Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells.

The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.

Insulin-like growth factor-1 regulates follicle selection of hens by promoting proliferation and inhibiting apoptosis of granulosa cells in prehierarchical follicles in vitro.

During the reproduction stage of poultry, a single follicle is selected from a cohort of 6-8 mm small yellow follicles to initiate rapid growth and final ovulation almost daily. In the process, follicle-stimulating hormone (FSH) plays a pivotal role by interacting with intraovarian factors, including insulin-like growth Factor 1 (IGF1). The objective of this study was to analyze whether IGF1 coordinates with FSH to affect the characteristics of granulosa cells from prehierarchical follicles. After treating granulosa cells with 50 ng/mL FSH and 200 ng/mL IGF1, we detected the proliferation and apoptosis of granulosa cells using flow cytometry. The percentage of G1 phase granulosa cells was increased, and the percentage of mitotic cells and apoptotic cells was reduced under IGF1 treatment. The expression levels of the steroidogenic acute regulatory protein gene, cytochrome P450 side-chain cleavage gene and 3ß-hydroxysteroid dehydrogenase gene, which are related to steroidogenic synthesis, were reduced by cotreatment with FSH and IGF1. The expression of the cell proliferation- or apoptosis-related genes cyclin dependent kinase 2, cyclin D2, B-cell leukemia/lymphoma 2, and BCL2 like 1 and the ratio of B-cell leukemia/lymphoma 2/BCL2-associated X were increased by treatment with IGF1. There was a decrease in the expression of caspase3 after treatment with FSH and IGF1. All these results showed that IGF1 reduced the expression of genes involved in progesterone synthesis, stimulated proliferation and inhibited apoptosis in granulosa cells. Thus, IGF1 may be one of the factors involved in affecting FSH responsiveness and maintaining the undifferentiated state of prehierarchical follicles before follicle selection.

Adjuvant Chemotherapy for Low-Clinical-Risk Breast Cancer Defined by Modified Version of Adjuvant! Online: A Propensity Score Matched SEER Analysis.

BACKGROUND: The purpose of this research was to investigate whether the modified version of Adjuvant! Online was able to omit chemotherapy (CT) for patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, and axillary node-negative breast cancer, who are defined as low clinical risk. METHODS: From 2010 to 2014, HR-positive, HER2-negative, and node-negative breast cancer patients aged 50 years and older were retrieved from the Surveillance, Epidemiology, and End Results (SEER) 18 database. The propensity score matching method was applied between the no-CT and CT groups. Overall survival (OS) was evaluated using Kaplan-Meier analysis and compared across groups using a log-rank test. RESULTS: A total of 48,857 patients were enrolled. After propensity score matching, the numbers of patients in the no-CT and CT groups were both 3,102. The median follow-up period was 37 months. The 5-year OS rates in the no-CT and CT groups were 92 and 91%, respectively (p = 0.066). In the subgroup with a tumor score (tumor size added to tumor grade) of 2-3, OS was significantly higher in the no-CT group than in the CT group (93 vs. 90%, p < 0.001). In the subgroup with a tumor score of 4, OS was not different between these 2 groups (92 vs. 93%, p = 0.47). CONCLUSION: This retrospective study provides evidence that CT may not be beneficial to patients 50 years of age or older with HR-positive, HER2-negative, axillary node-negative breast cancer and additionally defined as low clinical risk by a modified version of Adjuvant! Online.

Capecitabine in Combination with Standard (Neo)Adjuvant Regimens in Early Breast Cancer: Survival Outcome from a Meta-Analysis of Randomized Controlled Trials.

Capecitabine has been investigated in early breast cancer in several studies, but it was undefined that whether it could improve survival. To investigate whether the addition of capecitabine affected survival in patients with early breast cancer, a meta-analysis was conducted and overall survival (OS), disease-free survival (DFS), and toxicity were assessed. The PubMed, Embase databases and the Cochrane Central Register of Controlled Trials were searched for studies between January 2006 and April 2016. Hazard ratios (HRs) with their 95% confidence intervals (CIs), or data for calculating HRs with 95% CI were derived. Seven trials with 9097 patients, consisted of 4 adjuvant and 3 neoadjuvant studies, were included in this meta-analysis. Adding capecitabine showed no improvement in DFS (HR = 0.93; 95% CI, 0.85-1.02; P = 0.12), whereas a significant improvement in OS was observed (HR = 0.85; 95% CI, 0.75-0.96; P = 0.008). A sub-analysis of DFS showed that benefit of capecitabine derived from patients with triple negative subtype and with extensive axillary involvement. Safety profiles were consistent with the known side-effects of capecitabine, but more patients discontinued scheduled treatment in the capecitabine group. Combining capecitabine with standard (neo)adjuvant regimens in early breast cancer demonstrated a significantly superior OS, and indicated DFS improvement in some subtypes with high risk of recurrence. Selection of subtypes was a key to identify patients who might gain survival benefit from capecitabine.

Postoperative adjuvant therapy for resectable thoracic esophageal squamous cell carcinoma: a retrospective analysis of 426 cases.

The aim of this study was to evaluate the value of postoperative adjuvant therapy for resectable thoracic esophageal squamous cell carcinoma (ESCC) in China. We retrospectively analyzed 426 eligible patients seen between October 2007 and November 2011. Specifically, we assessed clinicopathological characteristics and the disease-free and overall survival rates. Of the 426 patients, 272 cases underwent surgery alone, and 154 cases received postoperative adjuvant therapy (67 cases with radiotherapy, 57 cases with chemotherapy, and 30 cases with simultaneous chemoradiotherapy). The median follow-up time was 48.0 months (23.0-72.0 months), and the median survival time was 48.4 months (1.0-72.0 months). We found a significant difference between the surgery-alone and adjuvant therapy groups in the status of lymph node (LN) metastasis (N stage; P < 0.01), but there were no differences between the two groups with regard to other clinicopathological characteristics, including age, sex, lesion location, T stage, differentiation grades, surgery approach, or average number of LN dissections. The 5-year disease-free survival (DFS) rates of the surgery-alone and adjuvant therapy groups were 48.9 and 37.1 %, respectively (P < 0.001); no significant difference was found in 5-year overall survival (OS) rate between the two groups (P > 0.05). A stratification analysis based on N stage suggested that the 5-year DFS and OS rates were similar in N0-N3 subgroups (P > 0.05), except that patients with surgery alone had a higher 5-year DFS than those with postoperative adjuvant therapy in N0 subgroup (P = 0.013). Our data suggest that patients with resectable thoracic ESCC may not benefit from postoperative adjuvant therapy. Further prospective studies are required to elucidate the utility of postoperative adjuvant therapy and to standardize individualized treatments for resectable ESCC.