This paper presents the results of the histochemical study on the activities ofATPase and succinic dehydrogenase and their distribution in mammary gland cancercell of mice by means of electron microscopy.It was found that the activity of ATPase activated by Mg~(++)were mainly locatedon the mitochondrial membrane,the nuclear membrane and the nucleolus,and that ofATPase activated by Ca~(++)were located on the cristae and matrix of mitochondria aswell as the outer nuclear membrane,and also evenly distributed in the chromatin.The activity of the succinic dehydrogenase is mainly located on the cristae andthe inner membrane of mitochondria.An alternitive responses of strong and weakactivities of the enzyme were shown between the outer and inner nuclear membranes.In the mean-while,the activity of the succinic dehydrogenase was also shown in ahigh level in the outer cytoplasmic membrane and the inner nuclear membrane.

Objective To optimize the extraction technology of Taxus x media by using the contents of Paclitaxel and 10-deacetylbaccatin(10-DAB),two representative active diterpene alkaloids of taxane type from T.x media,as evaluation standard.Methods The smashing tissue extraction(STE)of Paclitaxel and 10-DAB from T.x media,was investigated by comparing with ultrasonic extraction(UE)which was one of the modern technologies of extraction.Results STE was more efficient than UE,and the contents of 10-DAI3 and Paclitaxel in the extracts obtained by STE were higher than those by UE.Conclusion STE is a fast,high-performance,and energy-saving technology for the extraction of diterpene alkaloids of taxane type.STE also provides a simple,component-safe,workable,and highly efficient method for the extraction of active natural product.

Objective To detection the methylation of p16INK4A in primary hepatocellular carcinoma, a nested bisulfite sequencing and methylation-specific polymerase chain reaction (BS-MSP) protocol was designed and used.Methods Bisulfite-modified DNA were amplified to evaluate the quality of templates with a pair of bisulfite sequencing primers in the first round of PCR, then subjected to methylation assay with corresponding methylation or unmethylation specific PCR primers.Representative PCR products were sequenced to confirm its correctness.Results 3 of 40 cases (7.5%) were failed to assay due to poor quality of templates, and 29 of 37 cases (78%) were detected p16INK4A methylation.Sequencing results confirmed that templates were correctly amplified.Conclusion BS-MSP technique might be valuable for methylation study on carcinogenesis and clinical assay.

Objective To study the preparation of hepatitis C viruses (HCV) genotyping oligochip and its application in the detection of 76 hepatitis C patients.Methods Oligonucleotide probes and primers were designed in the 5’noncoding region and core region of HCV. The HCV typing chip was prepared by spotting the modified probes onto nylon membrane. Products of the second PCR were labeled with Dig-dUTP. Furthermore, 6 PCR products were sequenced.Results Using the chip,15 subtypes in 11 types of HCV were analyzed.Results of hybridization indicates that 76 hepatitis C patients were all positive and 20 health people were negative.Among 76 patients, 64 cases were 1b type, 11 cases were 2a type and 1 case was 3a type. Mix infection was not found. The results obtained by sequencing 6 samples and chip arraying were the same.Conclusion The HCV genotyping chip could be used in detecting serum HCV RNA and analyzing its genotypes.

ObjectiveTo establish a method for content determination of total iridoid compounds and baldrinal and 11-ethoxyviburtinal from Valerianae Jatamansi Rhizoma et Radix; To determine the contents of total iridoid compounds and baldrinal and 11-ethoxyviburtinal in Valerianae Jatamansi Rhizoma et Radix from three medicinal origins.Methods UV spectrophotometry was applied, 11-ethoxyviburtinal (cyclopentane-pyran-7-formaldehyde, 4-ethoxy methyl) was set as the reference substance, and the content of total iridoid compounds was determined at 288 nm. HPLC method was used to simultaneously determine the contents of baldrinal and 11-ethoxyviburtinal. The HPLC analysis was performed on a Phenomenex Luna C18 column (250 mm×4.6 mm, 5μm). The mobile phase was composed of acetonitrile-water in gradient elution at a flow rate of 0.95 mL/min. The detection wavelength was 288 nm and the column temperature was 30℃.Results The total iridoid compounds, baldrinal and 11-ethoxyviburtinal were in good linearity within the ranges of 2.088–14.616μg/μL, 74.88–224.64μg, and 41.6–249.6μg, respectively. This method was precise, and with good repeatability, stability and recovery rate.Conclusion The method is accurate, simple, rapid, which can be used for the quality control of Valerianae Jatamansi Rhizoma et Radix.

OBJECTIVETo establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.

METHODSFollowing bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.

RESULTSThe amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.

CONCLUSIONSYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.

Actins ; genetics ; Biopsy ; Calibration ; Carcinoma, Hepatocellular ; genetics ; pathology ; DNA Methylation ; Feasibility Studies ; Female ; Fluorescence ; Genes, p16 ; Humans ; Luminescent Measurements ; Male ; Middle Aged ; Nucleic Acid Denaturation ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Transition Temperature

Objective:To determine the relationship between uric acid (UA) and mild cognitive impairment (MCI), and its potential effect on inflammation.Methods:450 patients with MCI diagnosed by neuropsychological scale and 450 controls with normal cognitive function were included. All subjects were≥60 years old. There were 184 obese subjects in MCI group and 199 obese subjects in control group.Results:A correlation between increased serum UA level and decreased risk of MCI was found in all MCI patients and non-obese MCI patients ( OR: 0.60, 95% CI 0.45-0.78; OR: 0.42, 95% CI 0.29-0.62), but not in obese MCI patients ( OR: 0.86, 95% CI: 0.54-1.35). The levels of UA and hypersensitive C reactive protein (hs-CRP) in obese patients with MCI were higher than those in non-obese patients ( P<0.01). There was a linear positive correlation between serum UA and hs-CRP levels in obese patients with MCI ( r=0.505, P<0.01), but not in non-obese MCI patients ( r=0.053, P=0.385). Conclusion:A significant correlation between lower serum uric acid levels and higher risk of MCI in non-obese subjects was found. Inflammation caused by obesity may weaken this relationship.

Objective:To identify the characteristics of nailfold capillaroscopy (NFC) of systemic sclerosis (SSc) and investigate whether more severe peripheral microangiopathy at NFC were related to the development of SSc.Methods:① The study included 115 patients (60 cases with SSc and 55 patients with other connective tissue diseases). All patients were treated with neither prednisone nor immunosuppressive drugs within 3 months before enrollment. We collected the following data: age, disease duration, disease onset, mRSS, high-resolution chest tomography (HRCT), echocardiography, pulmonary function, nailfold capillaroscopy and routine laboratory assessments. ② All the NFC definitions were used for semi-quantitatively scoring and Cutolo's qualitative assessment. ③ The relationship between NFC changes and joint, visceral involvement and autoantibodies in SSc patients was analyzed. ④ T test, Rank sum test and chi-square test were applied to analyze data. Results:① According to Cutoloqualitative assessment of NFC, patients of SSc with active/late pattern ( n=52) were very common than other CTD ( n=21) ( Z=-3.853, P<0.01). ② According to semiquantitative assessment, the scores of loss of capillaries [(1.67±0.60) vs (0.72±0.46), t=8.347, P<0.01)], irregular enlarged capillaries [(1.22±0.88) vs (0.74±0.50), t=3.178, P<0.01)], hemorrhage [(0.30±0.39) vs (0.10±0.21), t=3.090, P<0.01)], disorganization of the microvascular array [(0.38±0.38) vs (0.18±0.32), t=2.729, P<0.01)] were significantly higher than CTD. ③ The NFC of SSc patients was significantly different from CTD. The number of capillary loss ( Z=-4.194, P<0.01), input capillary dimensions ( t=3.704, P<0.01), output capillary dimensions ( t=3.913, P<0.01), wide diameter of capillary ( t=4.586, P<0.01), tortuous capillaries ( Z=-2.677, P<0.01), gaint capillary ( χ2=8.040, P=0.013), effusion ( Z=-2.278, P=0.023) were more increased than CTD. ④ The NFC pattern of SSc with lung involvement were mainly active and late (66%, 33/50), whereas early and active pattern (60%, 6/10) for those without respiratory system involvement ( Z=10.114, P=0.045) . The NFC pattern of SSc patients with joint involvement were mainly active and late (75%, 12/16), whereas early and active (66%, 29/44) for those without joint involvement ( Z=5.550, P=0.057) . Conclusion:The NFC of SSc patients is significantly different from CTD. NFC may be a suitable tool for disease evaluation.

OBJECTIVETo understand HIV rival suppression and drug resistance (HIVDR) among AIDS patients who were receiving antiretroviral treatment (ART) in Dehong prefecture, Yunnan province.

METHODSAll AIDS patients who were aged over 15 years and with experience more than six months on ART by the end of 2012 in Dehong prefecture, were enrolled to receive testing for HIV viral load in plasma and genetic mutations associated with HIVDR.

RESULTSA total of 4 390 AIDS patients were qualified for the study according to the selection criteria, of whom 3 964 (90.3%) finally participated in the study. Among them, 2 307(58.2%) had CD₄(+) cell counts more than 350 cells/mm³. 3 169 (79.9%) patients showed undetectable plasma HIV viral load which was lower than the detection threshold. Those who had the following factors as:resided in Ruili city, being female, older than 45 years of age, married, heterosexually infected with HIV, having received ART more than 5 years, and CD₄(+) cell counts >500 cells/mm³, were more likely to have undetectable plasma virus load, with the differences statistically significant. 402 (10.1%) patients had plasma viral load ≥ 1 000 copies/ml, of whom 353 (87.8%) were successfully amplified and examined for HIVDR. Among them, 198 (56.1% ) were identified to bear genetic mutations associated with HIVDR. Most mutations were related to the resistance to nucleotide reverse transcriptase inhibitors (NNRTIs) or non-nucleotide reverse transcriptase inhibitors (NNRTIs), with M184V and K103N most frequently seen. 12 patients (3.4%) were found to have mutations resistant to protease inhibitors (PI). Data from multiple logistic regression analysis indicated that the period of receiving ART and the initial ART regimen could both significantly predict the occurrence of HIV resistance.

CONCLUSIONViral suppression was highly achieved among ART-prescribed AIDS patients in Dehong prefecture,Yunnan province. However, among those who did not show effective viral suppression, the proportion of HIVDR was high, underscoring the needs for health education so as to improve the adherence to drugs as well as for improving testing for viral load and HIVDR among AIDS patients.

Acquired Immunodeficiency Syndrome ; drug therapy ; Adolescent ; Adult ; Anti-HIV Agents ; therapeutic use ; Drug Resistance, Viral ; genetics ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Mutation ; Viral Load ; drug effects ; Young Adult

As known, the benefits of photothermal therapy (PTT) are greatly limited by the heat tolerance of cancer cells resulting from overexpressed heat shock proteins (HSPs). Then HSPs further trigger the formation of stress granules (SGs) that regulate protein expression and cell viability under various stress conditions. Inhibition of SG formation can sensitize tumor cells to PTT. Herein, we developed PEGylated pH (low) insertion peptide (PEG-pHLIP)-modified hollow copper sulfide nanoparticles (HCuS NPs) encapsulating the SG inhibitor ISRIB, with the phase-change material lauric acid (LA) as a gate-keeper, to construct a pH-driven and NIR photo-responsive controlled smart drug delivery system (IL@H-PP). The nanomedicine could specifically target slightly acidic tumor sites. Upon irradiation, IL@H-PP realized PTT, and the light-controlled release of ISRIB could effectively inhibit the formation of PTT-induced SG to sensitize tumor cells to PTT, thereby increasing the antitumor effect and inducing potent immunogenic cell death (ICD). Moreover, IL@H-PP could promote the production of reactive oxygen species (ROS) by tumor-associated macrophages (TAMs), repolarizing them towards the M1 phenotype and remodeling the immunosuppressive microenvironment. In vitro/vivo results revealed the potential of PTT combined with SG inhibitors, which provides a new paradigm for antitumor and anti-metastases.