Amplification of RAD54B promotes progression of hepatocellular carcinoma via activating the Wnt/β-catenin signaling

Liver cancer was reported to be the sixth most frequently diagnosed cancer, and hepatocellular carcinoma (HCC) accounts for 75%-85% of primary liver cancer. Nevertheless, the concrete molecular mechanisms of HCC progression remain obscure, which is essential to elucidate. The expression profile of RAD54B in HCC was measured using qPCR and western blotting. Moreover, the levels of RAD54B in paraffin-embedded samples were evaluated using immunohistochemistry (IHC). The effect of RAD54B on HCC progression was testified by in vitro experiments, and in vivo orthotopic xenograft tumor experiments. The mechanisms of RAD54B promoting HCC progression were investigated through molecular and function experiments. Herein, RAD54B are dramatically upregulated in HCC tissues and cell lines both on mRNA and protein levels, and RAD54B can servers as an independent prognostic parameter of 5-year overall survival and 5-year disease-free survival for patients with HCC. Moreover, up-regulation of RAD54B dramatically increases the capacity for in vitro cell viability and motility, and in vivo intrahepatic metastasis of HCC cells. Mechanistically, RAD54B promotes the HCC progression through modulating the wnt/β-catenin signaling. Notably, blocking the wnt/β-catenin signaling axis can counteract the activating effects of RAD54B on motility of HCC cells. Besides, further analysis illustrates that DNA amplification is one of the mechanisms leading to mRNA overexpression of RAD54B in HCC. Our findings indicate that RAD54B might be a promising potential prognostic marker and a candidate therapeutic target to therapy HCC.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Late‐life rapamycin treatment reverses age‐related heart dysfunction

Rapamycin has been shown to extend lifespan in numerous model organisms including mice, with the most dramatic longevity effects reported in females. However, little is known about the functional ramifications of this longevity‐enhancing paradigm in mammalian tissues. We treated 24‐month‐old female C57BL/6J mice with rapamycin for 3 months and determined health outcomes via a variety of noninvasive measures of cardiovascular, skeletal, and metabolic health for individual mice. We determined that while rapamycin has mild transient metabolic effects, there are significant benefits to late‐life cardiovascular function with a reversal or attenuation of age‐related changes in the heart. RNA‐seq analysis of cardiac tissue after treatment indicated inflammatory, metabolic, and antihypertrophic expression changes in cardiac tissue as potential mechanisms mediating the functional improvement. Rapamycin treatment also resulted in beneficial behavioral, skeletal, and motor changes in these mice compared with those fed a control diet. From these findings, we propose that late‐life rapamycin therapy not only extends the lifespan of mammals, but also confers functional benefits to a number of tissues and mechanistically implicates an improvement in contractile function and antihypertrophic signaling in the aged heart with a reduction in age‐related inflammation.     

LIN54 harboring a mutation in CHC domain is localized to the cytoplasm and inhibits cell cycle progression

The mammalian LIN complex (LINC) plays important roles in regulation of cell cycle genes. LIN54 is an essential core subunit of the LINC and has a DNA binding region (CHC domain), which consists of two cysteine-rich (CXC) domains separated by a short spacer. We generated various LIN54 mutants, such as CHC deletion mutant, and investigated their subcellular localizations and effects on cell cycle. Wild-type LIN54 was predominantly localized in the nucleus. We identified two nuclear localization signals (NLSs), both of which were required for nuclear localization of LIN54. Interestingly, deletion of one CXC domain resulted in an increased cytoplasmic localization. The cytoplasmic LIN54 mutant accumulated in the nucleus after leptomycin B treatment, suggesting CRM1-mediated nuclear export of LIN54. Point mutations (C525Y and C611Y) in conserved cysteine residues of CXC domain that abolish DNA binding activity also increased cytoplasmic localization. These data suggest that DNA binding activity of LIN54 is required for its nuclear retention. We also found that LIN54^C525Y and LIN54^C611Y inhibited cell cycle progression and led to abnormal nuclear morphology. Other CXC mutants also induced similar abnormalities in cell cycle progression. LIN54^C525Y led to a decreased expression of some G₂/M genes, whose expressions are regulated by LINC. This cell cycle inhibition was partially restored by overexpression of wild-type LIN54. These results suggest that abnormal cellular localization of LIN54 may have effects on LINC activity.     

Synthetic Lethal Targeting of Superoxide Dismutase 1 Selectively Kills RAD54B-Deficient Colorectal Cancer Cells

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.     

Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies

Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial–mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.     

瓜蚜为害对寄主植物主要营养物质和次生物质的影响

为明确瓜蚜Aphis gossypii Glover与其寄主植物相互作用关系及其机理,本文采用蒽酮比色法、考马斯亮蓝法、磷钼酸－磷钨酸比色法及索氏回流法,分别测定了瓜蚜为害对黄瓜、哈密瓜、南瓜、瓢葫芦和搅瓜等5种寄主植物叶片内可溶性总糖、可溶性蛋白质、单宁及黄酮含量的影响。结果表明,瓜蚜为害后,供试寄主植物叶片中可溶性糖和可溶性蛋白含量显著降低,而单宁和黄酮的含量明显上升。说明瓜蚜为害导致寄主植物营养质量下降,而次生代谢物质含量上升,从而对植食者产生诱导抗性。     

SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

A resource of genome‐wide single‐nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.     

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.