In the last decade, next-generation sequencing (NGS) technology, which is characterized by being high-throughput, rapid, sensitive, and accurate, has developed rapidly. Main components of NGS are platforms: 454 sequencing; illumina sequencing; ion torrent sequencing; SOLID sequencing. NGS is used widely for the human immunodeficiency virus (HIV)-1. In this review, we focus on applications of the dynamics of HIV-1 quasispecies.
Animals ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; High-Throughput Nucleotide Sequencing ; methods ; Humans

To investigate the effects of Tongxinluo capsule on sciatic nerve apoptosis in spontaneous type II diabetic KK/Upj-Ay mice, in order to explore its mechanism for improving diabetic peripheral neuropathy (DPN). KK/Upj-Ay mice were selected as the DPN animal model and randomly divided into the model, Tongxinluo low, middle and high group (1, 2, 4 g x kg(-1)). C57BL/6 mice were selected as the control group. Mice were given intragastrically for 12 weeks. Paw withdrawal latency, motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were detected. Apoptotic rate were detected by FCM. Bcl-2, Bax, Caspase-3 mRNA and protein expression in sciatic nerve were examined by Real-time PCR and Western blot. p38MAPK, p-p38MAPK expression were examined by Western blot. In this study,the authors found that Tongxinluo capsule could increase paw withdrawal latency, MNCV and SNCV. Apoptotic rate of sciatic, the expression of Bax and caspase-3 were lower, while Bcl-2 expression was higher in Tongxinluo group than those in model mice. The expression of p-p38MAPK significantly decreased in Tongxinluo group. The results showed that Tongxinluo capsule has protective effects on diabetic peripheral neuropathy of mice via inhibiting cell apoptosis and suppressing the expression of p-p38MAPK.
Animals ; Apoptosis ; drug effects ; Capsules ; administration & dosage ; Diabetic Neuropathies ; drug therapy ; physiopathology ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Sciatic Nerve ; cytology ; drug effects

Acupuncture Points ; Aged ; Aged, 80 and over ; Bloodletting ; Female ; Humans ; Middle Aged ; Pruritus ; therapy

AlM:To evaluate the early diagnosis of sub-clinic stage of diabetic retinopathy.
METHODS: This was cross sectional study, multifocal retina electroretinogram ( mf-ERG ) , contrast sensitivity ( CS) and central retinal artery color Doppler examination were recorded from 30 cases ( 30 eyes ) matched control subjects, 35 cases (35 eyes) with type 2 diabetes mellitus (DM) without diabetic retinopathy ( NDR) and 38 cases ( 38 eyes ) with non-prolifera tive diabetic retinopathy ( NPDR) . One-way ANOVA and SNK-q test were used for data analysis.
RESULTS: P1 response density of NDR patients were found decrease, N1 implicit time were delayed. Which were related with the degree of retinopathy (P<0. 05);CS of NDR patients were found significant in middle and high frequency ( P < 0. 05 ), NPDR patients were found significant in full frequency ( P<0. 05 ); Central retinal artery (CRA) blood flow in the control groups and NDR groups were not found statistically significant (P>0. 05), The differences between normal group, NDR group and NPDR group were found statistically significant (P<0. 05).
CONCLUSlON: mf-ERG and CS are sensitive indexes for early evaluation of visual function in patients with diabetes mellitus, with development of the disease, CRA blood flow also appears to decline.

Objective:To explore the hepatocyte insulin sensitivity of intrauterine growth retardation ( IUGR) rats and establish an insulin resistance cell model in vitro.Methods: An IUGR animal model was established by protein malnutrition during the mother pregnancy .On 60 d and 90 d after birth , the offspring rats were fasted for 12 hours and then their angular vein blood was collected to measure the fasting plasma glucose and fasting serum insulin level , then the insulin resistance index ( HOMA-IR) and insulin sensitivity index ( ISI) were calculated .The insulin sensitivity was evaluated by HOMA-IR and ISI.Primary hepatocytes from each group were respectively isolated by two-step perfusion with collage-nase and were defined as normal hepatocytes group and IUGR hepatocytes group .The normal hepatocyte group was divided into two groups: control group and insulin induction group .Insulin induction group was established by primary cultures of normal hepatocyte incubated with varying dilutions of insulin . CCK-8 was used to detect the viability of the cultured hepatocytes .Glucose oxidase-peroxidase method kit was used to measure glucose consumption of the hepatocytes .Results:HOMA-IR was significantly higher in IUGR rats than in the normal rats at the age of 60 days ( t=-17 .02 , P<0 .05 ) and 90 days ( t=-12.52, P<0.05).ISI was significantly lower than in the normal rats aged 60 days (t=5.61, P<0.05) and 90 days (t=12.42, P<0.05).There were no significant differences in hepatocyte viability among the control group , IUGR group and insulin induction group after incubation of 48 h on day 60 (F=1.34, P=0.29) and day 90 (F=0.22, P=0.81).The glucose consumption of the IUGR group and insulin induction group were significantly decreased compared with the control group on day 60 ( F=9.28, P=0.002) and day 90 (F=56.60, P<0.001), while there was no significant difference be-tween the IUGR group and insulin induction group (P=0.08, P=0.10).Conclusion:The insulin sen-sitivity of hepatocytes of IUGR rats decreased from adolescence to adulthood .High-dilution insulin may induce insulin resistance cell model in vitro.

OBJECTIVE: To investigate the expression of CDK4 and p16 in laryngeal squamous cell carcinoma (LSCC) tissues. METHOD: The expressions of CDK4 and p16 in 30 cases of LSCC tissues and 20 cases of edge tissues were detected by immunohistochemical technology SP method, and discuss their correlation with clincial pathology and clinical stage of LSCC. RESULT: The positive rates of CDK4 and p16 were 63. 3 %,46. 7% in LSCC tissues, and the positive rates were 25%, 90% in edge tissues. The expression of CDK4 in LSCC tissues was significantly higher than that in edge tissues(P<0. 05), which was not associated with the clincial pathology and clinical stage(P> 0. 05); The expression of p16 in LSCC tissues was significantly lower than that in edge tissues(P<0. 05), it was associated with the clincial pathology (P<0. 05), but not associated with clinical stage(P>0. 05) ;there is a negative correlation between CDK4 and p16 (r= -0. 786, P<0. 05). CONCLUSION Low expression of p16 and high expression of CDK4 may play an important role in the development of LSCC and the low expression of p16 in LSCC tissue could be used as important reference markers of malignant degree of tumour.
Biomarkers, Tumor ; Carcinoma, Squamous Cell ; metabolism ; Cyclin-Dependent Kinase 4 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; Head and Neck Neoplasms ; metabolism ; Laryngeal Neoplasms ; metabolism ; Prognosis ; Squamous Cell Carcinoma of Head and Neck

Objective To investigate the roles of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway and its regulatory protein peroxisome proliferator-activated receptor γ (PPAR γ) and phosphatase and tension homologue deleted on chromosome 10 (PTEN) in regulating insulin sensitivity in rats with fetal growth restriction (FGR).Methods Sixteen pregnant rats were randomly divided into two groups including FGR and control groups on the 12th day of pregnancy (eight in each group).The FGR group was given low protein diet (8％ of casein) and restriction diet to establish the neonatal rat model of FGR.All maternal rats after delivery and newborn rats after weaning on 21 days after born were fed with normal diet.Each time blood samples were collected from eight newborn rats of each group to measure levels of fasting plasma glucose (FPG) and fasting insulin(FINS) at the time points of 21 days,two and four months after birth.Then insulin resistance index (IRI) and insulin sensitivity index (ISI) were calculated to evaluate insulin sensitivity.Expression of PI3K,AKT,PPAR γγ,PTEN and glucose transporters 4 (GLUT4) in skeletal muscle at mRNA and protein levels were measured at 21 days,two and four months after birth with real time fluorescence polymerase chain reaction and Western blot,respectively.Relationships between the expression of key molecules of PI3K/AKT signaling pathway and insulin sensitivity were analyzed.T-test,and Pearson's correlation analysis were used for statistical analysis.Results (1) The average birth weight of newborn rats in the FGR group was lower than that of the control group [(4.37± 0.69) vs (7.03±0.55) g,t=-20.75,P＜0.05].The incidence of FGR in the FGR group was 93.33％ (70/75).(2) Compared with normal offspring,those in the FGR group showed significantly increased FPG [two months after birth:(5.53± 0.58) vs (7.49 ± 0.38) mmol/L,t=8.08;four months afterbirth:(6.35±0.66) vs (8.94±0.90) mmol/L,t=6.58],FINS [two months afterbirth:(9.18±0.66) vs (14.67± 1.90) mU/L,t=7.71;four months after birth:(33.08±2.76) vs (56.33±2.81) mU/L,t=16.71] and IR1 (two months after birth:2.25±0.31 vs 4.90±0.81,t=8.63;four months after birth:9.30±0.90 vs 22.44±3.10,t=1 1.51),but decreased ISI (two months after birth:0.020 ± 0.002 vs 0.009± 0.001,t=-10.1 4;four months after birth:0.005±0.000 vs 0.002 ±0.000,t=-14.91) at two and four months after birth (all P＜0.05).(3) Compared with normal offspring,those in the FGR group showed decreased expression of PI3K (21 days after birth:0.082±0.028 vs 0.019±0.004,t=-6.29;two months after birth:0.020±0.003 vs 0.010±0.005,t=-4.78;four months after birth:0.014±0.004 vs 0.003±0.001,t=-7.87) and GLUT4 (21 days after birth:0.132±0.057 vs 0.041 ±0.019,t=-4.32;two months after birth:0.183±0.084 vs 0.069±0.017,t=-3.74;four months after birth:0.248±0.069 vs 0.113±0.040,t=-4.74) at mRNA level at 21 days,two and four months after birth (all P＜0.05).Compared with normal offspring,decreased expression of PPAR γ (two months after birth:0.028±0.002 vs 0.012±0.005,t=-3.70;four months after birth:0.030±0.008 vs 0.012±0.005,t=-3.80) and increased expression of PTEN (two months after birth:0.020±0.004 vs 0.045±0.014,t=5.09;four months after birth:0.023±0.007 vs 0.034±0.009,t=2.57) at mRNA level were observed in offspring of the FGR group at two and four months after birth (all P＜0.05).(4) Compared with normal offspring,expression of PI3K protein (21 days after birth:0.22±0.01 vs 0.17±0.02,t=-6.62;two months after birth:0.27±0.03 vs 0.16±0.02,t=-7.25;four months after birth:0.18±0.01 vs 0.09±0.02,t=-9.79) and GLUT4 protein (21 days after birth:0.21 ±0.01 vs 0.03±0.01,t=-27.29;two months after birth:0.10±0.01 vs 0.06t±0.01,t=-3.90;four months after birth:0.13 ±0.01 vs 0.08± 0.02,t=-8.10) decreased in offspring in the FGR group at 21 days,two and four months after birth (all P＜0.05).Compared with normal offspring,those in the FGR group showed decreased expression of PPAR γ protein (two months after birth:0.10 ± 0.01 vs 0.07± 0.01,t =-7.29;four months after birth:0.09±0.01 vs 0.08±0.01,t=-2.83),but increased expression of PTEN at protein level (two months after birth:0.10±0.01 vs 0.15±0.02,t=6.01;four months after birth:0.09±±0.01 vs 0.13±0.02,t=5.51) at two and four months after birth (all P＜0.05).(5) The IRI levels in offsprings in the FGR group were negatively correlated with the expression of PI3K,GLUT4 and PPAR γ at protein level (two months after birth:r=-0.90,-0.92 and-0.79;four months after birth:r=-0.92,-0.75 and-0.73,all P＜0.05),but positively correlated with the expression of PTEN at protein level (r=0.87 and 0.86,both P＜0.05) at two and four months after birth.Conclusions The abnormal expression of the key molecules of PI3K/AKT signaling pathway precedes the decrease of insulin sensitivity in newborn rats with FGR and the expression regulatory protein PPAR γ and PTEN are also changed,suggesting that these molecules may induce the impairment of insulin sensitivity in rats with FGR and be involved in the development of insulin resistance.

BACKGROUND:There are numerous studies on bone marrow mesenchymal stem cells(BMSCs)from small animals such as rats and rabbits,but no few reports addressing BMSCs from big animals.OBJECTIVE:To observe in vitro cultured goat BMSCs,and to understand its biological properties.METHODS:A healthy Chinese goat aged ten months was obtained to extract 5 mL fresh bone marrow from the posterior superior iliac spine by puncture following anesthesia.Using the whole bone marrow method,the samples were incubated in a sterile plastic culture flask and added with DMEM/F12 containing 10% fetal bovine serum.Following 80% 90% confluence,cells were digested by trypsin.Cells at passage 3 in logarithmic phase were collected and frozen,and then recovered.Changes in cell morphology were observed using an inverted microscope.Cell growth curve was measured using MTT assay.The potential of osteogenic differentiation was examined utilizing Von Kossa's staining.RESULTS AND CONCLUSION:The primary cultured BMSCs were cultured with adherent growth.The cells were spindle form.Cell morphology following passage 3 was similar,showing long spindle shape.Following freezing and recovery,cell adherence was slower compared with subculture cells,and no significant difference was detected in cell morphology and viability compared with subculture cells.Growth cycle was similar in passage 3-passage 5 cells.BMSCs entered lag phase at days 2 and 3,logarithmic phase at day 3,and platform phase at days 6 and 7,and then growth speed was slow.Goat BMSCs were positive for Von Kossa's stain at 3 weeks following osteogenic induction.Results verified that cultured goat BMSCs showed strong genetic stability and proliferation ability,and differentiated into osteoblasts.

Objective To explore the effect of lysophosphatidic acid(LPA)on blood-brain barrier(BBB) permeability and its possible mechanism.Methods LPA or LPA+suramin(L+S)were stereotaxically injected into the right eaudate nucleus in SD rats in vivo.Evans blue(EB)was used to quantitatively measure the permeability of BBB at different time points.The expression of matrix metalloproteinase-9 was detected by immunohistochemistry technique.The pathological ultrastruetural changes of BBB were assessed by transmission electron microscopy.Results The BBB permeability began to increase after LPA administered into ipsilateral eaudate nucleus,and reached the peak at 24h.Then the permeability of BBB gradually lowered after 48h.In comparison with the same time points of control group,there were quite significant differences(P＜0.01).After L+S was injected,the change of BBB permeability had differences in comparison with those of LPA group in the same time points,(P＜0.05).MMP-9 positive cells were mainly vascular endothelial cells.The numbers of MMP-9 positive blood vessels grew at 6h in LPA group,and the expression of it reached maximum at 24h,then the number of it decreased at 48h,showing significant statistical differences in comparison with the L+S group(P＜0.01),It was observed microscopically that ultrastrueture of BBB of the LPA group was changed sharply,such as basement membrane roughed and fragmented,astroeyte end-feet swolled markedly and perivaseular space enlarged obviously.But there were no remarkable changes in BBB in L+S group.Conclusion LPA can induce increase of BBB permeability and its possible mechanism is the strong expression of MMP-9 protein produeted by endothelial cells through the mediation of LPA receptor,leading to degradation of basement membrane.

Circulating nitric oxide(NO)and endothelin (ET) levels were determined in 30 normal subjects and 63 diabetic patients.The results showed that:serum NO level was significantly higher in DM I group(68.66?12.37?mol/L)and DM Ⅱ group(63.43?11.09?mol/L)than in normal subjects(44. 92?9.04?mol/L,P0.05).Plasma ET level was markedly elevated in DM Ⅰ group(68.92?11.96ng/L,P