Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005-2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.

An assessment of prognostic immunity markers in breast cancer.

Tumor-infiltrating lymphocytes (TIL) and immunity gene signatures have been reported to be significantly prognostic in breast cancer but have not yet been applied for calculation of risk of recurrence in clinical assays. A compact set of 17 immunity genes was derived herein from an Affymetrix-derived gene expression dataset including 1951 patients (AFFY1951). The 17 immunity genes demonstrated significant prognostic stratification of estrogen receptor (ER)-negative breast cancer patients with high proliferation gene expression. Further analysis of blood and breast cancer single-cell RNA-seq datasets revealed that the 17 immunity genes were derived from TIL that were inactive in the blood and became active in tumor tissue. Expression of the 17 immunity genes was significantly (p < 2.2E-16, n = 91) correlated with TILs percentage on H&E in triple negative breast cancer. To demonstrate the impact of tumor immunity genes on prognosis, we built a Cox model to incorporate breast cancer subtypes, proliferation score and immunity score (72 gene panel) with significant prediction of outcomes (p < 0.0001, n = 1951). The 72 gene panel and its risk evaluation model were validated in two other published gene expression datasets including Illumina beads array data METABRIC (p < 0.0001, n = 1997) and whole transcriptomic mRNA-seq data TCGA (p = 0.00019, n = 996) and in our own targeted RNA-seq data TARGETSEQ (p < 0.0001, n = 303). Further examination of the 72 gene panel in single cell RNA-seq of tumors demonstrated tumor heterogeneity with more than two subtypes observed in each tumor. In conclusion, immunity gene expression was an important parameter for prognosis and should be incorporated into current multi-gene assays to improve assessment of risk of distant metastasis in breast cancer.

[Preliminary assessment of immune reconstitution after autologous peripheral hematopoietic stem cell transplantation (AHSCT)].

BACKGROUND & OBJECTIVES: Though high dose chemo-therapy combined with autologous hematopoietic stem cell transplantation (AHSCT) has made great progress on the treatment of chemo-sensitive malignant tumors, the relapse rate remains high. Successful immune reconstitution after AHSCT may reduce recurrence; therefore this study was to explore the characteristics of immune reconstitution after AHSCT and assess its feasibility in clinical use. METHODS: Twenty four cases after AHSCT were enrolled in our study. There were 19 Non-hodgkin Lymphoma (NHL), 3 Hodgkin Lymphoma (HD) and 2 rhabdomyosarcoma. Nineteen cases had achieved complete remission (CR) while 5 partial remission (PR) before AHSCT. All cases were administered Interleukin (IL)-2 and Interferon (IFN)-alpha after AHSCT. Some patients were given thymus factor and/or CIK infusion. Phenotypes of peripheral blood T, B, NK subsets and immunological profile of TH1/TH2 by intracellular staining of cytokines after PMA/ionomycin stimulation were evaluated. RESULTS: 75% of the cases achieved CR while 4.17% were progression of disease (PD) and 16.67% were relapsed during the median follow-up time of 12 (2-60) months. The changes of immune parameters after AHSCT were as followed: (1) CD4+T cells (normal control 33.5+/-6.9%) started to decrease dramatically one month after AHSCT, which was 2.5-13% (median rate 5.6%)in the 2nd month; and then slowly increased to 10-20% in the 7th month, but did not return back to normal even after one year in all patients. In addition, reversed ratio of CD4/CD8 lasted for a long period of time. B cells also began to decrease 1 month after AHSCT, and recovered to normal in the 4th month. But B cells remained 0% in the 6th month and 1% in 12th month in patients treated by rituximab before receiving AHSCT. The ratio of NK cells was 10-20% (higher than normal controls) in the 2nd month, then returned to normal thereafter. (2) The cytokine secretion by T cell: there were 48.79% patients whose TH1 was lower than normal controls or at the lower limit of normal range. All the patients with normal TH1 were treated by IFN-alpha or CIK cell infusion. TH2 was much higher than normal level among 68.29% cases and this abnormality lasted at least for 1 year in some cases. TH2 at normal range was only observed in cases receiving IFN-alpha treatment. Furthermore, IFN-alpha could significantly decrease TH2 level. (3) Increasing tendency of CD4+CD25+/CD4+, CD4+CD69+/CD4+ ratio was observed in patients received additional thymus factor treatment compared to those did not. CONCLUSIONS: Administration of CIK cells, thymus factor, IL-2 and IFN-alpha after AHSCT could improve the immunologic function of patients, and TH1/TH2 ratio may virtually reflect the immune status of patients. However more information is required to make prognostic assessments of immune reconstruction and the long-term survival rate.