Map-based cloning reveals the complex organization of the BnRf locus and leads to the identification of BnRf(b), a male sterility gene, in Brassica napus.

KEY MESSAGE: Sequencing of BAC clones reveals the complex organization of the BnRf locus and allowed us to clone BnRf (b) , which encodes a nucleus-localized chimeric protein BnaA7.mtHSP70-1-like. The male sterility in an extensively used genic male sterility (GMS) line (9012A) in Brassica napus was regarded to be conferred by BnMs3/Bnms3 and the multiallelic BnRf locus including three alleles. We previously mapped BnRf to a 13.8 kb DNA fragment on the B. napus chromosome A7. In the present study, we isolated bacterial artificial chromosome clones individually covering the restorer allele BnRf (a) and the male-sterile allele BnRf (b) , and revealed that the candidate regions of BnRf (a) and BnRf (b) show complex structural variations relative to the maintainer allele BnRf (c). By analyzing the recombination events and the newly developed markers, we delimited BnRf (a) to a 35.9 kb DNA fragment that contained seven predicted open-reading frames (ORFs). However, genetic transformation of the ORF G14 from both the male-sterile and restorer lines into wild-type Arabidopsis plants led to a stable male-sterile phenotype matching a 9012A-derived GMS line (RG206A); moreover, the male sterility caused by G14 could be fully recovered by the restorer gene BnMs3. These facts indicate that BnRf (b) corresponds to G14 while BnRf (a) likely associates with another flanking ORF. G14 encodes a nucleus-localized chimeric protein designated as BnaA7.mtHSP70-1-like. Ectopic expression of G14 in Arabidopsis negatively regulates some vital genes responsible for tapetum degeneration, and delayed programmed cell death of tapetum and led to the developmental arrest of tetrads. Our work not only presents new insights on the hereditary model of sterility control but also lays a solid foundation for dissecting the molecular basis underlying male sterility and restoration in 9012A.

Identification of potential pathogenic biomarkers in clear cell renal cell carcinoma.

The purpose of the present study was to screen potential pathogenic biomarkers of clear cell renal cell carcinoma (ccRCC) via microarray analysis. The mRNA and microRNA (miRNA) expression profiles of GSE96574 and GSE71302 were downloaded from the Gene Expression Omnibus (GEO) database, as well as the methylation profile of GSE61441. A total of 5 ccRCC tissue samples and 5 normal kidney tissue samples were contained in each profile of GSE96574 and GSE71302, and 46 ccRCC tissue samples and 46 normal kidney tissue samples were involved in GSE61441. The differentially expressed genes (DEGs) and the differentially expressed miRNAs (DEMs) were obtained via limma package in ccRCC tissues compared with normal kidney tissues. The Two Sample t-test and the Beta distribution test were used to identify the differentially methylated sites (DMSs). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs. The targets of the DEMs were screened with the miRWalk database, and the further combination analyses of DEGs, DEMs and DMSs were conducted. Additionally, reverse transcription PCR (RT-PCR) and methylation-specific PCR (MS-PCR) were performed to detect the mRNA level and methylation status of HAPLN1. The mRNA levels of hsa-miR-204 and hsa-miR-218 were tested by RT-PCR. A total of 2,172 DEGs, 202 DEMs and 2,172 DMSs were identified in RCC samples compared with normal samples. The DEGs were enriched in 1,015 GO terms and 69 KEGG pathways. A total of 10,601 miRNA-gene pairs were identified in at least 5 algorithms of the miRWalk database. A total of 143 overlaps were identified between the DEGs and the differentially methylated genes. Furthermore, the DEGs were involved in 851 miRNA-gene pairs, including 127 pairs in which the target genes were negatively associated with their corresponding DEMs and DMSs. HAPLN1 was lowly expressed and highly methylated in ccRCC tissues, while hsa-miR-204 and hsa-miR-218 were highly expressed. The results of the present study indicated that HAPLN1, hsa-miR-204 and hsa-miR-218 may be involved in the pathogenesis of ccRCC.

Outbreak by Ventilator-Associated ST11 K. pneumoniae with Co-production of CTX-M-24 and KPC-2 in a SICU of a Tertiary Teaching Hospital in Central China.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) often responsible for numerous hospital-associated outbreaks has become an important public health problem. From January 2013 to February 2014, a total of 41 non-duplicate K. pneumoniae isolates with carbapenem resistance, were collected at a tertiary teaching hospital in Nanchang, central China. Among 41 K. pneumoniae isolates, 28 were isolated from hospitalized patients including 19 from the patients in surgery intensive care unit (SICU) and 13 were isolated from ventilators. Twenty-four of 28 patients infected by CRKP have been submitted to mechanical ventilation using ventilator. More than 95% of the CRKP isolates were resistant to 13 antimicrobials tested. All CRKP isolates were confirmed as carbapenemase producer and were positive for bla KPC-2, with one positive for both blaKPC-2 and bla NDM-1. All carbapenemase-producing isolates harbored at least one of extended spectrum ß-lactamase genes tested, among which 95.1% (39/41) of the tested isolates were found to harbor both bla CTX-M-24 and bla KPC-2, Of note, one isolate harbored simultaneously two carbapenemase genes (bla KPC-2 and bla NDM-1) and two ESBL genes (bla CTX-M-3 and bla TEM-104). To the best of our knowledge, coexistence of bla KPC-2 and bla CTX-M-24 in one isolate is first reported. MLST results showed that 41 CRKP isolates belonged to four sequence types (STs) including ST11, novel ST1854, novel ST1855, and ST1224. PFGE results displayed three PFGE clusters. Thirty-eight ST11 CRKP isolates (92.7%, 38/41) including all 13 isolates from ventilators and 25 isolates from patients from seven wards (18 from SICU) belonged to same PFGE cluster, indicating these isolates were clonally related. Fifteen isolates have an identical undistinguished pattern (100% similarity) forming a single clonal population. Moreover, this clone was exclusively linked to the cases attended in SICU and linked to the Ventilators. Additionally, the other SICU cases were linked to closely related clones (similarity greater than 95%). These data indicated that the occurrence of a clonal outbreak associated with ventilators has been found. In conclusion, outbreak by ventilator-associated ST11 K. pneumoniae with co-production of CTX-M-24 and KPC-2 is found in a SICU of a tertiary teaching hospital in central China.