Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers

SUMMARY OBJECTIVE: The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. METHODS: DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. RESULTS: Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. CONCLUSION: Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

Construction of luminescent bacteriophage using CRISPR technology and its application in Escherichia coli indentification / 中华检验医学杂志

Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.

Progress of poly (ADP-ribose) polymerase inhibitors in treatment of colorectal cancer / 肿瘤研究与临床

Colorectal cancer is a highly malignant disease characterized by a poor prognosis. Although the targeted therapy based on chemotherapy improves the efficacy, the prognosis of advanced colorectal cancer patients is poor. Poly (ADP-ribose) polymerase (PARP) inhibitors, which act through a synergistic lethal mechanism, have been widely utilized in treating ovarian and breast cancer cases with homologous recombination deficiency and have demonstrated positive clinical outcomes. Recently, due to advancements in next-generation sequencing technology and the development of precision targeted therapy, an increasing number of clinical trials have started to investigate the potential of PARP inhibitors in colorectal cancer treatment. This review summarized the molecular mechanism of PARP inhibitors in the treatment of colorectal cancer and the application of several combined PARP inhibitors therapy regimens in colorectal cancer, in order to provide new insights for the treatment of colorectal cancer.

The relationship between HRD score based on GSS algorithm and clinicopathological characteristics, genomic mutations and prognosis in patients with high-risk and metastatic hormone-sensitive prostate cancer / 现代泌尿外科杂志

【Objective】 To statistically analyze the relationship between homologous recombination repair deficiency (HRD) score and clinicopathological characteristics, genomic mutations in patients with high-risk and metastatic hormone-sensitive prostate cancer (mHSPC) and the prognostic predictive value in mHSPC. 【Methods】 A total of 127 patients diagnosed with high-risk prostate cancer and mHSPC, treated at the Department of Urology of Chinese PLA General Hospital during Dec.2021 and Nov.2023 were enrolled.Homologous recombination repair (HRR) gene sequencing was performed, and the genomic scar score (GSS) algorithm were conducted to calculate the HRD score.The relationship between HRD scores and clinicopathological features, genomic alterations, and prognosis were analyzed. 【Results】 The median HRD score was 1.6(0.8, 5.2), 30(23.6%) patients’ HRD scores ≥10, and 11(8.7%) patients’ HRD scores ≥20.Clinicopathological features, including ISUP classification ≥4 (P=0.044) and metastatic status (P=0.008) were associated with high HRD score.Patients with mutations in the BRCA, TP53 and MYC systems had significantly higher HRD score than those with wild-type genes (P<0.05).In mHSPC, the risk of biochemical recurrence was 12.836 times higher in patients with HRD score ≥20 than in those with <20 [OR:12.836 (1.332-124.623), P=0.028]. 【Conclusion】 Baseline HRD score was lower in patients with high-risk prostate cancer and mHSPC.Patients with high HRD score may have higher histological grading (ISUP≥4) and later clinical stage.Further investigation is needed to determine the threshold of HRD scores as biochemical markers suggestive of a poor prognosis.

Construction,expression,purification and quality identification of kanamycin resistant strain of recombinant human interleukin-1 receptor antagonist / 中国生物制品学杂志

@#Objective To construct a recombinant human interleukin-1 receptor antagonist(rhIL-1Ra)strain of kanamycin resistance,express,purify and identify rhIL-1Ra protein in order to reduce the risks of β-lactam antibiotics.Methods The ampicillin-resistant rhIL-1Ra(A-rhIL-1Ra)plasmid and PET-28a plasmid were used as templates to prepare the linearized vector and kanamycin gene fragment by PCR.The kanamycin resistant rhIL-1Ra plasmid(K-rhIL-1Ra)was constructed by homologous recombination,which was transformed into E.coli BL21(DE3)to construct the recombinant engineered bacteria after correct sequencing.The engineered bacteria were induced by IPTG and then purified by CM Bestarose Fast Flow and DEAE Bestarose Fast Flow column chromatography.The purified products were collected,and then detected for the purity by 15% SDS-PAGE,size-exclusion high-performance liquid chromatography(SEC-HPLC),and reversed-phase high-performance liquid chromatography(RP-HPLC),determined for the relative molecular mass by tandem mass spectrometry,identified for the specificity by Western blot,measured for the biological activity by reporter gene assay,and compared for the related impurities by liquid chromatography-mass spectrometry(LC-MS)analysis.Results Colony PCR and sequencing results showed that K-rhIL-1Ra plasmid was constructed correctly.The expressed K-rhIL-1Ra protein had a relative molecular mass of about 17 000,mainly existing in soluble form,and the expression amount accounted for more than 30% of the total bacterial proteins.The purity of K-rhIL-1Ra protein purified by two steps was 97% and 99%,the monomer content was 99.33%and 100%,and the chromatographic purity was 91.86% and 96.96%,respectively.The mass spectral molecular mass was consistent with that of the standard(A-rhIL-1Ra protein),and the protein reacted specifically relative with mouse antihuman IL-1Ra monoclonal antibody.The biological activity of the purified K-rhIL-1Ra protein was 1.29 × 105U/mg,and the chemical modification types of related proteins were the same as those of the standard(A-rhIL-1Ra protein).Conclusion The K-rhIL-1Ra strain was successfully constructed,and the expressed and purified protein was in line with the characteristics and quality standards of rhIL-1Ra,which lays a foundation for the study of rhIL-1Ra strain change and comparability

Clinical Significance of FOXP3 Expression in BRCA1/2-Mutant Breast Cancer / 肿瘤防治研究

Objective To investigate the potential significance of FOXP3 expression in BRCA1/2-mutant breast cancer. Methods A total of 48 BRCA mutation carriers (16 with BRCA1 and 32 with BRCA2) and 78 age-matched non-carriers were included in this study. Immunohistochemistry was used to detect the expression of FOXP3 in breast cancer tissues. The FOXP3 RNA expression in 39 BRCA1, 36 BRCA2, and 948 non-carrier breast cancer patients from TCGA-BRCA and the correlation with homologous recombination deficiency scores were evaluated to validate the immunohistochemistry results. Results The FOXP3 positive rate was 43.8% (7/16) in BRCA1 mutation carriers, 59.4% (19/32) in BRCA2 mutation carriers, and 9.0% (7/78) in non-carriers. The FOXP3 positive rates in patients with BRCA1/2 mutant breast cancer were significantly higher than those in non-carriers (P=0.002; P<0.001). TCGA-BRCA results showed that the FOXP3 RNA level in BRCA1/2 mutant breast cancer was significantly higher than that in non-carriers (P=0.02, P=0.004). The FOXP3 RNA level was positively correlated with the homologous recombination deficiency score (Spearman R=0.30, P<2.2e-16). Conclusion Patients with BRCA1/2 mutant breast cancers have higher FOXP3 expression than non-carriers, and may be more sensitive to immunotherapy.

TRIP13 Enhances Radioresistance of Lung Adenocarcinoma Cells through the Homologous Recombination Pathway / 中国肺癌杂志

BACKGROUND@#Radiation therapy is one of the most common treatments for non-small cell lung cancer (NSCLC). However, the insensitivity of some tumor cells to radiation is one of the major reasons for the poor efficacy of radiotherapy and the poor prognosis of patients, and exploring the underlying mechanisms behind radioresistance is the key to solving this clinical challenge. This study aimed to identify the molecules associated with radioresistance in lung adenocarcinoma (LUAD), identified thyroid hormone receptor interactor 13 (TRIP13) as the main target initially, and explored whether TRIP13 is related to radioresistance in LUAD and the specific mechanism, with the aim of providing theoretical basis and potential targets for the combination therapy of LUAD patients receiving radiotherapy in the clinic.@*METHODS@#Three datasets, GSE18842, GSE19188 and GSE33532, were selected from the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (|log FC|>1.5, P<0.05) in each of the three datasets using the R 4.1.3 software, and then Venn diagram was used to find out the differentially expressed genes common to the three datasets. The screened differential genes were then subjected to protein-protein interaction (PPI) analysis and module analysis with the help of STRING online tool and Cytoscape software, and survival prognosis analysis was performed for each gene with the help of Kaplan-Meier Plotter database, and the TRIP13 gene was identified as the main molecule for subsequent studies. Subsequently, the human LUAD cell line H292 was irradiated with multiple X-rays using a sub-lethal dose irradiation method to construct a radioresistant cell line, H292DR. The radioresistance of H292DR cells was verified using cell counting kit-8 (CCK-8) assay and clone formation assay. The expression levels of TRIP13 in H292 and H292DR cells were measured by Western blot. Small interfering RNA (siRNA) was used to silence the expression of TRIP13 in H292DR cells and Western blot assay was performed. The clone formation ability and migration ability of H292DR cells were observed after TRIP13 silencing, followed by the detection of changes in the expression levels of proteins closely related to homologous recombination, such as ataxia telangiectasia mutated (ATM) protein.@*RESULTS@#Screening of multiple GEO datasets, validation of external datasets and survival analysis revealed that TRIP13 was highly expressed in LUAD and was associated with poor prognosis in LUAD patients who had received radiation therapy. And the results of gene set enrichment analysis (GSEA) of TRIP13 suggested that TRIP13 might be closely associated with LUAD radioresistance by promoting homologous recombination repair after radiation therapy. Experimentally, TRIP13 expression was found to be upregulated in H292DR, and silencing of TRIP13 was able to increase the sensitivity of H292DR cells to radiation.@*CONCLUSIONS@#TRIP13 is associated with poor prognosis in LUAD patients treated with radiation, possibly by promoting a homologous recombination repair pathway to mediate resistance of LUAD cells to radiation.

Construction and biological characterization of a Proteus mirabilis strain with modABC gene deletion / 南方医科大学学报

OBJECTIVE@#To construct a modABC gene knockout strain of Proteus mirabilis and explore the effect of modABC gene deletion on biological characteristics of Proteus mirabilis.@*METHODS@#Fusion PCR was used to obtain the fusion gene of modABC and the kanamycin-resistant gene Kn, which was ligated with the suicide vector pCVD442 and transduced into Proteus mirabilis. The modABC gene knockout strain of Proteus mirabilis was obtained after homologous recombination with the suicide vector. PCR and Sanger sequencing were used to identify genomic deletion of modABC gene in the genetically modified strain. The concentration of molybdate in the wild-type and gene knockout strains was determined using inductively coupled plasma mass spectrometry (ICP-MS), and their survival ability in LB medium was compared under both aerobic and anaerobic conditions.@*RESULTS@#PCR and sanger sequencing confirmed genomic deletion of modABC gene in the obtained Proteus mirabilis strain. The concentration of intracellular molybdenum in the modABC gene knockout strain was 1.22 mg/kg, significantly lower than that in the wild-type strain (1.46 mg/kg, P < 0.001). Under the aerobic condition, the modABC gene knockout strain grown in LB medium showed no significant changes in survival ability compared with the wild-type strain, but its proliferation rate decreased significantly under the anaerobic condition and also when cultured in nitrate-containing LB medium under anaerobic condition.@*CONCLUSION@#Homologous recombination with the suicide vector can be used for modABC gene knockout in Proteus mirabilis. modABC gene participates in molybdate uptake and is associated with anaerobic growth of Proteus mirabilis in the presence of nitrate.

The efficacy and safety comparison of radium-223 in the treatment of mCRPC patients with and without homologous recombination repair gene mutation / 中华泌尿外科杂志

Objective:To compare the efficacy and safety of radium-223 in the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients with and without homologous recombination repair (HRR) gene mutation.Methods:The clinical data of 27 patients with mCRPC bone metastases who received radium-223 therapy from April 2021 to November 2022 in Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine were retrospectively analyzed. Among the 27 mCRPC patients, 18 patients carrying HRR gene mutations belonged to the HRD(+ ) group, and 9 patients without HRR gene mutation belonged to the HRD(-) group. The age of patients in HRD(+ ) group was 69.5 (63.8, 77.0) years old, alkaline phosphatase (ALP) was 243.0 (82.8, 301.3) U/L, prostate specific antigen (PSA) was 71.6 (7.3, 329.8) ng/ml, pain score was 3.0 (1.0, 5.0) points. Eastern Cooperative Oncology Group (ECOG) score ranged from 0 to 1 points in 7 cases, and 2 points in 11 cases. In the HRD(-) group, the median age was 72.0 (64.5, 76.5) years old, ALP was 88.0 (67.5, 260.6) U/L, PSA was 19.1 (1.1, 117.8) ng/ml, and pain score was 2.0 (0, 4.5) points. The ECOG score ranged from 0 to 1 in 4 cases, and 2 in 5 cases in the HRD(-) group. There was no significant difference in the above general data between the two groups ( P>0.05). All patients received radium-223 treatment every 4 weeks, no more than 6 times. The changes of ALP, PSA, pain score and hematological adverse reactions were compared between the two groups. Results:In the HRD(+ ) group, the median number of radium-223 treatment was 4.5 (3.0, 5.3) couses, 4 patients (22.2%) completed 6 courses, and 6 patients died of prostate cancer during follow-up. In the HRD(-) group, the median number of radium treatment was 4.0 (2.5, 6.0) couses, 3 patients (33.3%) completed 6 courses, and 1 patient died of prostate cancer during follow-up. There was no significant difference in the number of radium treatment courses between the two groups ( P=0.320). ALP in HRD(+ ) group was 101.8 (61.3, 147.0) U/L after radium-223 treatment, which was significantly lower than that before treatment ( P=0.002). ALP in HRD(-) group was 73.0 (64.0, 113.5) U/L after radium-223 treatment, and it was not significantly different from that before treatment ( P=0.327). The rate of ALP response (ALP decrease >10%) in HRD(+ ) group was significantly higher than that in HRD(-) group [83.3% (15/18) vs. 44.4% (4/9), P=0.037]. PSA was 105.9(5.2, 798.4) ng/ml in HRD (+ ) group after radium-223 treatment, and was 25.6(0.8, 1 031.0) ng/ml in HRD(-) group, and they were not significantly different from that before treatment ( P=0.145, P=0.386). There were no significant differences in the rate of PSA response (PSA decrease>10%) between HRD(+ ) group and HRD(-) group [38.9% (7/18) vs. 22.2% (2/9), P=0.386]. The median pain score of HRD(+ ) group was 3.0 (0, 4.0) points after treatment, which was significantly lower than that before treatment ( P=0.028). The pain score of HRD(-) group was 1.0(0, 3.0) points after treatment, and it was not significantly different from that before treatment ( P=0.129). There was no significant difference in pain relief rate between HRD(+ ) group and HRD(-) group [66.7% (12/18) vs. 44.4% (4/9), P=0.411]. The incidence of at least one hematological adverse event during radium-223 treatment in the HRD(+ ) group was higher than that in the HRD(-) group [77.8% (14/18) vs. 33.3% (3/9), P=0.039]. There was no significant difference in the incidence of grade 1-2 hematological adverse events between the two groups [72.2%(13/18) vs. 33.3%(3/9), P=0.097]. Only 1 patient in the HRD(+ ) group experienced grade 3 anemia during treatment which was recovered after blood transfusion. Conclusions:Compared to mCRPC patients without HRR gene mutation, patients with HRR gene mutations had better ALP response and bone pain relief after radium-223 treatment. The overall incidence of adverse events in the HRD(+ ) group is higher than that in HRD(-) group, and there was no significant difference in grade 1-2 hematological adverse events between the two goups. It is necessary to expand the sample size to further verify the conclusion.

Genetic analysis of novel pathogenic gene HROB in a family with primary ovarian insufficiency / 浙江大学学报·医学版

A 13-year and 6-month-old girl attended the Hunan Children's Hospital due to delayed menarche. The laboratory test results indicated increased follicle-stimulating hormone and luteinizing hormone, decreased anti-Mullerian hormone, and pelvic ultrasound showed a cord-like uterus and absence of bilateral ovaries. Her 11-year and 5-month-old younger sister had the same laboratory and imaging findings, and both girls were diagnosed with primary ovarian insufficiency. Whole exome sequencing and Sanger sequencing confirmed that the proband and her sister carried heterozygous variants of HROB gene c.718C>T (p.Arg240*) and c.1351C>T (p.Arg451*), which were inherited from their parents respectively and consistent with autosomal recessive inheritance. Oral estradiol valerate at an initial dose of 0.125 mg/d was given to the proband, and the secondary sexual characteristics began to develop after 6 months.

DNA damage repair promotion in colonic epithelial cells by andrographolide downregulated cGAS‒STING pathway activation and contributed to the relief of CPT-11-induced intestinal mucositis

Gastrointestinal mucositis is one of the most debilitating side effects of the chemotherapeutic agent irinotecan (CPT-11). Andrographolide, a natural bicyclic diterpenoid lactone, has been reported to possess anti-colitis activity. In this study, andrographolide treatment was found to significantly relieve CPT-11-induced colitis in tumor-bearing mice without decreasing the tumor suppression effect of CPT-11. CPT-11 causes DNA damage and the release of double-stranded DNA (dsDNA) from the intestine, leading to cyclic-GMP-AMP synthase (cGAS)‒stimulator of interferon genes (STING)-mediated colitis, which was significantly decreased by andrographolide both in vivo and in vitro. Mechanistic studies revealed that andrographolide could promote homologous recombination (HR) repair and downregulate dsDNA‒cGAS‒STING signaling and contribute to the improvement of CPT-11-induced gastrointestinal mucositis. These results suggest that andrographolide may be a novel agent to relieve gastrointestinal mucositis caused by CPT-11.

MiR-375-3p enhanced radiosensitivity of colorectal cancer cells by inhibiting homologous recombination repair of DSBs / 中华放射医学与防护杂志

Objective:To investigate the effect of miR-375-3p on DNA damage repair and radioresistance of colorectal cancer cells.Methods:After overexpression of miR-375-3p in HCT116 and HT29, cell proliferation ability was detected by CCK-8 assay, clone formation ability was detected by clone formation assay, apoptosis was detected by Annexin V-FITC/PI double staining method and cell cycle distribution was detected by flow cytometry, and the formation of γ-H2AX foci were used to analyze homologous recombination (HR) repair efficiency. Bioinformatics was used to predict the downstream target genes of miR-375-3p in the HR repair pathway. A dual luciferase reporter gene assay was used to validate the regulation effect of miR-375-3p on Rad51 gene. The expression of miR-375-3p in HCT116 cells irradiated with 60Co γ-rays at 2 and 6 Gy was measured by RT-qPCR. The inhibition effect of miR-375-3p on the radiosensitivity of HCT116 cells was analyzed after irradiation with different doses of 0, 1, 2, 4 and 6 Gy. Results:Overexpression of miR-375-3p inhibited the proliferation and colony formation ability, induced G1 phase cycle arrest and cell apoptosis of colorectal cancer cells, enhanced DSBs formation, inhibited Rad51 expression, and significantly decreased HR repair efficiency ( t = 10.055, P < 0.05). Dual luciferase reporter gene assay demonstrated that miR-375-3p bound to Rad51 3′UTR region ( t = 5.013, P< 0.05). In addition, irradiation increased miR-375-3p expression, and inhibition of miR-375-3p expression reduced radiosensitivity of colorectal cancer cells ( t=6.460, 5.619, 10.150, P<0.05). Conclusions:miR-375-3p inhibited the homologous recombination repair efficiency of DSBs and enhanced the radiosensitivity of colorectal cancer cells.

Construction of hypervirulent Klebsiella pneumoniae strain with hfq gene deletion and study on its biological characteristics / 中华微生物学和免疫学杂志

Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.

Expression of DNA damage-binding protein 1 in hepatocellular carcinoma tissues and its effect on homologous recombination repair and targeted killing in SMMC-7721 cells / 肿瘤研究与临床

Objective:To investigate the expression of DNA damage repair factor DNA damage-binding protein 1 (DDB1) in hepatocellular carcinoma tissues, and the effect of DDB1 gene silencing on DNA repair and targeted killing in human hepatocellular carcinoma SMMC-7721 cells.Methods:The UALCAN platform was used to perform bioinformatics analysis on the expression of DDB1 in hepatocellular carcinoma tissues (371 cases) and paracancerous tissues (50 cases) in The Cancer Genome Atlas (TCGA) database and the correlation of DDB1 expression with the overall survival of liver cancer patients were analyzed by bioinformatics using the UALCAN platform. SMMC-7721 cells were transfected with small interfering RNA (siRNA) targeting DDB1 and negative control siRNA, which were DDB1 silencing group and negative control group, respectively. X-ray irradiation induced exogenous DNA double strand break (DSB) damage in the two groups of cells. Immunofluorescence staining (γH2AX was used for assessing cellular DSB damage, RPA32s33 and Rad51 were used for assessing homologous recombination repair) and Western blotting (were used to detect the level of RPA32s33 protein) were used to analyze the effect of DDB1 gene silencing on DSB damage repair. Sister chromosome exchange (SCE) experiment was used to analyze the frequency of SCE of homologous recombination of cells in DDB1 silencing group and negative control group. Tetramethylazozolium salt (MTT) method was used to analyze the killing effect of PARP inhibitor olapani (10 μmol/L), cisplatin (1 μg/ml) and olapani combined with cisplatin on SMMC-7721 cells in DDB1 silencing group and negative control group.Results:Bioinformatics analysis showed that the level of DDB1 mRNA in liver cancer tissues was higher than that in paracancerous tissues ( P < 0.001), and the overall survival of patients with high expression of DDB1 was worse than that of patients with low expression of DDB1 ( P = 0.029). When cultured for 4 hours after X-ray irradiation, the number of γH2AX foci in cells of the negative control group had mostly disappeared, and there were still more cells in DDB1 silencing group [(5.1±2.0) per cell vs. (13.4±2.0) per cell, t = -5.08, P = 0.007]. When cultured for 4 hours after X-ray irradiation, the number of RPA32s33 foci in the negative control group [(30.8±5.0) per cell vs. (13.2±1.6) per cell] and the number of Rad51 foci [(19.5±1.8) per cell vs. (8.3±3.3) per cell] were higher than those in the DDB1 silencing group, and the differences between the two groups were statistically significant (both P < 0.01). The frequency of SCE in the negative control group was higher than that in the DDB1 silencing group [(21.2±3.0)% vs. (11.2±1.6)%, t = 5.07, P = 0.007]. MTT assay showed that after olaparib treatment, the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(40.3±3.6)% vs. (79.8±1.3)%, t = 17.94, P < 0.001]. When treated with olapani combined with cisplatin, the survival rate of cells in the two groups further decreased, and the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(10.2±2.8)% vs. (29.6±3.4)%, t = 7.72, P = 0.002]. When treated with cisplatin alone, there was no significant difference in cell survival between DDB1 silencing group and negative control group [(41.9±5.1)% vs. (49.8±3.3)%, t = 2.71, P = 0.054]. Conclusions:The high expression of DDB1 in hepatocellular carcinoma tissues may be an important factor in the treatment resistance and poor prognosis of hepatocellular carcinoma. Knockdown of DDB1 gene expression can promote the sensitivity of SMMC-7721 cells to PARP inhibitors, and its mechanism may be related to the homologous recombination repair defect of SMMC-7721 cells caused by DDB1 silencing.

An efficient marker-free genome editing method for Aspergillus niger / 生物工程学报

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Progress of poly (adenosine diphosphate-ribose) polymerase inhibitors in treatment of colorectal cancer / 肿瘤研究与临床

Colorectal cancer is one of the most common malignant tumors, and its morbidity and mortality increase year by year. In recent years, some patients with colorectal cancer have benefited from precision targeted therapies, but the overall prognosis is still unsatisfactory. Treatment of homologous recombination deficiency represented by BRCA1/2 has become a hot research direction at present. With the wide application and exact curative effect of poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors in ovarian cancer, breast cancer, pancreatic cancer and other malignant tumors, PARP inhibitors are also gradually being used in colorectal cancer. This review summarizes the current research progress of PARP inhibitors in treatment of colorectal cancer.

Platinum in neoadjuvant therapy for triple-negative breast cancer / 中华内分泌外科杂志

Although adding platinum to taxane- and anthracycline-based neoadjuvant chemotherapy regimens for triple-negative breast cancer (TNBC) patients can significantly improve the pathological complete response (pCR) rate and long-term survival, it is associated with higher treatment-related adverse events (AEs) . Current researches focus on the response predictors to select patients who may benefit from platinum-based chemotherapy. Homologous recombination deficiency (HRD) can identify patients who truly need platinum drugs, that is, those with BRCA wild-type but HRD tumors. Results suggest that anthracycline-based chemotherapy is sufficient for BRCA mutation carriers and that non-HRD carriers will not benefit from the added carboplatin.

Screening of a Small Molecule Compound Library Identifies Potential Drugs that Synergize with PARP Inhibitors against Ovarian Cancer Cells / 中国生物化学与分子生物学报

PARP inhibitors are a new class of drugs that target cancer cells with defective DNA repair. Early trials have shown that PARP inhibitors have achieved satisfactory results, but the mechanism of resistance after drug treatment has not been fully revealed. Therefore, it is necessary to find more targeted drugs in combination with PARP inhibitors to kill tumor cells. In this paper, several potential drugs that can synergistically kill ovarian cancer cells with PARP inhibitors were identified based on the combined drug screening of 379 small molecule compound libraries and PARP inhibitor Niraparib through cell proliferation experiments, colony-formation survival experiments and immunofluorescence staining experiments. The results showed that there are eight small molecule compounds with good combination effects, including two small molecule inhibitors STF-118804 and Disulfiram that have been reported to have combined effects with PARP inhibitors. We selected GW441756, an inhibitor of tropomyosin receptor kinase A (TrKA), to verify a variety of tumor cells and explore the preliminary mechanism. The combined therapeutic effects of the Niraparib and the TrKA inhibitor increased the sensitivity of tumor cells to PARP inhibitors (P < 0. 05). Mechanistically, the number of γH2AX foci in the combined treatment group was significantly increased (P<0. 05), indicating that the TrKA inhibitor hindered the DNA damage repair ability. Moreover, combination therapy significantly reduced the formation of RAD51 foci (P<0. 05), a marker of homologous recombination repair (HRR), suggesting that TrKA inhibitors may inhibit DNA damage repair by inhibiting HRR efficiency. Overall, these results suggest that TrKA inhibitor can be used as a potential drug to kill ovarian cancer cells in combination with PARP inhibitors.

Comprehensive functional annotation of susceptibility variants identifies genetic heterogeneity between lung adenocarcinoma and squamous cell carcinoma / 医学前沿

Although genome-wide association studies have identified more than eighty genetic variants associated with non-small cell lung cancer (NSCLC) risk, biological mechanisms of these variants remain largely unknown. By integrating a large-scale genotype data of 15 581 lung adenocarcinoma (AD) cases, 8350 squamous cell carcinoma (SqCC) cases, and 27 355 controls, as well as multiple transcriptome and epigenomic databases, we conducted histology-specific meta-analyses and functional annotations of both reported and novel susceptibility variants. We identified 3064 credible risk variants for NSCLC, which were overrepresented in enhancer-like and promoter-like histone modification peaks as well as DNase I hypersensitive sites. Transcription factor enrichment analysis revealed that USF1 was AD-specific while CREB1 was SqCC-specific. Functional annotation and gene-based analysis implicated 894 target genes, including 274 specifics for AD and 123 for SqCC, which were overrepresented in somatic driver genes (ER = 1.95, P = 0.005). Pathway enrichment analysis and Gene-Set Enrichment Analysis revealed that AD genes were primarily involved in immune-related pathways, while SqCC genes were homologous recombination deficiency related. Our results illustrate the molecular basis of both well-studied and new susceptibility loci of NSCLC, providing not only novel insights into the genetic heterogeneity between AD and SqCC but also a set of plausible gene targets for post-GWAS functional experiments.

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.