ATRA promotes PD-L1 expression to control gastric cancer immune surveillance.

The vitamin A metabolite all-trans retinoic acid (ATRA) plays a key role in immune response, but effects of ATRA on cancer-associated immunity remains unclear. Previously, we have shown that ATRA regulates the expression of PD-L1 in gastric cancer (GC) cells. We herein reported the mechanism underlying ATRA-induced PD-L1 expression in GC cells and the effects of ATRA on cancer-associated immunosuppression in vitro and in vivo. ATRA enhanced PD-L1 expression through increasing its protein stability and protein synthesis, which was suppressed by JAK pan-inhibitor ruxolitinib (RUX) but enhanced in the combination with IFN-γ. In T-cell-mediated killing assay, the upregulation of PD-L1-induced by ATRA rendered GC cells strongly resistant to activated T-cell killing, which was reversed by RUX. In vivo, PD-L1 antibody restricted tumor growth, but ATRA antagonized PD-L1 antibody efficacy. Importantly, RUX not only inhibited the expression of PD-L1 induced by ATRA, but also resensitized GC cells to PD-L1 antibody. In conclusion, our study illustrated that ATRA attenuated the effect of PD-L1 blockade through upregulating PD-L1 and blocking PD-L1 expression is an important role for the generation of effective anti-tumor immune response in the combination of immunotherapy and chemotherapy or targeted therapy.

Theoretical analysis of gas-phase front-side attack identity S(N)2(C) and S(N)2(Si) reactions with retention of configuration.

Gas-phase front-side attack identity S(N)2(C) and S(N)2(Si) reactions, CH(3)X1 + X2(-) --> CH(3)X2 + X1(-) and SiH(3)X1 + X2(-) --> SiH(3)X2 + X1(-) (X = F, Cl), are investigated by the ab initio method and molecular face (MF) theory. The computations have been performed at the CCSD(T)/aug-cc-pVTZ//MP2/6-311++G(3df,3pd) and CISD/aug-cc-pVDZ levels. Front-side attack identity S(N)2 reactions for both SiH(3)X and CH(3)X have double-well potential energy surfaces (PESs), but their transition-state positions are different relative to the positions of reactants and products: it is lower for SiH(3)X, and it is higher for CH(3)X. The minimum energy path for an S(N)2(Si) reaction with retention of configuration proceeds from a stable pentacoordinated anion intermediate of C(s) symmetry (TBP) via a C(s) transition state (SP) to a complementary pentacoordinated intermediate (TBP) and finally up to separate products. Berry pseudorotation has been observed in the front-side attack identity S(N)2(Si) reactions with F(-) and Cl(-) along the intrinsic reaction coordinate (IRC) routes. In addition, the geometrical transformations of front-side attack identity S(N)2(C) and S(N)2(Si) reactions based on the IRC calculations at the MP2/6-311++G(3df, 3pd) level of theory are described compared with those of corresponding back-side attack reactions. The difference between front-side attack identity S(N)2(C) and S(N)2(Si) reactions has been demonstrated. In MF theory, the potential acting on an electron in a molecule (PAEM) is an important quantity; in particular, its D(pb) can measure the strength of a chemical bond in a molecule. It is found that the difference between D(pb) values of reactant and transition state may be related to the activation energy for front-side and back-side attack S(N)2(C) and S(N)2(Si) reactions, and the D(pb) curves along the IRC routes have features similar to those of the potential energy profiles for all of the back-side attack S(N)2 reactions and front-side attack S(N)2(Si) reaction with F(-). Furthermore, according to the MF theory, the spatial dynamic changing features of the molecular shapes and the face electron density are vividly depicted for the course of the reactions.

[Effects of Triptolide on Tc and Th Cell Excursion in Peripheral Blood of Nude Mice with Systemic Lupus Erythematosus BALB/c-un].

OBJECTIVE: To investigate the effect of triptolide on the excursion of Tc and Th cells in peripheral blood of systemic lupus erythematosus (SLE) BALB/c-un nude mice induced by pristane. METHODS: Eighteen female BALB/c-un nude mice were randomly divided into blank, SLE and triptolide group, each with 6 mice by random table method. Group SLE and group triptolide were established by single intraperitoneal injection of pristane, and blank group was used as blank control group. SLE model was established by single intraperitoneal injection. Triptolide group was fed with triptolide at the dose of 5 mg/(kg·d), and the blank group and SLE group were fed normally. Blood samples were collected from the caudal vein before treatment and 1, 3 and 6 months after treatment respectively. Fluorescence labeled flow cytometry was used to delect Tc and Th lymphocyte subsets at different stages of treatment. RESULTS: After treatment for 3 and 6 moths, the percentages of Tcl, Thl cells and CD8+, Tcl/Tc2, Thl/Th2 and CD4+/CD8+ all decreased in the group of triptolide, and the percentage of CD4+, Tc2 and Th2 cells increased (P＜0.05). CONCLUSION: The mechanism of triptolide in the treatment of SLE may be related with the excursion of Tc and Th cells to Tcl and Tc2 to maintain the relative homeostasis of Tc and Th cells at different stage, thus affecting the immune response and the inflammatory reaction.

Insight into markovnikov reactions of alkenes in terms of ab initio and molecular face theory.

Electrophilic additions of hydrogen halides to alkenes in the gas phase are investigated with a high-level ab initio method, MP2/6-311+G(3df,2p). Based on this, the interesting features of these reactions along the IRC routes are characterized by the molecular face (MF) theory. For an alkene at the initial state, if the representative electron density (ED) encoded on the molecular face (MF) of the Markovnikov (M) carbon atom (the carbon with more hydrogen atoms) is larger than that of the anti-Markovnikov (AM) carbon atom (the carbon with fewer hydrogen atoms), the electrophilic addition reaction is predicted to proceed along the Markovnikov addition route; in the reverse situtation, the anti-Markovnikov addition route would be slightly preferred. It is then demonstrated that for a series of alkenes, the difference between activation energies of Markovnikov and anti-Markovnikov addition routes [DeltaE(#) ((M-AM))] has a good linear correlation with sign(K(ED))K(2) (ED), where K(ED) is characteristic of the electron density (ED) at the pi region in the initial state of the alkenes. Interestingly, there is a good linear correlation between our sign (K(ED))K(2) (ED) and the absolute values of difference in the core ionization energy between M and AM carbon atoms obtained by others (L. J. Saethre, T. D. Thomas, S. Svensson J. Chem. Soc. P2 1997, 2, 749.) in terms of the experimental study. In addition, the spatial dynamic changing features of the MF faces and interesting pictures of the electron transfer are clearly shown during the course of the electrophilic addition reactions. These results indicate that not only regioselectivity, but also activation energy and reactivity correlate with the pi charge distribution in the initial state of the alkenes for electrophilic addition reactions.

[Correlations Between Substrate Structure and Microbial Community in Subsurface Flow Constructed Wetlands].

To identify the microbial factors that cause the differences in the purification performance of constructed wetlands with different substrate structures, the relationship between the substrate structure and the microbial community composition in horizontal subsurface flow constructed wetlands (HSSFCWs) was studied by high throughput sequencing. The results revealed that the purification performance of a six-layer constructed wetland (CW6), of which the permeability coefficient gradually increased from the surface layer to the bottom layer, was the highest among the three constructed wetland systems. The average concentrations of COD, TN, NO3--N, and NH4+-N in the effluent were 39, 11, 0.35, and 4 mg·L-1, respectively. The monolayer structure constructed wetland (CW1) had the worst purifying efficiency, with average effluent concentrations of 95, 21, 0.60 and 12 mg·L-1 for COD, TN, NO3--N, and NH4+-N, respectively. The results of the high-throughput sequencing showed that the number of microbial OTUs in multilayer structure wetlands was slightly lower than that in the monolayer structure wetland, but the relative abundance of the dominant phylum Proteobacteria and the nitrifying and denitrifying bacteria in the genus was significantly higher than the monolayer structure wetland. The results of PCA and heatmap indicated that there were significant differences in the spatial distribution of microbes in the genus of Proteobacteria in CW3 and CW6, which facilitated the degradation of pollutants. No significant differences were found in the community structure of CW1.

[Isolation of a novel peptide that binds to BLyS from a 12-mer phage display peptide library].

A 12-mer phage display peptide library was screened for specific binders against B lymphocyte stimulator (BLyS). After 3 rounds of panning, positive phage clones were enriched. ELISAs were used to identify positive phages, and the DNA encoding the positive peptide (RHKIQLRQNIIT) was cloned and expressed as a GST fusion protein in E. coli. After purification, the identity of the fusion protein was confirmed through its specific binding to BLyS by ELISA. We have obtained a peptide that can bind to BLyS and probably act as an antagonistic peptide against the natural BLyS receptor.

[Correlation of substrate structure and hydraulic characteristics in subsurface flow constructed wetlands].

The correlation of substrate structure and hydraulic characteristics was studied by numerical simulation combined with experimental method. The numerical simulation results showed that the permeability coefficient of matrix had a great influence on hydraulic efficiency in subsurface flow constructed wetlands. The filler with a high permeability coefficient had a worse flow field distribution in the constructed wetland with single layer structure. The layered substrate structure with the filler permeability coefficient increased from surface to bottom could avoid the short-circuited flow and dead-zones, and thus, increased the hydraulic efficiency. Two parallel pilot-scale constructed wetlands were built according to the numerical simulation results, and tracer experiments were conducted to validate the simulation results. The tracer experiment result showed that hydraulic characteristics in the layered constructed wetland were obviously better than that in the single layer system, and the substrate effective utilization rates were 0.87 and 0.49, respectively. It was appeared that numerical simulation would be favorable for substrate structure optimization in subsurface flow constructed wetlands.

[Effects of biochar on microbial ecology in agriculture soil: a review].

Biochar, as a new type of soil amendment, has been obtained considerable attention in the research field of environmental sciences worldwide. The studies on the effects of biochar in improving soil physical and chemical properties started quite earlier, and already covered the field of soil microbial ecology. However, most of the studies considered the soil physical and chemical properties and the microbial ecology separately, with less consideration of their interactions. This paper summarized and analyzed the interrelationships between the changes of soil physical and chemical properties and of soil microbial community after the addition of biochar. Biochar can not only improve soil pH value, strengthen soil water-holding capacity, increase soil organic matter content, but also affect soil microbial community structure, and alter the abundance of soil bacteria and fungi. After the addition of biochar, the soil environment and soil microorganisms are interacted each other, and promote the improvement of soil microbial ecological system together. This review was to provide a novel perspective for the in-depth studies of the effects of biochar on soil microbial ecology, and to promote the researches on the beneficial effects of biochar to the environment from ecological aspect. The methods to improve the effectiveness of biochar application were discussed, and the potential applications of biochar in soil bioremediation were further analyzed.

[Application of MALDI-TOF-MS in gene testing for non-syndromic hearing loss].

OBJECTIVE: To investigate the feasibility of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) , according to the genetic test of non-syndromic hearing loss (NSHL), and check using the direct sequencing. METHODS: Peripheral blood was collected from 454 NSHL patients. DNA samples were extracted and 20 loci of the four common disease-causing genes were analysed by MALDI-TOF-MS, including GJB2 (35delG, 167delT, 176_191del16, 235delC, 299_300delAT ), GJB3 (538C→T, 547G→A), SLC26A4 (281C→T, 589G→A, IVS7-2A→G, 1174A→T, 1226G→A, 1229C→T, IVS15+5G→A, 1975G→C, 2027T→A, 2162C→T, 2168A→G), and mitochondrial 12S rRNA (1494C→T, 1555A→G). Direct sequencing was also used to analyse the aforementioned 20 loci in order to validate the accuracy of MALDI-TOF-MS. RESULTS: Among the 454 patients, 166 cases (36.56%) of disease-causing mutations were detected, which included 69 cases (21.15%) of GJB2 gene mutation, four cases (0.88%) of GJB3 gene mutation, 64 cases (14.10%) of SLC26A4 gene mutation, and three cases (0.66%) of mitochondrial 12S rRNA gene mutation. Moreover, the results obtained from direct sequencing and MALDI-TOF-MS were consistent, and the results showed that the two methods were consistent. CONCLUSIONS: The MALDI-TOF-MS detection method was designed based on the hearing loss-related mutation hotspots seen in the Chinese population, and it has a high detection rate for NSHL related mutations. In comparison to the conventional detection methods, MALDI-TOF-MS has the following advantages: more detection sites, greater coverage, accurate, high throughput and low cost. Therefore, this method is capable of satisfying the needs of clinical detection for hearing impairment and it is suitable for large-scale implementation.

Application of phage-displayed single chain antibodies in western blot.

A phage display single chain fragment variable library constructed on pIII protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.

Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus.

Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, K(d)) of SARS-CoV E protein was 5.7 x 10(-8) M for B10 and 8.9 x 10(-8) M for C20. The affinity of A17 for N protein was 2.1 x 10(-6) M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.