Infection of MDM by HIV-1 PV.

<p>Monocytes were isolated from the peripheral blood of healthy donors and culture for 7 days in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and M-CSF (250 ng/ml) to allow the cell differentiation. The MDM cells were infected with HIV-1 PV at a multiplicity of infection (MOI) of ∼3 in the presence of 10 ng/ml of polybrene for 4 hr and cultured for 7 more days. MDM from the same donor treated with polybrene at 10 ng/ml and mock infected with the supernatant of HEK 293T/17 cells transfected with SG3ΔEnv only were used as control. The cells were analyzed with immunofluorescence staining (<b>A</b>) and Western blotting (<b>C</b>) for the viral p24 protein expression, and by HIV DNA PCR (<b>B</b>) for the presence of integrated proviral DNA.</p

HIV-1 PV infection suppresses expression of TRAIL decoy receptors and c-FLIP.

<p>The virus-infected MDM were collected and stained with PE-labeled anti- DR4, -DR5, -DcR1, -DcR2 antibody and the isotype control, respectively, and followed by flow cytometry. <b>A</b>, representative analysis of TRAIL receptor expression on mock- or virus-infected MDM, Open histograms with solid lines, mock-infected MDM stained with PE-labeled anti-TRAIL receptor mAbs; Open histograms with dashed lines, HIV-1 PV infected MDM stained with PE-labeled anti-TRAIL receptor mAbs; shaded histograms, mock-infected MDM stained with PE-labeled isotype control. <b>B</b>, statistical results from 12 samples. The MFI (mean fluorescent intensities) of TRAIL receptor was assessed by flow cytometry and analyzed by a two tailed, two sample student <i>t</i> test (<i>P</i> values were indicated). <b>C</b>, lentivirus-mediated over-expression of DR5-delta, DcR1 and DcR2 on MDM rescued rsTRAIL- or AD5-10-induced cell death. MDM cells were infected with lentivirus vectors expressing DR5-delta, DcR1 or DcR2, respectively. HIV-1 PV-infected MDM cells were treated with 500 ng/ml of rsTRAIL or 500 ng/ml of AD5-10 for 12 hr. The cell viability was examined by MTS assay. Data are representative of three independent experiments. <b>D</b>, expressions of Bcl-2 family proteins and c-FLIP in HIV-1 PV-infected MDM were analyzed by Western blot assay. Data is presented as mean ± S.D. of 12 independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01 compared with mock or HIV-1 PV.</p

RsTRAIL and AD5-10 induced caspase-dependent and -independent cell death.

<p><b>A-C</b>, Western blot analysis of caspase activation. HIV-1 PV infected MDM cells were treated with either 250 ng/ml rsTRAIL (A) or 250 ng/ml AD5-10 (B) or 250 ng/ml rsTRAIL plus 250 ng/ml AD5-10 (C) for 2, 4, 6 and 8 hr. The cells were lysed and the lysates were subjected to SDS-PAGE and immunoblotting with anti-caspase -3, -8, or -9 antibody. <b>D</b>–<b>E</b>, cell viability determined by MTS. HIV-1 PV infected MDM were treated with 500 ng/ml rsTRAIL (D) or 500 ng/ml AD5-10 (E) or 250 ng/ml rsTRAIL plus 250 ng/ml AD5-10 (F) in the absence or presence of 50 µM of Z-VAD-fmk for 12 hr. Values are mean of three independent experiments with error bar representing standard deviation of the mean. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; <i>ns</i>, no significant.</p

rsTRAIL and AD5-10 synergetically induces apoptosis in HIV-1 PV-infected MDM.

<p>HIV-1 PV-infected MDM cells were treated with increasing concentration of rsTRAIL (<b>A</b>) or AD5-10 (<b>B</b>). The cell viability was determined by MTS assay. <b>C and D</b>, the virus-infected MDM cells were treated either with 500 ng/ml of rsTRAIL or 500 ng/ml of AD5-10 for 3, 6, 12, and 24 hr. The virus-infected MDM cells were overnight incubated with or without 5 ng/ml of AD5-10 and rsTRAIL at indicated concentration (<b>E</b>), or 5 ng/ml of rsTRAIL and AD5-10 at indicated concentration (<b>F</b>). <b>G and H</b>, the virus-infected MDM cells were incubated with 500 ng/ml rsTRAIL and/or 500 ng/ml AD5-10 for 6 hr, and the apoptotic nucleosome was detected by Cell Death Detection ELISA kit. Values are represented results of three independent experiments with error bar representing standard deviation of the mean. *, <i>p</i><0.05; ***, <i>p</i><0.001; <i>ns</i>, no significant.</p

ROS generation and JNK phosphorylation involves in AD5-10 induced cell death.

<p><b>A</b>, HIV-1 PV- or mock-infected MDM cells were treated with 200 ng/ml rsTRAIL (left) or 200 ng/ml AD5-10 (middle) or 200 ng/ml rsTRAIL plus 200 ng/ml AD5-10 (right) for 15, 30, 60, 120, 240 min. ROS generation was examined by loading with DCFH and followed by flow cytometry. <b>B</b>, JNK phosphorylation was examined with indicated specific antibody (1∶1000 dilution, phosphor-SAPK/JNK(Thr183/Tyr185) and JNK antibody) in HIV-1 PV-infected MDM cells (DPI = 7) treated with 500 ng/ml rsTRAIL (bottom) or 500 ng/ml AD5-10 (middle) or 250 ng/ml rsTRAIL plus 250 ng/ml (top) for indicated time. Data was obtained from three independent assays and a typical experiment is presented.</p

Comparison of genetic diversity of <i>L</i><i>. dispar</i> in China.

<p>PP% is the percentage of polymorphic loci, Ne is the mean effective number of alleles, Na is the mean observed number of alleles, I is the mean Shannon’s information index and H is the mean Nei’s gene diversity.</p

Locations of 8 <i>L</i><i>. dispar</i> sampling sites in China.

<p>Locations of 8 <i>L</i><i>. dispar</i> sampling sites in China.</p

Molecular Determinants Conferring the Stoichiometric-Dependent Activity of α‑Conotoxins at the Human α9α10 Nicotinic Acetylcholine Receptor Subtype

α9α10 nicotinic acetylcholine receptors (nAChRs) putatively exist at different stoichiometries. We systematically investigated the molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR. Our results suggest that only Vc1.1 exhibits stoichiometric-dependent inhibition at the α9α10 nAChR. The hydrogen bond between N154 of α9 and D11 of Vc1.1 at the α9­(+)-α9(−) interface is responsible for the stoichiometric-dependent potency of Vc1.1

Immunofluorescent staining of TRPV1 and analysis of its expression pattern in DRG neurons at varying time points after the establishment of DMA.

<p><b>A–E</b>, representative photographs of TRPV1 staining in DRG of different groups. <b>F</b>, illustration of the percentage of TRPV1-IR neurons over total neurons in vehicle, DMA 7 d, 14 d, 21 d and 28 d group (n = 4–6). <b>G</b>, histogram for size distribution of TRPV1-IR neurons. Note the decreased distribution of TRPV1 in neurons with cross-sectional area larger than 400–500 and increased distribution of this protein within the range of 100–400 µm² (n = 4–6). <b>H</b>, comparison of the optical density due to TRPV1 immunostaining in small-sized (100–500 µm²) and medium-sized (500–1200 µm²) DRG neurons of different groups (n = 4–6). *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle control. Scale bar  = 100 µm.</p

Immunofluorescent staining of TRPV1 in plantar skin of hind paw with the progression of DMA.

<p>A–E, typical photograsph of TRPV1 immunoreactivities in vehicle and DMA model rats at varied time points. A′–C′, the magnified pictures from the rectangle areas in A–C. F, optical density analysis of TRPV1 immunoreactivities in epidermis and dermis displaying strong enhancement on DMA 7 d and 14 d. Sc, Ep and De are the abbreviation of stratum corneum, epidermis and dermis, respectively. Scale bar  = 20 µm</p