GPS-SUMO 2.0: an updated online service for the prediction of SUMOylation sites and SUMO-interacting motifs.

Small ubiquitin-like modifiers (SUMOs) are tiny but important protein regulators involved in orchestrating a broad spectrum of biological processes, either by covalently modifying protein substrates or by noncovalently interacting with other proteins. Here, we report an updated server, GPS-SUMO 2.0, for the prediction of SUMOylation sites and SUMO-interacting motifs (SIMs). For predictor training, we adopted three machine learning algorithms, penalized logistic regression (PLR), a deep neural network (DNN), and a transformer, and used 52 404 nonredundant SUMOylation sites in 8262 proteins and 163 SIMs in 102 proteins. To further increase the accuracy of predicting SUMOylation sites, a pretraining model was first constructed using 145 545 protein lysine modification sites, followed by transfer learning to fine-tune the model. GPS-SUMO 2.0 exhibited greater accuracy in predicting SUMOylation sites than did other existing tools. For users, one or multiple protein sequences or identifiers can be input, and the prediction results are shown in a tabular list. In addition to the basic statistics, we integrated knowledge from 35 public resources to annotate SUMOylation sites or SIMs. The GPS-SUMO 2.0 server is freely available at https://sumo.biocuckoo.cn/. We believe that GPS-SUMO 2.0 can serve as a useful tool for further analysis of SUMOylation and SUMO interactions.

Large-language models facilitate discovery of the molecular signatures regulating sleep and activity.

Sleep, locomotor and social activities are essential animal behaviors, but their reciprocal relationships and underlying mechanisms remain poorly understood. Here, we elicit information from a cutting-edge large-language model (LLM), generative pre-trained transformer (GPT) 3.5, which interprets 10.2-13.8% of Drosophila genes known to regulate the 3 behaviors. We develop an instrument for simultaneous video tracking of multiple moving objects, and conduct a genome-wide screen. We have identified 758 fly genes that regulate sleep and activities, including mre11 which regulates sleep only in the presence of conspecifics, and NELF-B which regulates sleep regardless of whether conspecifics are present. Based on LLM-reasoning, an educated signal web is modeled for understanding of potential relationships between its components, presenting comprehensive molecular signatures that control sleep, locomotor and social activities. This LLM-aided strategy may also be helpful for addressing other complex scientific questions.

GPS 6.0: an updated server for prediction of kinase-specific phosphorylation sites in proteins.

Protein phosphorylation, catalyzed by protein kinases (PKs), is one of the most important post-translational modifications (PTMs), and involved in regulating almost all of biological processes. Here, we report an updated server, Group-based Prediction System (GPS) 6.0, for prediction of PK-specific phosphorylation sites (p-sites) in eukaryotes. First, we pre-trained a general model using penalized logistic regression (PLR), deep neural network (DNN), and Light Gradient Boosting Machine (LightGMB) on 490 762 non-redundant p-sites in 71 407 proteins. Then, transfer learning was conducted to obtain 577 PK-specific predictors at the group, family and single PK levels, using a well-curated data set of 30 043 known site-specific kinase-substrate relations in 7041 proteins. Together with the evolutionary information, GPS 6.0 could hierarchically predict PK-specific p-sites for 44046 PKs in 185 species. Besides the basic statistics, we also offered the knowledge from 22 public resources to annotate the prediction results, including the experimental evidence, physical interactions, sequence logos, and p-sites in sequences and 3D structures. The GPS 6.0 server is freely available at https://gps.biocuckoo.cn. We believe that GPS 6.0 could be a highly useful service for further analysis of phosphorylation.

Boosting Ring Strain and Lewis Acidity of Borirane: Synthesis, Reactivity and Density Functional Theory Studies of an Uncoordinated Arylborirane Fused to o-Carborane.

Among the parent borirane, benzoborirene and ortho-dicarbadodecaborane-fused borirane, the latter possesses the highest ring strain and the highest Lewis acidity according to our density functional theory (DFT) studies. The synthesis of this class of compounds is thus considerably challenging. The existing examples require either a strong π-donating group or an extra ligand for B-coordination, which nevertheless suppresses or completely turns off the Lewis acidity. The title compound, which possesses both features, not only allows the 1,2-insertion of P=O, C=O or C≡N to proceed under milder conditions, but also enables the heretofore unknown dearomative 1,4-insertion of Ar-(C=O)- into a B-C bond. The fusion of strained molecular systems to an o-carborane cage shows great promise for boosting both the ring strain and acidity.

Synthesis of Iminoboryl o-Carboranes by Lewis Base Promoted Aminoborirane-to-Iminoborane Isomerization.

The iminoboryl o-carboranes (Me3Si)-Cb-B≡N-R (Cb = B10C2H10, 3a, R = SiMe3; 3b, R = tBu) have been successfully synthesized by tetrahydrofuran (THF)-promoted isomerization from the corresponding o-carborane-fused aminoboriranes Cb{BN(SiMe3)R} (2). The synthetic protocol of the previously reported borirane 2a was optimized. The borirane Cb{BN(SiMe3)tBu} (2b) and the iminoboranes 3a and 3b were fully characterized by NMR, IR, and single-crystal X-ray diffraction analyses. The borirane 2a isomerizes more readily than 2b. The kinetics study revealed a bimolecular mechanism between borirane and THF, which is in good agreement with the computationally proposed reaction pathway. The title compounds are thermally robust, but compound 3a dimerized in the presence of a catalytic amount of tBuNC to give the cyclodimer 4. Quick equilibrium between 4 and the isonitrile adduct 4·tBuNC was observed in solution.

HemI 2.0: an online service for heatmap illustration.

Recent high-throughput omics techniques have produced a large amount of biological data. Visualization of big omics data is essential to answer a wide range of biological problems. As a concise but comprehensive strategy, a heatmap can analyze and visualize high-dimensional and heterogeneous biomolecular expression data in an attractive artwork. In 2014, we developed a stand-alone software package, Heat map Illustrator (HemI 1.0), which implemented three clustering methods and seven distance metrics for heatmap illustration. Here, we significantly improved 1.0 and released the online service of HemI 2.0, in which 7 clustering methods and 22 types of distance metrics were implemented. In HemI 2.0, the clustering results and publication-quality heatmaps can be exported directly. For an in-depth analysis of the data, we further added an option of enrichment analysis for 12 model organisms, with 15 types of functional annotations. The enrichment results can be visualized in five idioms, including bubble chart, bar graph, coxcomb chart, pie chart and word cloud. We anticipate that HemI 2.0 can be a helpful web server for visualization of biomolecular expression data, as well as the additional enrichment analysis. HemI 2.0 is freely available for all users at: https://hemi.biocuckoo.org/.

Dynamic Experimental Study on the Paraffin Deposition Prevention Performance of Tungsten Alloy Coating Pipe in Simulating Vertical Wellbore.

In this study, a self-designed apparatus was used to provide a quantitative evaluation of the wax prevention effect of tungsten alloy-coated tubing compared with ordinary tubing in oil production. The paraffin deposition of both pipes at different temperatures and different flow rates was studied. The efficiency of paraffin deposition prevention of the tungsten alloy coating pipe was analyzed. The results show that using this apparatus can efficiently and accurately calculate the wax prevention rate and can accurately obtain the wax deposit and wax thickness of the inner wall. The paraffin deposition of both pipes reaches the highest point at 290.15 K, and it reduces with the increase of flow rate. The use of the tungsten alloy coating pipe results in about 30% reduction in paraffin deposition. It provides a promising method for the paraffin inhibition to extend the wax removal cycle.

[Immature dendritic cells phagocytosing human spleen cells treated by PUVA present the characteristics of regulatory dendritic cells].

Objective To investigate the effect of psoralen combined with A-band ultraviolet (PUVA)-treated human spleen lymphocytes on the phenotype and function of immature dendritic cells (imDCs). Methods Human peripheral blood mononuclear cells (PBMCs) were isolated and induced to produce DCs by interleukin-4 (IL-4) and recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF). On the sixth day, the imDCs were obtained and stimulated by lipopolysaccharide (LPS). One day later, mature DCs were harvested. Human spleen cells (SPs) were isolated and treated with PUVA to prepare apoptotic PUVA-SPs. Co-culture of imDCs with PUVA-SPs resulted in extracorporeal photochemotheraputic DCs (ecpDCs). Co-culture of imDCs with SPs resulted in SP-DCs. The expressions of CD11c, CD83 and CD86 were detected by flow cytometry. The levels of IL-10 and IL-12 in the supernatants of the above cells were determined by ELISA. Results The early apoptosis rate of PUVA-SPs was (94.21±3.75)%. There was no significant difference in the expressions of CD83 and CD86 between imDCs and ecpDCs. But the positive rates of CD83 and CD86 in ecpDCs were lower than those in DCs. However, the positive rates of CD83 and CD86 in SP-DCs were significantly higher than those of the imDCs. Conclusion The imDCs phagocytosing apoptotic human SPs present phenotype and function of regulatory DCs.

[Immature dendritic cells phagocytosing apoptotic human spleen cells treated with PUVA inhibits the maturation induced by LPS].

Objective To investigate whether lipopolysaccharide (LPS) can induce the maturation of immature dendritic cells (imDCs) which phagocytose apoptotic spleen lymphocytes. Methods Human peripheral blood mononuclear cells (PBMCs) were induced to produce DCs by interleukin 4 (IL-4) and recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF). Human spleen cells (hSPs) were isolated and treated with psoralen combined with ultraviolet A(PUVA) to obtain apoptotic PUVA-hSPs. Co-culture of imDCs with PUVA-hSPs resulted in extracorporeal photochemotherapeutic dendritic cells (ecpDCs). The imDCs and ecpDCs were collected and stimulated by 10 ng/mL LPS for 1 day. The expressions of CD11c, CD83 and CD86 were detected by flow cytometry. The level of IL-10 in the supernatants of the above cells was detected by ELISA. Results There was no significant difference in the expressions of CD83 and CD86 between ImDCs and ecpDCs. However, the positive rates of CD83 and CD86 in the imDCs stimulated by LPS were significantly higher than those in the ecpDCs treated by LPS. The level of IL-10 in imDCs culture supernatant was lower than that in ecpDCs. The level of IL-10 in LPS-stimulated imDCs was lower than that in LPS-stimulated ecpDCs. Conclusion Both imDCs and ecpDCs showed immature phenotype, but ecpDCs can inhibit the maturation of DC induced by LPS.

[Triplet anti-tumor therapy based on thymosin α-1 attenuates incidence of hepatoma and serum alpha-fetoprotein level in rat hepatoma model].

OBJECTIVE: To explore the impact of triple anti-tumor therapy based on thymosin α1 (Tα1) combined with Huaier granule(HG) and sirolimus on the level of serum alpha-fetoprotein (AFP) in rat models of liver cancer. METHODS: Ninety Sprague-Dawley rats were randomly divided into triple anti-tumor therapy group, Tα1 group, HG group, sirolimus group, positive control and blank control groups, with 15 rats in each group. Except the blank control group, the rats in the other groups were induced using diethylnitrosamine (DEN) to establish liver cancer models. After DEN treatment, the triple therapy group underwent 0.8 mg/kg Tα1 subcutaneous injection (from once a day for two weeks to twice a week since the third week), 0.35 g/kg HG gavage (three times a day) and 1 mg/kg sirolimus gavage (once a day). The dose of the rest single drug groups were the same with that of the triple therapy group. The positive control and blank control groups were not treated with the drugs. The treatment lasted 20 weeks. Then, the behavior of the rats were observed at the different time points, and the level of serum AFP in the rats were detected at 6, 16, 18, 20 weeks, respectively. RESULTS: The typical symptoms of liver cancer were seen in the DEN-induced rats at 16 weeks. Since the tenth week, 6 rats died one after another. Pathological section of rat liver tissue suggested that the rat models were established successfully. According to the incidence rate of liver cancer and the survival rate at 20 weeks, the triple anti-tumor therapy was significantly superior to the single drug treatments. In addition, the triple anti-tumor therapy significantly reduced the level of serum AFP in the rats. CONCLUSION: The triple anti-tumor therapy can significantly prolong the survival time of rats with liver cancer, decrease the cancer incidence rate and the level of serum AFP.

[Expression of T lymphocyte cytokines in peripheral blood of renal transplant recipients].

OBJECTIVE: To evaluate the clinical values of T-lymphocyte cytokines in renal transplant acute rejection. METHODS: A total of 31 recipients with renal transplantation and 15 healthy volunteers from January 2010 to January 2012 were enrolled and divided into acute rejection group (n = 11) and stable renal allograft function group (n = 20) according to the inclusion criteria. Peripheral blood was collected from the patients before transplantation, 1, 7, 14, 28 day after transplantation and acute rejection onset. Cytometric bead array (CBA) was used to monitor the levels of interleukin-17 (IL-17), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin(IL)-10, IL-6, IL-4 and IL-2. The associations of the changes and levels of cytokines in 3 groups were examined with Pearson correlation analysis. RESULTS: The levels of IL-17A, TNF, IL-10 and IL-2 in recipients before transplantation were (3.40 ± 1.29) , (1.79 ± 0.53) , (2.73 ± 0.65) and (1.79 ± 1.02) ng/L respectively and decreased significantly compared to healthy volunteers ((4.52 ± 2.01), (3.36 ± 1.09) , (3.91 ± 0.42) , (3.12 ± 1.07) ng/L respectively, all P < 0.05). However the levels of IFN-γ, IL-6 and IL-4 showed no significant changes between two groups (all P > 0.05). In acute rejection group after transplantation, the levels of IL-17A, IFN-γ, IL-10 and IL-6 were (9.47 ± 4.75) , (5.01 ± 2.23) , (12.20 ± 5.79) , (6.55 ± 3.45) ng/L respectively and increased significantly compared to the renal allograft function group ((4.39 ± 1.26), (2.90 ± 0.87),(5.68 ± 2.25) and (2.10 ± 0.70) ng/L respectively, all P < 0.05); the level of IL-17A was correlated with those of IFN-γ and IL-4 (Pearson r = 0.519, 0.395, both P < 0.01), the level of IFN-γ was correlated with those of IL-4 and IL-2 (r = 0.276, 0.335, all P < 0.05) , the level of TNF was correlated with that of IL-4 (r = 0.423, P < 0.05) and the level of IL-10 was correlated with that of IL-6 (r = 0.361, P < 0.05). CONCLUSIONS: Cytokines play an important role in acute rejection of renal transplant. And further understanding of its mechanism may provide experimental and preventive rationales.

[The modified extracorporeal photochemotherapy promotes apoptosis of spleen lymphocytes].

OBJECTIVE: To explore the efficacy of the modified extracorporeal photochemotherapy (ECP) in improving the apoptotic rate of lymphocytes in vitro. METHODS: The spleens which were obtained from liver transplantation donor under aseptic condition were used as experimental materials. Splenic lymphocytes (SPs) suspensions were prepared by modified and traditional ECP method, respectively. And then the isolated SPs were treated by the irradiation of 8-methoxypsoralen (8-MOP) combined with ultraviolet A (UVA) named PUVA, 8-MOP and UVA, and compared with a blank group meanwhile. The treated SPs were cultured overnight in an incubator at 37 Degrees Celsius, in a humidified atmosphere of 50 mL/L CO2 for 6-8 hours. The morphological changes of cells were observed using an inverted microscope, the apoptotic rates of SPs were detected by flow cytometry, and the difference between groups was analyzed finally. RESULTS: The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the modified ECP method were respectively (95.33±3.03)% and (97.10±2.12)% after treated by PUVA, (23.39±4.55)% and (36.32±6.63)% after treated by 8-MOP, and (66.98±3.60)% and (68.65±4.35)% by UVA. Compared with control group (12.82±1.86% and 13.4±2.65%), there were statistically significant differences (P<0.01). The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the traditional ECP method were respectively (79.73±4.21)% and (82.70±4.13)%, (61.42±2.28)% and (68.91±2.18)%, (19.30±1.78)% and (28.06±1.88)%, (10.84±0.98)% and (12.77±1.22)%, and the statistical comparisons between groups also had significant difference (P<0.01). In addition, there was a significant difference in the early and total apoptosis between the modified and traditional ECP (P<0.01), but no obvious variation in the end-stage apoptosis in the two groups (P>0.05). CONCLUSION: The modified ECP method can promote apoptosis of SPs in vitro conveniently, safely and efficiently, especially in the early stage. This can lay a foundation for the further study on dendritic cell immunomodulation induced by ECP method.

[Frequency analyses of human cytomegalovirus specific CD8(+) T cell in kidney transplantation].

OBJECTIVE: To establish a new detection method for cytomegalovirus (CMV) specific cytotoxic CD8(+)T cells and examine its proportion and significance in peripheral blood from kidney transplant recipients. METHODS: A total of 30 recipients of kidney transplantation from January 2009 to December 2010 for the first time were enrolled. And 10 healthy volunteers were selected as health control group. Tetramer technology was applied. The proportions of CMV antigen specific T cells expressed in each group were detected directly by flow cytometry. RESULTS: The positive rate of CMV antigen specific CTL, CMV-pp65 specific CD8(+)T cell was between 0.16%-7.21% in kidney transplant recipients (n = 30) and health control group (n = 10). The proportions of CMV antigen specific CTL were 2.95% ± 0.62% in CMV+ group. And it was significantly higher than that in CMV-group (1.17% ± 0.45%) and health control group (0.65% ± 0.17%) (P = 0.003,0.006). In CMV+ group, the proportion of CMV antigen specific CTL was 2.95% ± 0.62% in CMV-pp65 positive phase and decreased significantly to 1.50% ± 0.32% after turning into negative phase. In CMV+ group (n = 15), the proportion of CMV antigen specific CTL was positively related with the number of CMV-pp65 positive cells (Pearson test, r = 0.871, P < 0.01). CONCLUSIONS: The proportion of specific CTL is an important guide for evaluating and judging the prognosis of CMV infection. And it may provide rationales for future targeted therapy in kidney transplant recipients.

HLA-G expression in the peripheral blood of live kidney transplant recipients.

BACKGROUND: The human leukocyte antigen-G (HLA-G) has been considered to be an important tolerogeneic molecule playing an essential role in maternal-fetal tolerance, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. The aim of this study was to investigate the clinical relevance of the expression of membrane HLA-G (mHLA-G), intracellular HLA-G (iHLA-G), and soluble HLA-G (sHLA-G) in the peripheral blood of live kidney transplant recipients. METHODS: We compared the expression of the three HLA-G isoforms in three groups, healthy donors (n=20), recipients with acute rejection (n=19), and functioning transplants (n=30). Flow cytometry was used to detect the expression of mHLA-G and iHLA-G in the T lymphocytes of peripheral blood from subjects in the three groups. Enzyme-linked immunosorbent assays were used to detect sHLA-G in the plasma from the three groups. RESULTS: There were no significant differences in mHLA-G and intracellular HLA-G among the three groups, but the sHLA-G plasma level was higher in the functioning group than in the acute rejection or healthy group. We found a subset of CD4(+)HLA-G(+) and CD8(+)HLA-G(+) T lymphocytes with low rates of mHLA-G expression in the peripheral blood of kidney transplantation recipients. Intracellular expression of HLA-G was detected in T lymphocytes. However, there was no correlation between acute rejection and the mHLA-G or intracellular HLA-G expression. CONCLUSION: sHLA-G was the major isoform in the peripheral blood of live kidney transplant recipients and high sHLA-G levels were associated with allograft acceptance.

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4+CD25+Foxp3+ regulatory T cells and down-regulates cardiac allograft rejection.

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-gamma by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4(+)CD25(high)Foxp3(+) regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

Assessment of validity of INR system for patients with liver disease associated with viral hepatitis.

International Normalized Ratio (INR), which standardizes prothrombin time (PT) during oral anticoagulation, has been extended to standardize PT in liver diseases and is included in all prognostic models of survival, the classification of CHILD-Pugh or Meld. However, the mechanisms of PT prolongation in liver diseases differ from those involved in oral anticoagulation. Our aim was to assess the validity of the INR system for patients with liver disease associated with viral hepatitis. We prospectively collected blood samples from 61 patients with liver disease associated with viral hepatitis; control patients were on warfarin (n = 20). PTs were measured on a STA-R coagulometer with six thromboplastin reagents, and INRs were calculated using instrument-specific ISIs. Simultaneously, we selected 15 pairs of patients in the study population and in the control population such that INR values for each patient pair are almost equal. For these 15 pairs of patients, we performed factor assays and measured the coagulant activities of factors II, V, VI, and X and fibrinogen. Analysis of results for the control population confirms the validity of the INR system for patients on oral anticoagulants in that there was no significant difference between the reported INRs for the six different thromboplastin reagents. Conversely, for the study population, there was a significant difference between the INR results using the different reagents. Results for fibrinogen and factors V, VII, and X showed significant differences between the two groups; however, control and patient results for factor II were not statistically different. The INR system is not valid for comparison of patients with liver disease associated with viral hepatitis because different reagents do not yield the same INR for the same sample.

Comparison of modes of prothrombin time reporting in patients with advanced liver disease associated with viral hepatitis.

BACKGROUND: Prothrombin time is widely utilized for evaluation of liver disease severity. However, its standardization of modes of reporting is not established for universal purpose. Variability in thromboplastin reagents leads to large intralaboratory and interlaboratory differences in PT results. OBJECTIVE: The aim of this study was to establish standardization of modes of PT reporting by the interchangeability analysis of prothrombin time in patients with advanced liver disease associated with viral hepatitis measured with different thromboplastin reagents by the use of various methods of expression, i.e. prothrombin time (PTs), prothrombin activity percentage (PTp), prothrombin time ratio (PTr) and International Normalized Ratio (INR). METHODS: we prospectively collected blood samples from 61 patients with advanced liver disease associated with viral hepatitis, control patients were on warfarin (n = 20). PT was measured on a STA-R with six thromboplastin reagents. PT was expressed in PTs, PTr, PTp, and INR. Neoplastin was selected as reference reagent for comparison of methods of reporting. RESULTS: The study revealed the closest agreement of the results in study population between Neoplastin and the other five reagents, and the regression lines of these reagents were close to each other, when the results were expressed in PTp while INR, PTs and PTr is not valid for comparison of patients with liver disease. In patients on oral anticoagulant therapy, only INR standardize PT results. CONCLUSION: we conclude that, in patients with liver disease, only activity percentage expression may provide a common international scale of PT reporting.

[Immune regulation effect of rat dendritic cells phagocytosing photochemotherapy-treated allogeneic cells on syngeneic T cells].

The aim of this study was to investigate the immune regulatory effect of dendritic cells phagocytosing photochemotherapy-treated allogeneic spleen lymphocytes on syngeneic T cells. DA rat spleen lymphocytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DCs were co-cultured with PUVA-treated DA spleen lymphocytes (PUVA-SP), and the surface markers (MHC-II, CD86 and CD40) of treated DC were detected by flow cytometry. CFSE-labeled PUVA SP were incubated with LEW DCs and the phagocytosis of DCs on PUVA-SP was observed by using fluorescent microscope. The ability of DC phagocytosing allogeneic PUVA-SP (PUVA-SP DC) to stimulate the proliferation of LEW T cells was analyzed by mixed leukocyte reactions (MLR). The production of IL-4, IL-10, IL-2, IFN-gamma in MLR culture supernatant was determined by luminex method. The results indicated that the PUVA treatment effectively induced early apoptosis of DA rat spleen lymphocytes. After co-culture, DC efficiently phagocytosed allogeneic PUVA-SP and still maintained an immature phenotype with low levels of MHC II, CD40 and CD86. PUVA-SP DC induced LEW T cell hyporesponsiveness to DA rat antigen, and led to skewing of T cell cytokine expression toward Th2 (IL-10 and IL-4). It is concluded that the PUVA-SP DC effectively down-regulate T cell response to alloantigen and induce Th2 immune deviation in vitro.

Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide.

Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.

OBJECTIVES: To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA by increasing the number and affinity of the pac site. METHODS: The plasmid pET-MS2-pac was constructed with one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified. RESULTS: The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions. CONCLUSIONS: The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences.