Increased yield performance of mutation induced Soybean genotypes at varied agro-ecological conditions / Aumento no desempenho de rendimento de genótipos de soja induzidos por mutação em condições agroecológicas variadas

Abstract In soybean breeding program, continuous selection pressure on traits response to yield created a genetic bottleneck for improvements of soybean through hybridization breeding technique. Therefore an initiative was taken to developed high yielding soybean variety applying mutation breeding techniques at Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh. Locally available popular cultivar BARI Soybean-5 was used as a parent material and subjected to five different doses of Gamma ray using Co60. In respect to seed yield and yield attributing characters, twelve true breed mutants were selected from M4 generation. High values of heritability and genetic advance with high genotypic coefficient of variance (GCV) for plant height, branch number and pod number were considered as favorable attributes for soybean improvement that ensure expected yield. The mutant SBM-18 obtained from 250Gy provided stable yield performance at diversified environments. It provided maximum seed yield of 3056 kg ha-1 with highest number of pods plant-1 (56). The National Seed Board of Bangladesh (NSB) eventually approved SBM-18 and registered it as a new soybean variety named 'Binasoybean-5' for large-scale planting because of its superior stability in various agro-ecological zones and consistent yield performance.

Resumo No programa de melhoramento da soja, a pressão pela seleção contínua para a resposta das características de rendimento criou um gargalo genético para melhorias da soja por meio da técnica de melhoramento por hibridação. Portanto, foi desenvolvida uma variedade de soja de alto rendimento, aplicando técnicas de reprodução por mutação, na Divisão de Melhoramento de Plantas, no Instituto de Agricultura Nuclear de Bangladesh (BINA), em Bangladesh. A cultivar popular BARI Soybean-5, disponível localmente, foi usada como material original e submetida a cinco doses diferentes de raios gama usando Co60. Em relação ao rendimento de sementes e às características de atribuição de rendimento, 12 mutantes genuínos foram selecionados a partir da geração M4. Altos valores de herdabilidade e avanço genético com alto coeficiente de variância genotípico (GCV) para altura da planta, número de ramos e número de vagens foram considerados atributos favoráveis ​​ao melhoramento da soja, garantindo, assim, a produtividade esperada. O mutante SBM-18, obtido a partir de 250Gy, proporcionou desempenho de rendimento estável em ambientes diversificados e produtividade máxima de sementes de 3.056 kg ha-1 com o maior número de vagens planta-1 (56). O Conselho Nacional de Sementes de Bangladesh (NSB) finalmente aprovou o SBM-18 e o registrou como uma nova variedade de soja, chamada 'Binasoybean-5', para plantio em larga escala por causa de sua estabilidade superior em várias zonas agroecológicas e desempenho de rendimento consistente.

Increased yield performance of mutation induced Soybean genotypes at varied agro-ecological conditions

Abstract In soybean breeding program, continuous selection pressure on traits response to yield created a genetic bottleneck for improvements of soybean through hybridization breeding technique. Therefore an initiative was taken to developed high yielding soybean variety applying mutation breeding techniques at Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh. Locally available popular cultivar BARI Soybean-5 was used as a parent material and subjected to five different doses of Gamma ray using Co60. In respect to seed yield and yield attributing characters, twelve true breed mutants were selected from M4 generation. High values of heritability and genetic advance with high genotypic coefficient of variance (GCV) for plant height, branch number and pod number were considered as favorable attributes for soybean improvement that ensure expected yield. The mutant SBM-18 obtained from 250Gy provided stable yield performance at diversified environments. It provided maximum seed yield of 3056 kg ha-1 with highest number of pods plant-1 (56). The National Seed Board of Bangladesh (NSB) eventually approved SBM-18 and registered it as a new soybean variety named Binasoybean-5 for large-scale planting because of its superior stability in various agro-ecological zones and consistent yield performance.

Resumo No programa de melhoramento da soja, a pressão pela seleção contínua para a resposta das características de rendimento criou um gargalo genético para melhorias da soja por meio da técnica de melhoramento por hibridação. Portanto, foi desenvolvida uma variedade de soja de alto rendimento, aplicando técnicas de reprodução por mutação, na Divisão de Melhoramento de Plantas, no Instituto de Agricultura Nuclear de Bangladesh (BINA), em Bangladesh. A cultivar popular BARI Soybean-5, disponível localmente, foi usada como material original e submetida a cinco doses diferentes de raios gama usando Co60. Em relação ao rendimento de sementes e às características de atribuição de rendimento, 12 mutantes genuínos foram selecionados a partir da geração M4. Altos valores de herdabilidade e avanço genético com alto coeficiente de variância genotípico (GCV) para altura da planta, número de ramos e número de vagens foram considerados atributos favoráveis ao melhoramento da soja, garantindo, assim, a produtividade esperada. O mutante SBM-18, obtido a partir de 250Gy, proporcionou desempenho de rendimento estável em ambientes diversificados e produtividade máxima de sementes de 3.056 kg ha-1 com o maior número de vagens planta-1 (56). O Conselho Nacional de Sementes de Bangladesh (NSB) finalmente aprovou o SBM-18 e o registrou como uma nova variedade de soja, chamada Binasoybean-5, para plantio em larga escala por causa de sua estabilidade superior em várias zonas agroecológicas e desempenho de rendimento consistente.

Progress on the relationship of aldehyde dehydrogenase 2 with human diseases and its small-molecule activators / 药学实践杂志

Aldehyde dehydrogenase 2 (ALDH2) is one of important factors against from the damage under oxidative stress in human body. A high proportion of East Asians carry ALDH2 inactive mutation gene. There are many diseases closely related to ALDH2, such as cardiovascular diseases, neurodegenerative diseases and liver diseases. Recent studies also have found that ALDH2 is associated with ferroptosis. Therefore, ALDH2 has becoming a potential target for the treatment of the above related diseases. Several types of small molecule activators with potential value of clinical application have been reported. The research progress on the structure and function of ALDH2 , the relationship with human diseases and its activators were summarized in this paper.

The regulatory relationship between RagA and Nprl2 in Drosophila gut development / 生物工程学报

The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells / 中华肿瘤杂志

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Prokaryotic expression and activity detection of insulin-degrading enzyme mutant T142A / 中国生物制品学杂志

@#Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate cleavage sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min~(-1) and the Michaelis constant(K_m) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_m was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

Targeting metabolic vulnerability in mitochondria conquers MEK inhibitor resistance in KRAS-mutant lung cancer

MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEK inhibitor therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEK inhibitors resistance in KRAS-driven NSCLC.

Berberine targets the electron transport chain complex I and reveals the landscape of OXPHOS dependency in acute myeloid leukemia with IDH1 mutation / 中国天然药物

Metabolic reprogramming, a newly recognized trait of tumor biology, is an intensively studied prospect for oncology medicines. For numerous tumors and cancer cell subpopulations, oxidative phosphorylation (OXPHOS) is essential for their biosynthetic and bioenergetic functions. Cancer cells with mutations in isocitrate dehydrogenase 1 (IDH1) exhibit differentiation arrest, epigenetic and transcriptional reprogramming, and sensitivity to mitochondrial OXPHOS inhibitors. In this study, we report that berberine, which is widely used in China to treat intestinal infections, acted solely at the mitochondrial electron transport chain (ETC) complex I, and that its association with IDH1 mutant inhibitor (IDH1mi) AG-120 decreased mitochondrial activity and enhanced antileukemic effect in vitro andin vivo. Our study gives a scientific rationale for the therapy of IDH1 mutant acute myeloid leukemia (AML) patients using combinatory mitochondrial targeted medicines, particularly those who are resistant to or relapsing from IDH1mi.

Role of Iron Uptake Systems in Coordinating Iron Homeostasis in NaCl-resistant Mutant of the Cyanobacterium Nostoc muscorum

Aims: To investigate the effect of NaCl stress on parent Nostoc muscorum and its spontaneously occurring mutant clone showing resistance to growth inhibitory action of NaCl in terms of various physiological parameters. We have further analyzed the role of iron uptake systems in providing a resistant phenotype. Place and Duration of Study: Division of Microbiology, Department of Botany, Government Motilal Science College, Bhopal 462008 (M.P.) India. This work was carried out between August 2021 to May 2022. Methodology: We have examined the various physiological parameters viz. growth, specific growth rate, photosynthetic O2 evolution, and nitrogenase activity as per the prescribed protocol. Further, DNA microarray analysis was carried out using the Agilent platform. Results: NaCl stress adversely affected growth, photosynthetic O2 evolution, and nitrogenase activity of the wild-type Nostoc muscorum, while NaCl-resistant mutant remains unaffected under a given stress. Microarray data analysis identified 24 ORF related to the uptake of iron with fold regulation ?2 in the mutant strain. These ORFs belonging to the ABC-type ferric iron transporter that plays a significant role in the iron acquisition were identified in the mutant strain. Conclusion: The mechanism of iron homeostasis in the NaCl-resistant mutant has been explained. The results presented are essential to explain the regulatory role of the iron uptake system in stressed conditions.

Effect of IGF1Rβ Subunit Mutants on Proliferation, Migration and Apoptosis of Human Osteosarcoma Cells / 肿瘤防治研究

Objective To investigate the effect of IGF1R β subunit mutants sb-IGF1R and ma-IGF1R on the biological behavior of osteosarcoma 143B cells. Methods We designed and constructed sb-IGF1R and ma-IGF1R fragments. They were cloned into adenovirus AdEasy shuttle plasmid, to obtain Ad-sbIGF1R and Ad-maIGF1R. We observed the proliferation, migration and apoptosis of the osteosarcoma cells transfected with Ad-sbIGF1R, Ad-maIGF1R and Ad-IGF1R. The Ad-sbIGF1R, Ad-maIGF1R and Ad-GFP nude mouse models were constructed to evaluate the tumor growth in vitro. Results By plasmid PCR, IGF-1R β subunit mutant was overexpressed in osteosarcoma cells. Ad-sbIGF1R and Ad-maIGF1R significantly inhibited the proliferation and migration of osteosarcoma cells, and promoted cell apoptosis, and inhibited tumor growth in subcutaneous tumor-bearing nude mouse models. Conclusion IGF1R β subunit mutants inhibit the proliferation and migration of osteosarcoma cells and induce cell apoptosis.

Mutation Genes in Common Tumors of Digestive System and Druggability of New Targets / 肿瘤防治研究

Objective To establish a druggability evaluation method for new targets of anti-tumor drugs by analyzing the mutation genes of common tumors in the digestive system. Methods We collected the mutant gene data of the five common tumors of the digestive system (esophageal cancer, gastric cancer, colorectal cancer, liver cancer and pancreatic cancer) in the Integrative Onco Genomics database, and screened out the genes with higher mutation rates in each tumor. We evaluated the druggability of these genes or their encoded proteins, and discovered the potential targets for the new anti-tumor drugs. Results A total of five tumors, 35 cohorts and 5445 tumor samples were collected in this study. The top 10 mutation genes were selected for further analysis. The canSAR database was used to analyze the druggability of unpublished mutant genes or their encoded proteins, and a total of 17 potential therapeutic drug targets were screened out. Conclusion A method for evaluating druggability of targets based on mutant genes or their encoded protein is established in this study. The application of this method can provide a reference for discovering new anti-tumor therapeutic target, saving the cost and time of target screening in new drug development.

The relationship between IDH, P53 mutations, MGMT methylation with characteristics in high grade glioma patients / La relación entre IDH, mutaciones P53, metilación de MGMT con las características de pacientes con glioma de alto grado

ABSTRACT Introduction: Some gene mutations in high grade glioma patients have many implications in prognosis and treatment response. Objectives: To describe the characteristics and associations of IDH, TP53 gene mutations and MGMT methylation status with some characteristics and treatment response in patients with high grade glioma. Methods: A descriptive, prospective, uncontrolled study was conducted, in 52 patients with high-grade glioma. Research variables include age, sex, Karnofsky score, the rate of IDH, P53 mutation, MGMT methylation; the relationship between genes mutation with some characteristics and response to treatment according to the RECIST classification. Results: For IDH gene mutation, grade III patients (23.1%) have a higher positive rate than grade IV (11.5 %); for P53 gene mutation, grade III patients (55.6 %) have a higher positive rate than grade IV (44.1 %); the rate of MGMT promoter methylation occurred in the study group of patients with the rate of 42.3 %. There is a relationship between IDH gene mutation with pathological results and malignancy in studied patients. Patients with the mutant expression of the IDH gene, p53, MGMT methylation status had better RECIST responses than patients without these expressions. Conclusion: High-grade glioma mainly occurs in men, over 40 years old. The presence of mutations in IDH, P53 genes, and MGMT methylation status was a beneficial factor for treatment response as assessed by RECIST.

RESUMEN Introducción: Algunas mutaciones genéticas en pacientes con glioma de alto grado tienen implicaciones en el pronóstico y respuesta al tratamiento. Objetivos: Describir las características y asociaciones de IDH, mutaciones del gen TP53 y estado de metilación de MGMT con algunas características y respuesta al tratamiento en pacientes con glioma de alto grado. Métodos: Se realizó un estudio descriptivo, prospectivo no controlado, en 52 pacientes con glioma de alto grado. Las variables investigadas fueron: edad, sexo, puntuación de Karnofsky, tasa de IDH, mutación P53, estado de metilación de MGMT, relación entre la mutación de genes con algunas características y la respuesta al tratamiento según la clasificación RECIST. Resultados: Mutación del gen IDH: los pacientes grado III (23,1 %) tienen una tasa positiva más alta que los grado IV (11,5 %). Mutación del gen P53: los grado III (55,6 %) tienen una tasa positiva más alta que los grado IV (44,1 %). La tasa de metilación del promotor de MGMT se produjo con una tasa del 42,3 %. Existe relación entre la mutación del gen IDH con los resultados patológicos y la malignidad. Los pacientes con la expresión mutante del gen IDH, p53, estado de metilación de MGMT tuvieron mejores respuestas RECIST. Conclusión: El glioma de alto grado se presenta principalmente en hombres, mayores de 40 años. La presencia de mutaciones en los genes IDH, P53 y el estado de metilación de MGMT fue un factor beneficioso para la respuesta al tratamiento según lo evaluado por RECIST.

Strategic Exploration of Targeted Therapy for BRAF Non-V600E Mutant Lung Cancer / 中国肺癌杂志

BACKGROUND@#Dabrafenib+Trametinib/Dabrafenib targeted therapy has been approved for V-RAF murine sarcoma viral oncogene homolog B1 with amino acid substitution for valine at position 600 (BRAF V600E) in lung cancer patients, however, the targeted therapy strategy for lung cancer patients with BRAF non-V600E mutations has not been determined yet. This study intends to explore the efficacy of targeted therapy for BRAF non-V600E mutant lung cancer, and provide a reference for clinical treatment.@*METHODS@#Computer search of PubMed, Cochrane Library, Embase, Web of Science, Clinicaltrials.gov, CBM, CNKI, Wanfang database. Collect the relevant literature relevant on the targeted therapy of BRAF non-V600E mutant lung cancer, and conduct a descriptive analysis of the included literature.@*RESULTS@#There were 10 articles that met the inclusion criteria, including 3 cohort studies and 7 case reports. 18 patients with BRAF non-V600E mutant lung cancer were ineffective to vermurafenib; 1 patient obtained partial response (PR) after applying vermurafenib, 5 patients did not respond to BRAF inhibitors; 9 patients showed a potential clinical benefit rate of 34% after monotherapy with trametinib; 7 patients have different degrees of benefit from dabrafenib and trametinib on progression-free survival (PFS); 1 patient is effective to sorafenib.@*CONCLUSIONS@#At present, there is no standard treatment specification for BRAF non-V600E mutation targeted therapy. The challenge lies in the heterogeneous mutation of BRAF gene. Different mutation types respond differently to targeted therapy. In addtion, real-world research evidence is scarce, so it is necessary to carry out further large-sample high-quality research to provide reference for clinical practice.

Arabidopsis CK2 family gene CKB3 involved in abscisic acid signaling / Gene da família Arabidopsis CK2 CKB3 envolvido na sinalização de ácido abscísico

CKB3 is a regulatory (beta) subunit of CK2. In this study Arabidopsis thaliana homozygous T-DNA mutant ckb3 was studied to understand the role of CKB3 in abscisic acid (ABA) signaling. The results shown: CKB3 was expressed in all organs and the highest expression in the seeds, followed by the root. During seed germination and root growth the ckb3 mutant showed reduced sensitivity to ABA. The ckb3 mutant had more stomatal opening and increased proline accumulation and leaf water loss. The expression levels of number of genes in the ABA regulatory network had changed. This study demonstrates that CKB3 is an ABA signaling-related gene and may play a positive role in ABA signaling.

CKB3 é uma subunidade reguladora (beta) de CK2. Neste estudo, o mutante homozigoto ckb3 de Arabidopsis thaliana foi estudado para entender o papel da CKB3 na sinalização de ácido abscísico (ABA). Os resultados apresentados: CKB3 foi expresso em todos os órgãos e a maior expressão nas sementes, seguida pela raiz. Durante a germinação das sementes e o crescimento radicular, o mutante ckb3 mostrou sensibilidade reduzida ao ABA. O mutante ckb3 teve mais abertura estomática e aumento do acúmulo de prolina e perda de água nas folhas. Os níveis de expressão do número de genes na rede reguladora da ABA haviam mudado. Este estudo demonstra que CKB3 é um gene relacionado à sinalização ABA e pode desempenhar um papel positivo na sinalização ABA.

Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza / 中国中药杂志

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

Effect of amino acid site modification on stability of foot-and-mouth disease virus-like particles / 生物工程学报

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.

Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175 / 口腔疾病防治

Objective@#To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function.@*Method @# Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated.@* Results @# The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001).@* Conclusion@# The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.

Progress in the construction and screening of random mutation library / 生物工程学报

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.

Inhibitory Effect and Target Prediction of Genistein on the Growth of Human Nasopharyngeal Carcinoma CNE 1 Cells / 中国药房

OBJECTIVE：To study the inhibi tory effects of genistein on the growth of human nasopharyngeal carcinoma. CNE 1 cells and predict its potential target. METHODS ：CCK-8 method was used to test the effects of 0（blank control ），12.5，25，50， 100，150 µmol/L genistein on the proliferation of CNE 1 cells after treated for 24，48，72 h. Flow cytometry was carried out to detect the effects of 0（blank control ），15，30，60 µmol/L genistein on the cell cycle and ap optosis of CNE 1 cells after treated for 24 h. Scratch test was used to investigate the effects of 0（blank control ）， 10， 20， 30 µmol/L genistein on themigration ability of CNE 1 cells after treated for 24 h. High （No.18210156） throughput sequencing was conducted to discover the differential genes in CNE 1 cells after treated with 0（blankcontrol），30 µmol/L genistein for 24 h. RT-qPCR assay was adopted to verify the mRNA expression of related differential genes in above trials. RESULTS ： Compared with blank control，12.5，25，50，100，150 µmol/L genistein sho wed significant inhibitory effect on the proliferation of CNE 1 cells（P＜ 0.01），in a concentration- time-effect manner ；15，30 µmol/L genistein could arrest CNE 1 cell cycle at G 0/G1 stage（P＜0.05 or P＜ 0.01）；30，60 µmol/L could arrest CNE 1 cell cycle at G 2/M stage and promoted cell apoptosis （P＜0.05 or P＜0.01）. 10，20，30 µmol/L genistein could significantly inhibit the migration ability of CNE 1 cells（padj＜0.01）. High throughput sequencing revealed a total of 2 271 differentialgenes（P＜0.05），1 154 of which were up-regulated while 1 117 of which were down-regulated ；8 potential target genes ，including p53，p21，STC2，FGF2，CDK6，CYCLIN D ，PI3K，AKT，were screened by cell experiment. After validated by RT-qPCR assay ，mRNA expression of p53，p21，STC2，FGF2，CDK6，CYCLIN D and AKT were significantly down-regulated（P＜0.05），which consistent with the sequencing results. CONCLUSIONS ：Genistein can effectively inhibit the growth of human nasopharyngeal carcinoma CNE 1 cells，the mechanism of which may associated with inhibiting the expression of mutant gene p53，restoring the function of wild-type P 53 protein and inhibiting the activity of PI 3K/Akt pathway.

Recent advancement in targeting the KRAS-G12C mutant for cancer therapy / 药学学报

RAS, as a well-known proto-oncogene, is the most frequently mutated oncogene in human cancers, yet tremendous efforts over the past 30 years have failed to develop effective therapies for RAS-mutant cancer. Recently, specifically targeting the KRAS-G12C mutant, a frequently occurring KRAS mutation in human cancers, has shown promise in conquering KRAS-mutant cancers, and has inspired interest in this direction. We herein review the very recent progress achieved in the development of covalent inhibitors towards KRAS-G12C mutant, in combinational therapies and in proteolysis-targeting chimeras (PROTACs)-based approaches to disrupt KRAS-G12C protein. We provide insights for drug discovery against KRAS-G12C-mutated tumors and discuss the potential challenges in this field.