Efficacy Analysis of Combinatorial siRNAs against HIV Derived from One Double Hairpin RNA Precursor

Combinatorial small interfering RNA duplexes (siRNAs) have the potential to be a gene therapy against HIV-1, and some studies have reported that transient combinatorial siRNA expression represses HIV replication, but the effects of long-term siRNA expression on HIV replication have not been studied in detail. In this study, HIV-1 replication under the influence of stable combinatorial siRNA expression from a single RNA transcript was analyzed. First, a series of cassettes encoding short hairpin RNA (shRNA)/long hairpin RNA (lhRNA)/double long hairpins (dlhRNA) was constructed and subjected to an analysis of inhibitory efficacy. Next, an optimized dlhRNA encoding cassette was selected and inserted into lentiviral delivery vector FG12. Transient dlhRNA expression reduced replication of HIV-1 in TZM-bl cells and CD4+ T cells successfully. HIV-1 susceptible TZM-bl cells were transducted with the dlhRNA expressing lentiviral vector and sorted by fluorescence-activated cell sorting to obtain stable dlhRNA expressing cells. The generation of four anti-HIV siRNAs in these dlhRNA expressing cells was verified by stem–loop RT-PCR assay. dlhRNA expression did not activate a non-specific interferon response. The dlhRNA expressing cells were also challenged with HIV-1 NL4-3, which revealed that stable expression of combinatorial siRNAs repressed HIV-1 replication for 8 days, after which HIV-1 overcame the inhibitory effect of siRNA expression by expressing mutant versions of RNAi targets. The results of this evaluation of the long-term inhibitory effects of combinatorial siRNAs against HIV-1 provide a reference for researchers who utilize combinatorial RNA interference against HIV-1 or other error-prone viruses

Synthesis and in Vitro Antiproliferative Evaluation of Some B-norcholesteryl Benzimidazole and Benzothiazole Derivatives

Taking orostanal (a compound from a Japanese marine sponge, Stelletta hiwasaensis) as a lead compound, some novel B-norcholesteryl benzimidazole and benzothiazole derivatives were synthesized. The antiproliferative activity of the compounds against human cervical carcinoma (HeLa), human lung carcinoma (A549), human liver carcinoma cells (HEPG2) and normal kidney epithelial cells (HEK293T) was assayed. The results revealed that the benzimidazole group was a better substituent than benzothiazole group for increasing the antiproliferative activity of compounds. 2-(3β′-Acetoxy-5β′-hydroxy-6′-B-norcholesteryl)benzimidazole (9b) with the structure of 6-benzimidazole displays the best antiproliferative activity to the cancer cells in all compounds, but is almost inactive to normal kidney epithelial cells (HEK293T). The assay of compound 9b to cancer cell apoptosis by flow cytometry showed that the compound was able to effectively induce cancer cell apoptosis. The research provided a theoretical reference for the exploration of new anti-cancer agents and may be useful for the design of novel chemotherapeutic drugs

Porphyromonas gingivalis infection reduces regulatory T cells in infected atherosclerosis patients.

Increasing evidence has shown periodontal pathogen Porphyromonas gingivalis (P.gingivalis) infection contributes to atherosclerosis (AS) progression. P.gingivalis fimbriae act as an important virulence factor in AS. Regulatory T cells (Tregs) may play a crucial role in autoimmune response during this process. However, whether P.gingivalis infection is associated with Tregs dysregulation during AS is still unknown and the prevalence of different P.gingivalis FimA genotypes during this process is unclear. Here we analyzed the distribution of Tregs and in P.gingivalis-infected atherosclerotic patients to reveal the relationship between P.gingivalis infection and Tregs reduction/dysfunction and to elucidate their role in periodontitis-AS interaction. FimA genotype was also examined to determine the prevalence of fimbriae. Our results showed that P.gingivalis infection reduced Tregs in atherosclerotic patients compared with non-atherosclerotic patients and health controls. Concentration of TGF-β1, which plays an important role in the development of Tregs, also decreased in P.gingivalis infected patients. Furthermore, type II FimA seems to show higher prevalence than the other five detected types. The population of Tregs further decreased in patients with type II FimA compared with the other types. P.gingivlias FimA genotype II was the dominant type associated with decreased Treg population. These results indicate that P.gingivalis infection may be associated with Tregs dysregulation in AS; type II FimA may be a predominant genotype in this process

Soil Respiration at Different Stand Ages (5, 10, and 20/30 Years) in Coniferous (Pinus tabulaeformis Carrière) and Deciduous (Populus davidiana Dode) Plantations in a Sandstorm Source Area

Understanding the effects of stand age and forest type on soil respiration is crucial for predicting the potential of soil carbon sequestration. Thus far, however, there is no consensus regarding the variations in soil respiration caused by stand age and forest type. This study investigated soil respiration and its temperature sensitivity at three stand ages (5, 10, and 20 or 30 years) in two plantations of coniferous (Pinus tabulaeformis Carrière) and deciduous (Populus davidiana Dode) species using an automated chamber system in 2013 in the Beijing-Tianjin sandstorm source area. Results showed that mean soil respiration in the 5-, 10-, and 20/30-year-old plantations was 3.37, 3.17, and 2.99 μmol·m−2·s−1 for P. tabulaeformis and 2.92, 2.85, and 2.57 μmol·m−2·s−1 for P. davidiana, respectively. Soil respiration decreased with stand age for both species. There was no significant difference in soil respiration between the two plantation species at ages 5 and 10 years (p > 0.05). Temperature sensitivity of soil respiration, which ranged from 1.85–1.99 in P. tabulaeformis and 2.20–2.46 in P. davidiana plantations, was found to increase with stand age. Temperature sensitivity was also significantly higher in P. davidiana plantations and when the soil water content was below 12.8%. Temperature sensitivity incorporated a combined response of soil respiration to soil temperature, soil water content, soil organic carbon, and fine root biomass and, thus, provided an ecological metric for comparing forest carbon dynamics of these species

The percentage of CD4⁺FOXP3⁺ T cells in Pg-AS patients, Pg patients, and HCs.

<p>A. Representative profiles of Pg-AS, Pg and HC for detecting CD4⁺FOXP3⁺ T cells by flow cytometry within the CD4⁺ gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 236A/E7) and Fixation/Permeabilization buffer (no. 00-5521) from eBioscience. B. Representative profiles of Pg-AS, Pg and HC for detecting CD4⁺FOXP3⁺ T cells by flow cytometry within the CD4⁺ gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 259D/C7) and fixation permeabilization reagents (no. 560098) from BD Bioscience. C. The percentage of CD4⁺FOXP3⁺ T cells stained with eBioscience reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. D. The percentage of CD4⁺FOXP3⁺ T cells stained with BD reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. Boxes with the 25th to 75th percentiles and the lines with the 5th to 95th percentiles are presented.*<i>P</i><0.0167. HC = health control; Pg = <i>P.gingivalis</i> infected patients; Pg-AS = <i>P.gingivalis</i> infected atherosclerosis patients.</p

Distribution of 6 FimA types in Pg-AS and Pg patients.

<p>Pg = <i>P.gingivalis</i> infected patients; Pg-AS = <i>P.gingivalis</i> infected atherosclerosis patients.</p

Demographic characteristics and clinical parameter of the study population.

<p>Continuous variables expressed as mean ± standard deviation, percentage or number. *<i>P</i><0.01 Pg and Pg-AS vs. control; ^#<i>P</i><0.01 Pg-AS vs. Pg. HC = health control; Pg = <i>P.gingivalis</i> infected patients; Pg-AS = <i>P.gingivalis</i> infected atherosclerosis patients.</p

Plasma TGF-β1 concentration in Pg-AS patients, Pg patients, and HCs.

<p>A. Plasma TGF-β1 concentrations in HC (n = 29), Pg (n = 32), Pg-AS (n = 40) groups were presented. Boxes with the 25th to 75th percentiles and the lines with the 5th to 95th percentiles are shown.*<i>P</i><0.0167. B. Spearman correlation between frequencies of circulating TGF-β1 and CD4⁺CD25⁺FOXP3⁺/CD4⁺ T cells in these groups (n = 101). HC = health control; Pg = <i>P.gingivalis</i> infected patients; Pg-AS = <i>P.gingivalis</i> infected atherosclerosis patients.</p