, thyroid ultrasonography was performed, during which 2.4 ml of sulphur hexafluoride microbubbles for injection and 5 ml of 0.9% sodium chloride injection were given intravenously. Ten minutes later, the patient developed fever, confusion, profuse sweating, red skin rash, and blood pressure 80/60 mmHg, and anaphylactic shock caused by sulphur hexafluoride microbubbles for injection was considered. The patient was given intravenous injection of dexamethasone 10 mg, intravenous infusion of adrenaline 1 mg dissolved in 0.9% sodium chloride injection 250 ml, intramuscular injection of promethazine 25 mg, and mask oxygen. About 3 minutes later, the patient′s consciousness gradually turned clear, and the blood pressure rose (103/72 mmHg); 1.5 hours later, the patient′s consciousness was completely recovered, the blood pressure was 143/86 mmHg, the heart rate was 98 beats/min, and the blood oxygen saturation was 0.98. Twenty-four hours later, the rash was basically disappeared, and the patient′s condition was stable.

OBJECTIVES: To establish a novel method for the separation of sperm cells in mixed stain, and to evaluate its application value. METHODS: Totally 40 mixed stain samples were collected from sexual assault cases. Sperm cells were separated by the conventional differential lysis method and the nylon membrane bushing separation technique, respectively. The DNA of sperm cells was extracted with the silicon membrane kit （Forensic DNA Extraction Kit for Soft Tissues）. The PCR amplification was performed using AmpFℓSTR® Identifiler® Plus kit, and the products were electrophoresed by 3500xL genetic analyser. The results of two separation methods were then compared. RESULTS: Complete and single-source male STR genotypes could be obtained from all the 40 mixed stain samples except three samples with minimal residual of female DNA by the nylon membrane bushing separation technique. The STR genotypes of sperm cells could not be detected in 25 samples, which were obtained in 15 samples （seven were of incomplete male STR genotypes, six with residual of female DNA, two were complete and single-source STR genotypes of sperm cells）. CONCLUSIONS The nylon membrane bushing separation technique developed in present study can be used in the separation of sperm cells in mixed stain, especially for the extraction of a small amount of sperm from a large quantity of female cells, which is inexpensive, rapid and simple.
Coloring Agents ; DNA/genetics* ; DNA Fingerprinting ; Genotype ; Humans ; Male ; Microsatellite Repeats ; Nylons ; Polymerase Chain Reaction ; Semen ; Sex Offenses ; Spermatozoa

Objective To detect mutations in the filaggrin (FLG) gene and expressions of FLG and loricrin in patients with ichthyosis vulgaris (Ⅳ),and to investigate their clinical significance.Methods Tissue specimens were resected from the skin lesions of 10 patients with Ⅳ and normal skin of 14 healthy human controls,and immunohistochemical SP method was used to detect the expressions of filaggrin and loricrin.The expression intensity was determined by the Image-Pro Plus (IPP) software,and expressed as positive units (PU).Blood samples were collected from 10 patients of Han nationality with Ⅳ and 100 healthy human controls followed by DNA extraction.PCR and DNA sequencing were performed to detect the presence of 13 mutations (3321delA,441delA,1249insG,E1795X,S3296X,R501X,2282de14,R2447X,S2889X,7945delA,3702delG,Q2417X,R4307X) in the FLG gene.Results FLG was mainly expressed in the cytoplasm of keratinocytes in the stratum corneum,granular layer,prickle layer and basal layer,and loricrin was observed in the cytoplasm and nuclei of keratinocytes in the granular layer,prickle layer and basal layer,in both the lesional and normal skin.Compared with the normal skin,the lesional skin showed significantly weaker expressions of FLG (0.208 2 ± 0.008 0 vs.0.230 0 ± 0.0228,t =3.30,P ＜ 0.01) and loricrin (0.137 0 ± 0.011 2 vs.0.149 3 ± 0.007 3,t =3.07,P ＜ 0.01).Sequencing analysis identified two mutations,including 3321delA in 7 patients and 441delA in 2 patients.No mutations were detected in the healthy controls.Conclusions The 3321delA and 441delA mutations in the FLG gene may represent the most frequent genetic cause of Ⅳ in patients of Han nationality.The low expressions of FLG and loricrin may be associated with the impairment of skin barrier function in patients with Ⅳ.

AIM: To observe the effect of hemin on the expression of heme oxygenase-1(HO-1) in rat vascular smooth muscle cells(VSMCs). METHODS: Wistar rat aortic VSMCs were cultured in vitro and induced to proliferate by angiotensinⅡ(AngⅡ),hemin(a substrate and inducer of HO-1) and zinc protoporphyrin-Ⅸ(ZnPP,an inhibitor of HO-1)were added to induce and inhibit the expression of HO-1,respectively.The expression of HO-1 mRNA and protein were detected using reverse transcription polymerase chain reaction (RT-PCR)and Western blot,respectively. The relative amount of CO released into the media was quantitated as carbon monoxide hemoglobin(COHb)by enzyme-linked immunosorbent assay(ELISA),the proliferation of VSMCs was detected by MTT. RESULTS: The expression of HO-1 mRNA and protein in VSMCs and the amount of COHb in the media were increased significantly by hemin.Meanwhile the proliferation of VSMCs was suppressed markedly. CONCLUSION: The change of HO-1 mRNA and protein expression is the molecular base of the antiproliferation of endogenous CO .The HO-CO system might play a significant role in the development of cardiovascular diseases characterized by proliferation of VSMCs.