Spiroarborin, an ent-Clerodane Homodimer from Callicarpa arborea as an Inhibitor of the Eleven-Nineteen Leukemia (ENL) Protein by Targeting the YEATS Domain.

A spiro ent-clerodane homodimer with a rare 6/6/6/6/6-fused pentacyclic scaffold, spiroarborin (1), together with four new monomeric analogues (2-5), were isolated from Callicarpa arborea. Their structures were elucidated by comprehensive spectroscopic data analysis, quantum-chemical calculations, and X-ray diffraction. A plausible biosynthetic pathway of 1 was proposed, and a biomimetic synthesis of its derivative was accomplished. Compound 1 showed a potent inhibitory effect by directly binding to the YEATS domain of the 11-19 leukemia (ENL) protein with an IC50 value of 7.3 µM. This gave a KD value of 5.0 µM, as recorded by a surface plasmon resonance binding assay.

Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

Mo-doping heterojunction: interfacial engineering in an efficient electrocatalyst for superior simulated seawater hydrogen evolution.

Exploring economical, efficient, and stable electrocatalysts for the seawater hydrogen evolution reaction (HER) is highly desirable but is challenging. In this study, a Mo cation doped Ni0.85Se/MoSe2 heterostructural electrocatalyst, Mox-Ni0.85Se/MoSe2, was successfully prepared by simultaneously doping Mo cations into the Ni0.85Se lattice (Mox-Ni0.85Se) and growing atomic MoSe2 nanosheets epitaxially at the edge of the Mox-Ni0.85Se. Such an Mox-Ni0.85Se/MoSe2 catalyst requires only 110 mV to drive current densities of 10 mA cm-2 in alkaline simulated seawater, and shows almost no obvious degradation after 80 h at 20 mA cm-2. The experimental results, combined with the density functional theory calculations, reveal that the Mox-Ni0.85Se/MoSe2 heterostructure will generate an interfacial electric field to facilitate the electron transfer, thus reducing the water dissociation barrier. Significantly, the heteroatomic Mo-doping in the Ni0.85Se can regulate the local electronic configuration of the Mox-Ni0.85Se/MoSe2 heterostructure catalyst by altering the coordination environment and orbital hybridization, thereby weakening the bonding interaction between the Cl and Se/Mo. This synergistic effect for the Mox-Ni0.85Se/MoSe2 heterostructure will simultaneously enhance the catalytic activity and durability, without poisoning or corrosion of the chloride ions.

Metatranscriptome of human lung microbial communities in a cohort of mechanically ventilated COVID-19 Omicron patients.

The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) infected a substantial proportion of Chinese population, and understanding the factors underlying the severity of the disease and fatality is valuable for future prevention and clinical treatment. We recruited 64 patients with invasive ventilation for COVID-19 and performed metatranscriptomic sequencing to profile host transcriptomic profiles, plus viral, bacterial, and fungal content, as well as virulence factors and examined their relationships to 28-day mortality were examined. In addition, the bronchoalveolar lavage fluid (BALF) samples from invasive ventilated hospital/community-acquired pneumonia patients (HAP/CAP) sampled in 2019 were included for comparison. Genomic analysis revealed that all Omicron strains belong to BA.5 and BF.7 sub-lineages, with no difference in 28-day mortality between them. Compared to HAP/CAP cohort, invasive ventilated COVID-19 patients have distinct host transcriptomic and microbial signatures in the lower respiratory tract; and in the COVID-19 non-survivors, we found significantly lower gene expressions in pathways related viral processes and positive regulation of protein localization to plasma membrane, higher abundance of opportunistic pathogens including bacterial Alloprevotella, Caulobacter, Escherichia-Shigella, Ralstonia and fungal Aspergillus sydowii and Penicillium rubens. Correlational analysis further revealed significant associations between host immune responses and microbial compositions, besides synergy within viral, bacterial, and fungal pathogens. Our study presents the relationships of lower respiratory tract microbiome and transcriptome in invasive ventilated COVID-19 patients, providing the basis for future clinical treatment and reduction of fatality.

[Expression of c-erbB2 protein and its relation to prognosis in 284 primary breast cancer patients].

OBJECTIVE: To investigate the expression of oncoprotein c-erbB2 in primary breast cancer and to analyze its relation to its prognosis. METHODS: Immunohistochemical staining for c-erbB2 was performed on paraffin-embedded specimens of primary breast cancer from 284 patients, and the relation to its prognosis was statistically analyzed. RESULTS: Positive expression rate of c-erbB2 was 26.8% (76/284) in 284 primary breast cancer patients. Expression of c-erbB2 was positively correlated with the status of lymph node metastasis (P = 0.003). Univariate analysis indicated that c-erbB2 expression is a significant prognostic factor for the disease-free survival (DFS) (P = 0.024) and overall survival (OS) (P = 0.002), while multivariate analysis demonstrated that c-erbB2 is an independent prognostic factor for OS (P = 0.023). Moreover, tumors with c-erbB2 positive expression are more tend to metastasis to other viscera than those with c-erbB2 negative. c-erbB2 expression has different prognostic values for patients with different status of estrogen receptor (ER) and lymph node metastasis. CONCLUSION: c-erbB2 expression is an independent prognostic factor for total survival time in primary breast cancer patients, and its prognostic values are different according to the different ER status and lymph node metastasis.

[Molecular design and construction of IL6D24-PE40KDEL, a novel recombinant interleukin6-pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemias expressing interleukin6 receptors].

OBJECTIVE: A novel recombinant interleukin6-Pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemic cells highly expressing IL6R has been molecularly designed and constructed in this study. METHODS: Firstly, REDLK at C-terminus of Pseudomonas exotoxin PE40 was replaced with KDEL using point mutagenesis technology. Secondly, a cDNA encoding interleukin-6 devoid of N-terminal 24 amino acids [IL6D24] was fused to 5' terminus of PE40KDEL DNA by the method of gene splicing by overlap extension, which could generate recombinant IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL fusion genes. Thirdly, recombinant fusion genes IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL were ligated into the EcoR I and Sma I cloning sites in the pBV220 plasmids respectively and then transformed into E.coli HB101 cells. The expressed recombinant exotoxin fusion proteins were purified to electrophoresis purity by Mono Q column chromatography. Its selectively killing was tested by the MTS colorimetric method using both U937 and CEM cells lines. RESULTS: Recombinant exotoxin fusion proteins IL6D24-PE40KDEL was expressed as a form of inclusion bodies at higher level of 40% approximately of total proteins in bacterial cells. Western blot showed that the purified products could react specifically with IL6 monoclonal antibody and PEA antiserum, respectively. IL6D24-PE40KDEL was selectively cytotoxic to U937 cells expressing IL6R-positive with ID50 of 250 ng/ml, and but not CEM cells expressing IL6R-negative. CONCLUSIONS: Two novel recombinant interleukin6-Pseudomonas exotoxin fusion proteins IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL have been successfully constructed and they can selectively kill the leukemic cells expressing highly IL6R.

[Prokaryotic expression of HLA-B * 2705 heavy chain and identification for its activity].

In order to investigate the expression of heavy chain of HLA-B * 2705 in prokaryotic system and identify its activity, the extra-membrane gene fragment of HLA-B * 2705 was amplified from full-length HLA-B*2705 cDNA by PCR and cloned into pGEM-T vector. After identification by sequencing, the prokaryotic expressing vector pET32a (+)-B * 2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLA-B * 2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLA-B * 2705 fusion protein are obtained as basis for the further studies.

[Establishment of purification procedure for recombinant fusion protein B7-2-PE40KDEL].

This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.

[A preliminary study on the expression and biological function of recombinant human SCF-TPO fusion protein].

OBJECTIVE: To study the expression of recombinant human SCF-TPO fusion protein and its biological function. METHODS: Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography. RESULTS: The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml. CONCLUSIONS: Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.

[Fusion construction, prokaryotic expression and structure characteristics prediction of bimolecular thrombopoietin].

AIM: Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity. METHODS: The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software. RESULTS: The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure. CONCLUSION: We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.

[Biochemical and physical properties for a recombinant IL6 Pseudomonas exotoxin fusion protein IL6D24-PE40KDEL].

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.

[A New Criterion for Donor and Recipient Selection in Hematopoietic Stem Cell Transplantation - the Matching of Three-Dimensional Structure of HLA Molecular Modeling]

The purpose of the research is to provide a new standard for matching of HLA three-dimensional structure, and summarize the major permissible mismatch and immunogenic mismatch antigens. The molecular modeling method was used to create HLA molecular structures by Swiss Model Server, and the comparison of the differences among the alleles was done by SPDV software with the function of iterative magic fit. The results were recorded by relative mean square deviation (RMSD, nm). The differences among alleles were scattered below 0.06 nm for HLA-A and -B molecules, and below 0.03 nm for HLA-DRB1 molecules. On the basis of the statistical analysis, when RMSD is greater than 0.04 nm for -A and -B molecules and 0.02 nm for -DRB1 molecules, the difference is meaningful and can be related with graft versus host disease. When RMSD is lower than 0.02 nm for -A and -B molecules and 0.01 nm for -DRB1 molecules, the difference is decided unmeaningful. From the data, the permissible mismatch and immunogenic mismatch alleles within HLA-A, HLA-B and HLA-DRB1 molecules were summarized. Three-dimensional structure matching is a new area in the transplantation field, much research should be done in the future.

[Selection of donor in mismatched hematopoietic stem cell transplantation by CREG, residue match and HLA three-dimensional structure].

After search at hematopoietic stem cell banks for transplant donors, there may be several donors matched with given standards. To determine the most appropriate donor for a specific patient, the potential donors were analyzed and compared by three methods. The first is cross-reactive group (CREG) antigens, which defined as the public antigens that shared specific serological reaction patterns. The second is residue match theory, which concerned the three residues oriented upward toward the T-cell receptor. The third is comparing HLA three-dimensional structure models. The results of the three methods were not completely accorded in our case. However, some less matched donors could be excluded from the candidates and the range of selection was further reduced. It is concluded that combined application of three methods would contribute in selecting donor for hematopoietic stem cell transplantation in clinics.

[Expression of alpha subunit for IL-6 receptor at mRNA and protein levels in human leukemic cells].

To probe into the expression of alpha subunit for IL-6R at both mRNA and protein levels in human leukemic cells and to discuss its implication in targeted treatment for leukemia with recombinant IL-6-PE40 exotoxin fusion protein mediated by IL-6/IL-6R system, semi-quantitative RT-PCR, sequencing and FCM were used to analyze the gene and protein expression levels of alpha subunit for IL-6R in leukemic cells. Our results showed that not only mRNA but also protein of alpha subunit for IL-6R are highly expressed in the myelogenous leukemic cell lines, the relative expression levels of mRNA were KG-1(1.22) > (1.02) > U266(1.00) > U937(0.99) > HL-60(0.76). Among lymphoblastic leukemic cell lines, Raji expressed a certain amount of alpha subunit mRNA (0.77), whereas its alpha subunit protein was not detected. Expression of alpha subunit mRNA and protein were negative in lymphoblastic leukemic cell lines, HuT28 and CEM, and chronic myelocytic leukemic cell line K562. These results correlate with those of FCM highly. Noteworthily, normal human peripheral blood mononuclear cells expressed hardly protein of IL-6R alpha subunit. So this study provides sufficient experimental evidence that the targeted treatment by recombinant IL-6-PE40 can specifically kill leukemic cells highly expressing IL-6R without toxicity to normal hematopoietic cells.