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Chromosomal abnormalities (CAs) can cause spontaneous miscarriage and increase the incidence of subsequent pregnancy loss and other complications. Presently, CAs are detected mainly by array comparative genomic hybridization (CGH) and single nucleotide polymorphism microarrays. The present study developed a low-coverage next-generation sequencing method to detect CAs in spontaneous miscarriage and assess its clinical performance. In total, 1,401 patients who had experienced an abortion were enrolled in the present study and divided into two groups. In group I, 437 samples that had been previously validated by array CGH were used to establish a method to detect CAs using a semiconductor sequencing platform. In group II, 964 samples, which were not verified, were assessed using established methods with respect to clinical significance. Copy number variant (CNV)-positive and euploidy samples were verified by array CGH and short tandem repeat profiling, respectively, based on quantitative fluorescent PCR. The low-coverage sequencing method detected CNVs >1 Mb in length and a total of 3.5 million unique reads. Similar results to array CGH were obtained in group I, except for six CNVs 1 Mb, with advantages of requiring less input DNA and lower cost.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent one of the successful novel approaches to targeted cancer treatment. Indeed, the US Food and Drug Administration (FDA) has recently approved PARP inhibitors for the treatment of breast and ovarian cancers. Despite the proven efficacy of these agents, certain challenges remain with their use. Among the most important are primary and secondary resistance. Here, we review the mechanism of action of PARP inhibitors and their ability to exploit certain inherent deficiencies among malignant cells to improve cell killing, with a focus on deficiencies in homologous recombination among cells with BRCA1 and BRCA2 mutations. Moreover, we discuss the different mechanisms of resistance including development of secondary resistance and strategies to overcome them. Finally, we discuss the limitations of novel therapeutic interventions and possible future studies to exploit biochemical pathways in order to improve therapeutic efficacy of PARP inhibitors.

Background Chemotherapy-induced peripheral neuropathy is not only one of the most common causes of dose reduction or discontinuation of cancer treatment, but it can also permanently decrease the quality of life of cancer patients and survivors. Notably, Sirt2 protects many organs from various injuries, including diabetic peripheral neuropathy. As demonstrated previously by our laboratory and others, the overexpression of Sirt2 can improve cisplatin-induced neuropathy, although the mechanism is still unclear. Results In this study, the underlying mechanism by which Sirt2 protects neurons from cisplatin-induced injury was explored using the RNAseq technique in cultured rodent neurons. Sirt2 status was modified by genetic knockout (Sirt2/KO) and was then reconstituted in Sirt2/KO cells (Sirt2/Res). We observed 323 upregulated genes and 277 downregulated genes in Sirt2-expressing cells (Sirt2/Res) compared to Sirt2-deficient cells (Sirt2/KO). Pathway analysis suggested that Sirt2 may affect several pathways, such as MAPK, TNF, and cytokine–cytokine interaction. Furthermore, cisplatin-induced changes to the transcriptome are strongly associated with Sirt2 status. Cisplatin induced distinctive transcriptome changes for 227 genes in Sirt2-expressing cells and for 783 genes in Sirt2-deficient cells, while changes in only 138 of these genes were independent of Sirt2 status. Interestingly, changes in the p53 pathway, ECM–receptor interactions, and cytokine–cytokine receptor interactions were induced by cisplatin only in Sirt2-deficient cells. Conclusions This study demonstrated that Sirt2 regulates the transcriptome in cultured rodent neuronal cells. Furthermore, Sirt2-associated transcriptome regulation may be an important mechanism underlying the role of Sirt2 in organ protection, such as in cisplatin-induced neuronal injury. Sirt2 may be a potential target for the prevention and treatment of chemotherapy-induced neuropathy.

SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. Using murine allograft melanoma models, our results suggest increased systemic expression of SIRT2 promotes tumor progression. In this study, SIRT2-overexpressing mice exhibited enhanced tumor growth and larger tumor volumes compared to their wild-type littermates. Mechanistically, systemic overexpression of SIRT2 reduces the number of tumor-infiltrating natural killer (NK) cells and suppresses NK cell function and proliferation within the tumor microenvironment (TME). Furthermore, despite the enhancing effect of NK cell depletion on tumor volume and growth rate in wild-type littermate mice, this effect was diminished in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increases NK cell tumor infiltration and suppresses allograft melanoma tumor growth. The findings of this study identify a dynamic functional interaction between systemic SIRT2 and NK cell activity, which controls melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could present a new opportunity to mediate immunotherapy response and resistance.

Sugar metabolism is the most important and core one which drives plant growth and development. Invertases are key enzymes that regulate sugar metabolism. A still-growing number of studies have revealed that invertases play a crucial role in various aspects of plant growth and development. Crop yield is the product of sugar metabolism; it could be deduced that invertase also regulated the yield formation. So we have done a series of research on soluble acid invertase in sweet sorghum from enzyme activity to gene cloning and functional marker development. In this paper, we sequenced full length of SAI-1 gene in 69 grain sorghum parent lines, trying to see how it differs in their gene sequences and their distribution in related hybrid varieties released in the past. To our surprise, the result showed that B-lines and restore lines (R-line) have almost different SAI-1 haplotype distribution. The change of haplotype of SAI-1 gene is associated with yield gain as with grain sorghum breeding progress, which proved that SAI-1 may take a very important role in yield formation. And we also found the SAI-1 gene tends to become shorter as with the breeding advance, which means short sequence in introns, while exon remains unchanged leading to higher gene efficiency. The best SAI-1 haplotype combination of sorghum hybrid was also found for different planting regions. These findings are of great significance for improving breeding efficiency, understanding heterosis, and germplasm enhancement. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01231-2.

MAIN CONCLUSION The sorghum soluble acid invertase gene SbSAI-2 was cloned and the function verified in Pichia pastoris and rice, showing the SbSAI-2 affects composition and content of sugar in stem juice. Sugar metabolism is one of the most important metabolic processes in plants, in which soluble acid invertase plays a key role. However, the structure and function of the soluble acid transferase gene in sorghum are still fully unclear. In this study, SbSAI-2 was cloned from the sorghum variety BTx623, and two transcripts were found through sequence analysis, with only one transcript translated into an active protein. There is 72% homology between SbSAI-2 and OsVIN2. The construction of Osvin2 mutant lines and SbSAI-2-1 overexpression lines in Oryza sativa L. japonica. cv. Nipponbare were produced to clarify the invertase functionality. While the invertase activity in the stem of the Osvin2 mutant line was reduced, with no significant difference (P > 0.05), and the contents of fructose and glucose in stem tissue did not change significantly (P > 0.05), and the content of sucrose increased by 38.89% (P

Ginsenosides F4 and Rg6 (GF4 and GRg6), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F4 and Rg6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F4 and Rg6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F4 and Rg6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F4 and Rg6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3−M5, M7−M9 and N1 (N5), N2, N4 (N9), N7−N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization,...

Ethnopharmacological relevance Nauclea officinalis Pierrc ex Pitard. is a Chinese medicinal herb that contains high level of alkaloids which is the most abundant and active constituent. Strictosamide isolated from Nauclea officinalis Pierrc ex Pitard. showed significant effects on inflammatory response, compared with pumiloside, 3- epi -pumiloside, vincosamide, 3α,5α-tetrahydrodeoxycordifoline lactam and naucleamide A-10-O-β-D-glucopyranoside of this plant. Aim of study we investigated the biological activities of the six compounds mentioned-above, and the underlying molecular mechanism exerted by the most potent one, strictosamide. Materials and methods The effects of strictosamide and other five compounds on the inhibitory activity of nitric oxide (NO) were screened by Griess test. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in media were detected by using Enzyme-linked immunosorbent (ELISA) kits. The effects on the mRNA expression of nitric oxide synthase (iNOS), TNF-α and IL-1β of strictosamide were further investigated by RT-qPCR. Western blot assay was conducted to illustrate the effects of strictosamide on iNOS and phosphorylation of p65, inhibitor of NF-κB (IκB)-α, IκB-kinase (IKK)-α as well as p-extracellular signal-regulated kinase (ERK), p-c-jun N-terminal kinase (JNK) and p-p38 in the protein levels. Results Strictosamide potently suppressed the productions of NO, TNF-α and IL-1β in LPS-induced RAW 264.7 macrophages, and it dose-dependently alleviated the LPS-simulated protein level of iNOS as well as the mRNA expressions of iNOS, TNF-α and IL-1β. In addition, molecular data revealed that strictosamide markedly decreased the expressions of p-p65, p-IκBα and p-IKKα. Furthermore, strictosamide significantly attenuated LPS-induced the phosphorylation of p38, ERK and JNK. Conclusions At present study, the results indicated that the anti-inflammatory activity of strictosamide was associated with the restraint of NO, TNF-α and IL-1β via negative regulation of both NF-κB and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells.

Effect of laparoscopic myomectomy on the serum levels of interleukin-6 (IL-6) and total antioxidant capacity (TAC), and the ovarian function of patients with uterine fibroids was studied. Ninety patients with uterine fibroids admitted to The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, from March 2013 to July 2014, were randomly divided into the experimental group (n=45) and the control group (n=45). The experimental group was treated with laparoscopic myomectomy, and the control group was treated with abdominal myomectomy. ELISA was used for detecting IL-6 and TAC content. Radioimmunoassay (RIA) was used for detecting serum ovarian function indicators, including estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Patients in the experimental group had significantly lower intraoperative blood loss, postoperative exhaust time, number of days to recovery and return to work, number of intraoperative fibroids removed, and length of stay than those in the control group (P

Resistance to genotoxic therapies is a primary cause of treatment failure and tumor recurrence. The underlying mechanisms that activate the DNA damage response (DDR) and allow cancer cells to escape the lethal effects of genotoxic therapies remain unclear. Here, we uncover an unexpected mechanism through which pyruvate kinase M2 (PKM2), the highly expressed PK isoform in cancer cells and a master regulator of cancer metabolic reprogramming, integrates with the DDR to directly promote DNA double-strand break (DSB) repair. In response to ionizing radiation and oxidative stress, ATM phosphorylates PKM2 at T328 resulting in its nuclear accumulation. pT328-PKM2 is required and sufficient to promote homologous recombination (HR)-mediated DNA DSB repair through phosphorylation of CtBP-interacting protein (CtIP) on T126 to increase CtIP's recruitment at DSBs and resection of DNA ends. Disruption of the ATM-PKM2-CtIP axis sensitizes cancer cells to a variety of DNA-damaging agents and PARP1 inhibition. Furthermore, increased nuclear pT328-PKM2 level is associated with significantly worse survival in glioblastoma patients. Combined, these data advocate the use of PKM2-targeting strategies as a means to not only disrupt cancer metabolism but also inhibit an important mechanism of resistance to genotoxic therapies.

Unrepaired DNA double-strand breaks (DSBs) are lethal. The present study compared the extent of DSBs, neuronal apoptosis, and status of two major DSB repair pathways - homologous combinational repair (HR) and nonhomologous end-joining (NHEJ) - in hippocampus of 5–6 month and 16–18 month-old wild-type and APP/PSEN1 mice fed control diet or high fat diet (60% fat from lard). We performed immunohistochemical staining and quantification for nuclear foci formation of γ-H2AX for DSBs, RAD51, and 53BP1, which represent the functional status of HR and NHEJ, respectively. Increased γ-H2AX and caspase-3 staining indicated greater DSBs and associated neuronal apoptosis in APP/PSEN1 mice at both ages studied. RAD51-positive foci were fewer in APP/PSEN1 indicating that HR processes may be diminished in these mice, although NHEJ (53BP1 staining) appeared unchanged. High fat diet in young wild-type mice led to similar changes to those observed in APP/PSEN1 mice (γ-H2AX and caspase-3 staining, and fewer RAD51-positive foci). Overall, these data suggest that APP/PSEN1- and high fat diet-associated early accumulation of DSBs and neuronal cell death, resulted at least in part, from inhibition of HR, one of the major DSB repair pathways.

Idiopathic asthenospermia is the most common type of male infertility. Although the mechanisms causing asthenospermia are complex, recent studies have indicated an important role of cation channel of sperm (CATSPER) gene downregulation or abnormality in the etiology of idiopathic asthenospermia. In the present study, 192 patients with idiopathic asthenospermia and 288 healthy controls were enrolled, and a flight mass spectrometry using Sequenom’s MassArray biochip system was applied for genotyping 16 CATSPER gene SNPs reported in the human single nucleotide polymorphism (SNP) database. Our results indicated a correlation between CATSPER1 SNPs and idiopathic asthenospermia. In particular, the exonal SNP rs1893316 in CATSPER1 significantly correlated with idiopathic asthenospermia risk and is a potential important factor in determining an individual’s genetic susceptibility to idiopathic asthenospermia. These finding will help to further elucidate the role of CATSPER1 in idiopathic asthenospermia pathogenesis.

The production capacity and yield of neodymium (Nd) in China have ranked the first in the world. Because of its unique biophysical and biochemical properties, Nd compounds have entered into the agricultural environment greatly to promote plant growth. Mitochondria play a crucial role in respiration and metabolism during the growth of plants. However, little is known about the mechanism by which Nd act at the mitochondrial level in plant cells. In this study, rice mitochondrial swelling, collapsed transmembrane potential and decreased membrane fluidity were examined to be important factors for mitochondria permeability transition pore (mPTP) opening induced by Nd(III). The protection of cyclosporin A (CsA) and dithiothreitol (DTT) could confirm that Nd(III) could trigger mPTP opening. Additionally, mitochondrial membrane breakdown observed by TEM and the release of cytochrome c (Cyt c) could also elucidate the mPTP opening from another point of view. At last, the study showed that Nd(III) could restrain the mitochondrial membrane lipid peroxide, so it might interact with anionic lipid too. This detection will be conductive to the safe application of Nd compounds in agriculture and food industry.

Exposure to high levels of estrogen is considered an important risk factor for susceptibility to breast cancer. Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk, but no comprehensive study has been performed among Han Chinese women. In the present study, 32 single-nucleotide polymorphisms (SNPs) in estrogen-related genes were genotyped using the MassARRAY IPLEX platform in 1076 Han Chinese women. Genotypic and allelic frequencies were compared between case and control groups. Unconditional logistic regression was used to assess the effects of SNPs on breast cancer risk. Associations were also evaluated for breast cancer subtypes stratified by estrogen receptor (ER) and progesterone receptor (PR) status. Case-control analysis showed a significant relation between heterozygous genotypes of rs700519 and rs2069522 and breast cancer risk (OR = 0.723, 95% CI = 0.541–0.965, p = 0.028 and OR = 1.500, 95% CI = 1.078–2.087, p = 0.016, respectively). Subgroup comparisons revealed that rs2446405 and rs17268974 were related to ER status, and rs130021 was associated with PR status. Our findings suggest that rs700519 and rs2069522 are associated with susceptibility to breast cancer among the Han Chinese population and have a cumulative effect with three other identified SNPs. Further genetic and functional studies are needed to identify additional SNPs, and to elucidate the underlying molecular mechanisms.

Recent genome-wide association studies (GWASs) have identified 15q25.1 as a lung cancer susceptibility locus. Here, we sought to explore the direct carcinogenic effects of genetic variants in this region on the risk of developing lung adenocarcinoma (ADC). Five common SNPs (rs8034191, rs16969968, rs1051730, rs938682, and rs8042374) spanning the 15q25.1 locus were assayed in a case-control study examining a cohort of 301 lung ADCs and 318 healthy controls. Stratification analysis by gender, smoking status, and tumor, node, metastasis (TNM) classification, was performed. In addition, sections from ADC tissue and normal tissue adjacent to tumors were stained with an anti-CHRNA3 (cholinergic receptor nicotinic α3) antibody by immunohistochemistry in 81 cases. Our results demonstrate that rs8042374, a variant of the CHRNA3 gene, is associated with an increased risk of ADC with an OR of 1.76 (95% CI: 1.17–2.65, p = 0.024). This variant was linked to a greater risk of ADC in female nonsmokers (OR (95% CI): 1.81 (1.05–3.12), p = 0.032) and female stage I + II cases (OR (95% CI): 1.92 (1.03–3.57), p = 0.039). Although located within the same gene, rs938682 showed protective effects for smokers, stage III + IV cases, and male stage III + IV cases. Additionally, the CHRNA3 protein level in ADC tissue was slightly higher than in the surrounding normal lung tissue, based on immunohistochemical analysis. Our results suggest that the CHRNA3 polymorphism functions as a genetic modifier of the risk of developing lung ADC in the Chinese population, particularly in nonsmoking females.

DNA damage repair plays essential roles in drug resistance, especially resistance to Poly (ADP-ribose) polymerase (PARP) inhibitors in the clinic. A subset of DNA repair proteins such as Breast cancer gene 1 (BRCA1), BRCA2 and RecA homolog (RAD51) are client proteins of heat shock protein 90 (Hsp90). Clearance of these DNA repair proteins by inhibition of Hsp90 is a promising strategy for overcoming resistance to PARP inhibitors. Here we report the pharmacological analysis of the highly potent second-generation Hsp90 inhibitor, ganetespib. Methods Nuclear BRCA1, BRCA2, and RAD51 expression in breast cancer cells were detected by subcellular fractionation and western blot analysis. Formation of nuclear RAD51 and γ-H2AX foci was analyzed by immunofluorescent staining. The cytotoxicity of ganetespib and ABT-888 in breast cancer cells were evaluated by cell proliferation, colony survival, and apoptosis assay. To investigate the efficacy of this therapy in vivo, SCID mice bearing MCF7 xenografts were treated with ganetespib and ABT-888, both as single agents and in combination. Results Ganetespib significantly destabilized nuclear BRCA1, BRCA2, and RAD51, and efficiently disrupted homologous recombination-mediated DNA double-strand break repair in breast cancer cells. The synergistic antitumor effects of ganetespib and the PARP inhibitor, ABT-888 were observed, and concurrent treatment with both inhibitors synergistically inhibited xenograft tumor growth. Importantly, the combined treatment was well tolerated, without significant loss of body weight or major histological changes in the breast cancer xenograft model. Conclusion These data provide a novel strategy for the treatment of breast cancer with wild type BRCA1 using combination therapy targeting Hsp90 to overcome resistance to PARP inhibitors.

Sweet sorghum [Sorghum bicolor (L.) Moench] accumulates large amounts of sugars in the stem parenchyma and can be used to manufacture sucrose and alcohol. Soluble acid invertase (SAI) plays an important role in sucrose accumulation in the sweet sorghum stem. Characterization of SAI genes and development of functional markers are important for marker-assisted selection in breeding programs. Here, the full-length genomic DNA sequence of a SAI gene (SAI-1) was cloned by in silico cloning and experimentally validated. The SAI-1 gene comprises three exons and two introns, totaling 4,966 bp, with an open reading frame of 2,025 bp encoding a polypeptide of 674 amino acids. The protein encoded by SAI-1 (molecular weight 73.4 kDa) contains a glycosyl hydrolase family 32 conserved domain, which catalyzes the hydrolysis of sucrose. Four allelic variants at the SAI-1 locus, SAI-1a, SAI-1b, SAI-1c and SAI-1d (GenBank accession nos. JX535515, JX535516, JX535517 and JX535518), were identified in 165 sweet sorghum cultivars. A co-dominant marker, SBX1, was developed to discriminate these variants. The mean Brix value from cultivars with a 141-bp (SAI-1c) PCR product was higher than in cultivars with a 136-bp (SAI-1b) product, and the mean Brix value from cultivars with a 136-bp (SAI-1b) product was significantly higher than those with a 132-bp (SAI-1a) product. The results showed that a domain of ATTGA repeat sequences in the second intron might be an important regulatory site, and that the functional marker SBX1 was closely related to the Brix value, and could therefore be used in sweet sorghum breeding programs.

Multiple endocrine neoplasia type 1 is a familial cancer syndrome resulting from loss-of-function mutations in the MEN1 gene. We previously identified the tumor suppressor MEN1 as a gene required for oncogene-induced senescence in melanocytes, raising the possibility that MEN1 is a melanoma tumor suppressor. Here we show that MEN1 expression is lost in a high percentage of human melanomas and melanoma cell lines. We find that melanocytes depleted of MEN1 are deficient in homologous recombination (HR)-directed DNA repair, which is accompanied by increased nonhomologous end-joining activity. Following DNA damage, MEN1 levels increase as a result of phosphorylation by the DNA damage kinase ATM/ATR. Most importantly, we show that MEN1 functions by directly stimulating the transcription of several genes, including BRCA1, RAD51, and RAD51AP1, that encode proteins involved in HR. MEN1 and its coactivator, the mixed-lineage leukemia histone methyltransferase, are recruited to the BRCA1, RAD51, and RAD51AP1 promoters by estrogen receptor 1, resulting in increased histone H3-lysine 4 trimethylation and transcription. Collectively, our results indicate that MEN1 is a melanoma tumor suppressor that functions by stimulating the transcription of genes involved in HR-directed DNA repair.

ObjectivesTo investigate the relationships between single-nucleotide polymorphisms (SNPs) inNOTCH4and brain-derived neurotrophic factor (BDNF) with schizophrenia among Han Chinese in Southern China.MethodsTwoNOTCH4SNPs (rs520688 and rs415929) and twoBDNFSNPs (rs2030324 and rs12273539) were examined in 464 schizophrenics and 464 healthy controls from Hunan province in South China, using the Sequenom MassARRAY®iPLEX System.ResultsIn the study population, rs520688 and rs2030324 were significantly associated with schizophrenia. A decreased risk of schizophrenia was associated with the rs520688 GA genotype (p= 0.035), whereas an increased risk of schizophrenia was associated with the rs2030324 CC/CT genotype (p= 0.044). The genotype distributions of rs415929 inNOTCH4and rs12273539 inBDNFdid not differ significantly between the case and control groups. Although no allele–allele interactions were detected between rs520688 and rs2030324, recombination analysis revealed a combined effect of the two on the susceptibility to schizophrenia, with GA-TT decreasing and CT/CC-GG/GA increasing the risk of schizophrenia.ConclusionIn conclusion, rs520688 inNOTCH4and rs2030324 inBDNFare significantly associated with schizophrenia among Han Chinese in Southern China. The two had a combined effect on the susceptibility to schizophrenia among Han Chinese in Southern China, but this may not be caused by an allele–allele interaction.

In recent years, there have been multiple breakthroughs in our understanding of lung cancer biology. Despite significant advances in molecular targeted therapies, DNA-damaging cytotoxic therapies will remain the mainstay of lung cancer management for the near future. Similar to the concept of personalized targeted therapies, there is mounting evidence that perturbations in DNA repair pathways are common in lung cancers, altering the resistance of the affected tumors to many chemotherapeutics as well as radiation. Defects in DNA repair may be due to a multitude of mechanisms including gene mutations, epigenetic events, and alterations in signal transduction pathways such as epidermal growth factor receptor and phosphoinositide 3-kinase/AKT. Functional biomarkers that assess the subcellular localization of central repair proteins in response to DNA damage may prove useful for individualization of cytotoxic therapies including poly(adenosine diphosphate-ribose) polymerase inhibitors. A better mechanistic understanding of cellular sensitivity and resistance to DNA damaging agents should facilitate the development of novel, individualized treatment approaches. Absolute resistance to radiation therapy, however, does not exist. To some extent, radiation therapy will always have to remain unselective and indiscriminant to eradicate persistent, drug-resistant tumor stem cell pools.

Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction. Results A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B. Conclusion A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.

DNA hypomethylation and/or hypermethylation are presumed to be early events in carcinogenesis, and one or more DNA methyltransferases (DNMTs) have been suggested to play roles in carcinogenesis of gastric cancer (GC). However, there have been no systematic studies regarding the association between DNMT gene polymorphisms and GC risk. Here, we examined the associations of 16 single nucleotide polymorphisms (SNPs) from DNMT1 (rs2114724, rs2228611, rs2228612, rs8101866, rs16999593), DNMT2 (rs11695471, rs11254413), DNMT3A (rs1550117, rs11887120, rs13420827, rs13428812, rs6733301), DNMT3B (rs2424908, rs2424913, rs6087990) and DNMT3L (rs113593938) with GC in the Southern Chinese population. We assessed the associations of these 16 SNPs with GC in a case-control study that consisted of 242 GC cases and 294 controls, using the Sequenom MALDI-TOF-MS platform. Association analyses based on the χ2 test and binary logistic regression were performed to determine the odds ratio (OR) and 95% confidence interval (95%CI) for each SNP. We found that rs16999593 in DNMT1, rs11254413 in DNMT2 and rs13420827 in DNMT3A were significantly associated with GC susceptibility (OR 1.45, 0.15, 0.66, respectively; 95% CI 1.00–2.11, p = 0.047; 0.08–0.27, p < 0.01; 0.45–0.97, p = 0.034, respectively, overdominant model). These results suggested that DNMT1, DNMT2 and DNMT3A may play important roles in GC carcinogenesis. However, further studies are required to elucidate the mechanism.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ., Author Summary The cell itself damages its own DNA throughout the cell cycle as a result of oxidative metabolism, and this damage creates barriers for replication fork progression. Thus, DNA replication is not a smooth and continuous process, but rather one of stalls and restarts. Therefore, proper replication fork restart is crucial to maintain the integrity of the cell’s genome, and preventing its own death or immortalization. To restart after stalling, the replication fork subverts a DNA repair pathway termed homologous recombination. Using any other pathway for fork repair will result in an unstable genome. How the homologous recombination repair pathway is initiated at the replication fork is not well defined. In this study we demonstrate the previously uncharacterized EEPD1 protein is a novel gatekeeper for the initiation of this fork repair pathway. EEPD1 promotes 5’ end resection, the initial step of homologous recombination, which also prevents alternative fork repair pathways that lead to unstable chromosomes. Thus, EEPD1 protects the integrity of the cell genome by promoting the safe homologous recombination fork repair pathway.

SummaryHdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

The tumor suppressor BRCA1 is a nuclear shuttling protein. However, the role of BRCA1 localization in the control of its functions remains to be elucidated. Given the central role of BRCA1 in DNA damage repair, we hypothesized that depletion of nuclear BRCA1 would compromise its nuclear function in DNA repair and thereby result in enhanced cytotoxic response to DNA damage. In this study, we showed that repair of DNA double-strand breaks required BRCA1 in the nucleus. In addition, sequestering BRCA1 in the cytosol enhanced the cytotoxic response to ionizing radiation or cisplatin in human breast and colon cancer cells. However, further genetic dissection of the mechanism of this enhanced cytotoxicity using BRCA1 mutants deficient in double-strand break repair unexpectedly revealed a dissociation of BRCA1's function in DNA repair from its effects on cellular sensitivity to DNA damage. Interestingly, we observed a dependence of the DNA damage–induced cell killing on the translocation and accumulation of BRCA1 in the cytosol. Together, these data suggest a novel role of cytoplasmic translocation of BRCA1, not only in controlling its DNA repair functions, but also in the regulation of cell death processes following DNA damage. Further dissection of the mechanism of cytotoxicity induced by BRCA1 cytoplasmic translocation revealed the involvement of the apoptotic pathway. We propose that the status of BRCA1 nuclear/cytoplasmic shuttling might provide a molecular marker to predict tumor response and a potential novel target to sensitize cancer cells to DNA damage–based therapy. Cancer Res; 70(15); 6258–67. ©2010 AACR.

The tumor suppressor, breast cancer susceptibility gene 1 (BRCA1), plays an integral role in the maintenance of genome stability and, in particular, the cellular response to DNA damage. Here, the emerging role of BRCA1 in nonhomologous end-joining-mediated DNA repair following DNA damage will be reviewed, as well as the activation of apoptotic pathways. The control of these functions via DNA damage-induced BRCA1 shuttling will also be discussed, in particular BRCA1 shuttling induced by erlotinib and irradiation. Finally, the potential targeting of BRCA1 shuttling as a novel strategy to sensitize cells to DNA damage will be entertained.

A novel halophilic, filamentous actinobacterium, designated TRM 40136(T), was isolated from a hypersaline habitat in Xinjiang Province, north-west China. The strain is aerobic, Gram-positive, halophilic, and the optimum NaCl concentration for growth is 10-15% (w/v). The whole-cell sugar pattern consists of xylose, glucose and arabinose. The predominant menaquinone is MK-6 (51.2%) and the major fatty acids are anteiso-C15:0 (35.2%), anteiso-C17:0 (15.9%) and iso-C15:0 (13.7%). The phospholipid pattern consists of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and two unknown phospholipids. The G + C content of the genomic DNA is 68.9 mol%. Phylogenetic analysis showed that strain TRM 40136(T) had 16S rRNA gene sequence similarity of 96.1% with the closest described species Actinopolyspora mortivallis, and it can be distinguished from all species in the genus Actinopolyspora by using these data of polyphasic taxonomy study. On the basis of the polyphasic evidence, the strain TRM 40136(T) should be designated as a novel species of the genus Actinopolyspora for which the name Actinopolyspora xinjiangensis sp.nov. is proposed. The type strain is TRM 40136(T) (=CCTCC AA 209080(T) = KCTC 19656(T)).

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine-arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.

The epidermal growth factor receptor (EGFR) family has been implicated in several cancers, including breast, and its members have become the target of novel cancer therapies. In this report, we show a novel link between erlotinib, a potent EGFR inhibitor, DNA damage, and homology-directed recombinational repair (HDR) in human breast cancer cells. Erlotinib suppresses HDR. This is not secondary to erlotinib-mediated changes in cell cycle and is associated with increased γ-H2AX foci, which is an in situ marker of chromosomal double-strand breaks. Both Rad51 and BRCA1 are essential components of the HDR machinery. Consistent with decreased HDR in erlotinib-treated cells, erlotinib also attenuates DNA damage-induced Rad51 foci and results in cytoplasmic retention of BRCA1. As BRCA1 is a shuttling protein and its nuclear function of promoting HDR is controlled by its subcellular localization, we further show that targeted translocation of BRCA1 to the cytoplasm enhances erlotinib sensitivity. These findings suggest a novel mechanism of action of erlotinib through its effects on the BRCA1/HDR pathway. Furthermore, BRCA1/HDR status may be an innovative avenue to enhance the sensitivity of cancer cells to erlotinib. [Cancer Res 2008;68(22):9141–6]

In this study, we examine the potential role of receptor-associated protein 80 (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), in DNA damage response and double-strand break (DSB) repair. We show that following ionizing radiation and treatment with DNA-damaging agents, RAP80 translocates to discrete nuclear foci that colocalize with those of γ-H2AX. The UIMs and the region of amino acids 204 to 304 are critical for the relocalization of RAP80 to ionizing radiation–induced foci (IRIF). These observations suggest that RAP80 becomes part of a DNA repair complex at the sites of IRIF. We also show that RAP80 forms a complex with the tumor repressor BRCA1 and that this interaction is mediated through the BRCA1 COOH-terminal repeats of BRCA1. The UIMs are not required for the interaction of RAP80 with BRCA1. Knockdown of RAP80 in HEK293 cells significantly reduced DSB-induced homology-directed recombination (HDR). Moreover, inhibition of RAP80 expression by small interfering RNA increased radiosensitivity, whereas increased radioresistance was observed in human breast cancer MCF-7 cells with overexpression of RAP80. Taken together, our data suggest that RAP80 plays an important role in DNA damage response signaling and HDR-mediated DSB repair. We further show that RAP80 can function as a substrate of the ataxia-telangiectasia mutated protein kinase in vitro, which phosphorylates RAP80 at Ser205 and Ser402. We show that this phosphorylation is not required for the migration of RAP80 to IRIF. [Cancer Res 2007;67(14):6647–56]

The purpose of this study is to identify the immunosuppressive effects of cyclophosphamide (a known immunosuppressant) in male Wistar rats which were administered orally for 30 days repeatedly, and to find a proper dose of cyclophosphamide at which the immunosuppressive effects is identified. The dose of cyclophosphamide is 2, 5, 10 mg/kg bw, respectively. The results showed that 10 mg/kg cyclophosphamide treatment induced decreases in body weight, body weight gain, relative weight of spleen and thymus, antibody plaque-forming cells, delayed-type hypersensitivity reaction, natural killer cell activity, lipopolysaccharide-induced B-cell proliferation, and Concanavalin A-induced T-cell proliferation. The histopathology observation on spleen and thymus revealed as decreased cellularity. But, no significant clinical symptoms of toxicities and stress were observed and no rats died. These results indicated that the immunosuppressive effects of 10 mg/kg cyclophosphamide dosed for 30 days orally were identified in male Wistar rats. Based on the results, it is considered that the dose of 10 mg/kg of cyclophosphamide orally for 30 days in male Wistar rats could be used as the dose of positive control group for chemicals immunotoxicity assessment tests. This study also proved that the testing procedures and the experimental techniques established in our laboratory for immunotoxicity tests in male Wistar rats are reliable and feasible.

The tumor suppressor gene BRCA1 maintains genomic integrity by protecting cells from the deleterious effects of DNA double-strand breaks (DSBs). Through its interactions with the checkpoint kinase 2 (Chk2) kinase and Rad51, BRCA1 promotes homologous recombination, which is typically an error-free repair process. In addition, accumulating evidence implicates BRCA1 in the regulation of nonhomologous end-joining (NHEJ), which may involve precise religation of the DSB ends if they are compatible (i.e., error-free repair) or sequence alteration upon rejoining (i.e., error-prone or mutagenic repair). However, the precise role of BRCA1 in regulating these different subtypes of NHEJ is not clear. We provide here the genetic and biochemical evidence to show that BRCA1 promotes error-free rejoining of DSBs in human breast carcinoma cells while suppressing microhomology-mediated error-prone end-joining and restricting sequence deletion at the break junction during repair. The repair spectrum in BRCA1-deficient cells was characterized by an increase in the formation of >2 kb deletions and in the usage of long microhomologies distal to the break site, compared with wild-type (WT) cells. This error-prone repair phenotype could also be revealed by disruption of the Chk2 phosphorylation site of BRCA1, or by expression of a dominant-negative kinase-dead Chk2 mutant in cells with WT BRCA1. We suggest that the differential control of NHEJ subprocesses by BRCA1, in concert with Chk2, reduces the mutagenic potential of NHEJ, thereby contributing to the prevention of familial breast cancers. (Cancer Res 2006; 66(3): 1401-8)

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were −0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within populations were highly significant (P < 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm) was 1.68, which indicated that the gene permutation and interaction among populations was relatively high. (Managing editor: Wei WANG)

The effects of three uncoupled metabolic systems (conventional activated sludge process with the addition of 3,3', 4',5-tetrachlorosalicylanilide [TCS], oxic-settling-anaerobic [OSA] process modified by insertion of a sludge-holding tank in the sludge return line, and TCS and OSA combined process) on reducing excess sludge production were studied. Compared with the control conventional activated sludge process, the most effective system was the combined process, which could reduce excess sludge production by 46.90%. The 180-d operation results confirmed that TCS is an effective chemical uncoupler in reducing the sludge yield but that it had an adverse effect on substrate removal capability, effluent nitrogen concentration, and sludge settleability. The OSA process decreased excess sludge production by only 26% but had less adverse effect on effluent quality and could improve sludge settleability. The effluent total phosphorous concentration of the three systems was slightly lower than of the control unit. Microbial populations were monitored by both microscopic and molecular biologic analysis method (polymerase chain reaction [PCR]-denaturing gradient gel electrophoresis [DGGE]). The presence of TCS caused metazoans to disappear and decreased the number and activity of protozoa. PCR amplification of 16S rRNA and sequent DGGE analysis found a shift in the diversity of the predominant species. The results imply that OSA combined with the chemical uncoupler process may effectively reduce excess sludge yield and not affect process performance significantly.

There is an urgent need to develop new antibacterial agents because of multidrug resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of high-yielding benzaldehyde Schiff bases were synthesized, and their structures were determined by NMR and MS spectra data. The structure-microbicidal activity relationship of derivatives was investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The active compounds were selective for certain active groups. The polar substitution of the R2 group of the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus; specific active group at the R3 or R4 groups of the acylhydrazone Schiff bases could improve their inhibitory activity against these three tested organisms. The antibacterial mechanism of the active benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E. coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for prophylactic or therapeutic intervention of bacterial infections.

Sera of cancer patients may contain antibodies that react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs). The present study aimed to determine whether a mini-array of multiple TAAs would enhance antibody detection and be a useful approach in esophageal cancer detection and diagnosis. Our mini-array of multiple TAAs consisted of eleven antigens, p53, pl6, Impl, CyclinB1, C-myc, RalA, p62, Survivin, Koc, CyclinD1 and CyclinE full-length recombinant proteins. Enzyme-linked immunosorbent assays (ELISA) were used to detect autoantibodies against eleven selected TAAs in 174 sera from patients with esophageal cancer, as well as 242 sera from normal individuals. In addition, positive results of ELISA were confirmed by Western blotting. In a parallel screening trial, with the successive addition of antigen to a final total of eleven TAAs, there was a stepwise increase in positive antibody reactions. The eleven TAAs were the best parallel combination, and the sensitivity and specificity in diagnosing esophageal cancer was 75.3% and 81.0%, respectively. The positive and negative predictive values were 74.0% and 82.0%, respectively, indicating that the parallel assay of eleven TAAs raised the diagnostic precision significantly. In addition, the levels of antibodies to seven antigens, comprising p53, Impl, C-myc, RalA, p62, Survivin, and CyclinD1, were significantly different in various stages of esophageal cancer, which showed that autoantibodies may be involved in the pathogenesis and progression of esophageal cancer. All in all, this study further supports our previous hypothesis that a combination of antibodies might acquire higher sensitivity for the diagnosis of certain types of cancer. A customized mini-array of multiple carefully-selected TAAs is able to enhance autoantibody detection in the immunodiagnosis of esophageal cancer and autoantibodies to TAAs might be reference indicators of clinical stage.

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.

The specific mechanism by which low-risk genetic variants confer breast cancer risk is currently unclear, with contradictory evidence on the role of single nucleotide polymorphisms (SNPs) in TOX3/LOC643714 as a breast cancer susceptibility locus. Investigations of this locus using a Chinese population may indicate whether the findings initially identified in a European population are generalizable to other populations, and may provide new insight into the role of genetic variants in the etiology of breast cancer. In this case-control study, 623 Chinese female breast cancer patients and 620 cancer-free controls were recruited to investigate the role of five SNPs in TOX3/LOC643714 (rs8051542, rs12443621, rs3803662, rs4784227, and rs3112612); Linkage disequilibrium (LD) pattern analysis was performed. Additionally, we evaluated how these common SNPs influence the risk of specific types of breast cancer, as defined by estrogen receptor (ER) status, progesterone receptor (PR) status and human epidermal growth factor receptor 2 (HER2) status. Significant associations with breast cancer risk were observed for rs4784227 and rs8051542 with odds ratios (OR) of 1.31 ((95% confidence intervals (CI), 1.10–1.57)) and 1.26 (95% CI, 1.02–1.56), respectively, per T allele. The T-rs8051542 allele was significantly associated with ER-positive and HER2-negative carriers. No significant association existed between rs12443621, rs3803662, and rs3112612 polymorphisms and risk of breast cancer. Our results support the hypothesis that the applicability of a common susceptibility locus must be confirmed among genetically different populations, which may together explain an appreciable fraction of the genetic etiology of breast cancer.

Mitochondria play an important role in plant growth and development, cooperating with the endoplasmic reticulum and nucleus. Gadolinium, one of the rare earth elements, is an inhibitor of stretch-activated calcium channels located on the endoplasmic reticulum and plasma membrane and has no effect on nuclear calcium variation in plant cells. We analyzed the effects of Gd3+ on mitochondria function by monitoring mitochondrial swelling, changes of membrane fluidity, and transmembrane potential collapse and by observing mitochondrial ultrastructure. We found that high concentration of Gd3+ induces rice mitochondrial dysfunction through mitochondrial permeability transition (MPT). The protection of DTT and EDTA demonstrate that Gd3+ blocks the inner membrane ion channel through thiol chelation.

The tumor-suppressor breast cancer susceptibility gene 1 (BRCA1) is a nuclear-cytoplasmic shuttling protein that when in the nucleus is required for DNA repair whereas when in the cytoplasm is important in activating cell death processes. Although BRCA1 mutations have been shown to be associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC), its role in disease progression is yet to be determined. We hypothesized that BRCA1 expression pattern could be used as a prognostic biomarker.Sixty-seven patients who underwent resections for PDAC were included. A tissue microarray was constructed, stained with antibodies to BRCA1, and scored for intensity and subcellular location. Univariate and multivariate statistical analyses were performed.An increase in cytosolic BRCA1 distribution was associated with higher pathologic stage (P = 0.006). Nuclear-cytosolic BRCA1 distribution was associated with a decrease in recurrence-free survival with a hazards ratio of 1.4 (P = 0.059). Decreased BRCA1 intensity was associated with higher pathologic stage (P = 0.027), but BRCA1 intensity was not associated with overall survival or recurrence-free survival.Our results demonstrate a possible association of BRCA1 expression pattern with pathologic stage, implying a potential role of BRCA1 in PDAC development and progression.

A novel halophilic, filamentous actinobacterium, designated strain TRM 40139T, was isolated from a hypersaline habitat in Xinjiang Province, north-west China. Its taxonomic status was determined using a polyphasic approach. Phylogenetic analysis based on the almost-complete 16S rRNA gene sequence of the strain showed that it formed a well-separated sub-branch within the radiation of the genus Actinopolyspora and the organism was related most closely to the type strains of Actinopolyspora alba (97.6 % similarity), Actinopolyspora xinjiangensis (97.6 %) and Actinopolyspora erythraea (97.1 %). However, it had relatively lower mean DNA–DNA relatedness values with the above strains (36.4, 31.3 and 26.1 %, respectively). Optimal growth occurred at 35 °C, at pH 7.0 and in the presence of 12 % (w/v) NaCl. The whole-cell sugar pattern consisted of xylose, glucose, ribose and arabinose. The major fatty acids were iso-C16 : 0 (28.0 %) and anteiso-C17 : 0 (27.6 %). The diagnostic phospholipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and two unknown phospholipids. The predominant menaquinones were MK-9(H4) (49.8 %) and MK-10(H4) (24.2 %). The G+C content of the genomic DNA was 66.4 mol%. Strain TRM 40139T therefore represents a novel species of the genus Actinopolyspora , for which the name Actinopolyspora lacussalsi sp. nov. is proposed. The type strain is TRM 40139T ( = KCTC 19657T = CCTCC AA 2012020T).

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G1 phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G1. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G1 phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G2/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G1.

Soft tissue sarcomas (STS) are a heterogeneous group of tumors originating from mesodermal tissues such as skeletal muscle, adipose, and fibrous tissue. The propensity of STS to metastasize, particularly to the lung which occurs in approximately 20% of all cases, is the major source of mortality associated with this disease. Currently, themolecular mechanisms that drive STS metastasis are poorly understood greatly limiting our ability to determine which STS will metastasize and impeding the development of targeted therapies for the treatment of STS metastases. In this study we utilized analysis of publicly available gene expression data to identify loss of PPP2R1B expression as a strong prognosticator of STS metastasis. In vitro analyses using SW872 liposarcoma (LPS) and HT1080 fibrosarcoma cell lines determined that PPP2R1B plays an essential role in the dephosphorylation and inactivation of RALA, a major downstream effector of the Ras pathway. RALA was found to be essential for invasion of STS cells in modified Boyden chamber assays as knockdown of RALA by either siRNA or shRNA significantly decreased invasion through matrigel. Analysis of publicly available gene expression data revealed that RALA expression correlated with more aggressive LPS subtypes and was prognostic of LPS metastasis. Furthermore, we identify the transmembrane matrix metalloprotease MMP14 (MT1-MMP) as a key downstream effector of RALA mediated invasion in STS. Stable knockdown of RALA in SW872 and HT1080 cells resulted in significant mislocalization of MMP14 and reduced MMP14 enzymatic activity. MMP14 expression was also significantly prognostic of metastasis in a publically available LPS cohort. Finally, we demonstrate that a three gene signature comprised of PPP2R1B, RALA, and MMP14 can retrospectively stratify LPS patients into groups of low, moderate, or high risk for metastatic recurrence. To the best of our knowledge this is the first demonstration of a vital role for PPP2R1A and RALA in STS invasion and metastasis and these data demonstrate a novel role for RALA in the localization and activity of MMP14. In conclusion, we have identified the PPP2R1B-RALA-MMP14 axis as driver of STS metastasis with potential clinical value as both a prognostic marker and therapeutic target. Citation Format: Steven T. Sizemore, Fen Xia, Arnab Chakravarti. The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1634.

PARP inhibitors have gained recent attention due to their highly selective killing of BRCA1/2-mutated and DNA double-strand break (DSB) repair–deficient tumors. Unfortunately, the majority of sporadic breast cancers carry wild-type BRCA1/2 and are proficient in DSB repair. We and others have shown that BRCA1 is a nuclear/cytoplasm shuttling protein that is transiently exported from the nucleus to the cytosol upon various stimuli. Thus, we hypothesized that depletion of nuclear BRCA1 would compromise DSB repair and subsequently render sporadic tumors susceptible to PARP inhibition. Indeed, in human sporadic breast cancer cells with functional BRCA1 and proficient DSB repair, a transient nuclear depletion of BRCA1 and subsequent homologous recombination repair deficit was induced with either truncated BRCA1 or irradiation. This rendered these human sporadic breast cancer cells susceptible to PARP inhibition. These observations were confirmed genetically using mislocated BRCA1 mutants as well as in vivo in mice bearing breast tumor xenografts. These data support the potential strategy of targeting BRCA1 location to convert BRCA1-proficient sporadic tumors to be susceptible to the synthetic lethal combination with PARP inhibitors. Cancer Res; 72(21); 5547–55. ©2012 AACR.

Objective: Data from a recent genome-wide association studiesy of gastric cancer (GC) and oesophageal squamous cell carcinoma in Chinese living in the Taihang Mountains of north-central China suggest that 1q22 and 10q23 are susceptibility-associated regions for GC. However, this has not been confirmed in southern Chinese populations. The aim of this study was to investigate whether these polymorphisms at 1q22 and 10q23 are associated with the risk of GC in a southern Chinese population. Methods: We selected seven top significant associated single nucleotide polymorphisms (SNPs) at 1q22 and 10q23 and conducted a population-based case- control study in a southern Chinese population. Genotypes were determined using MassARRAYTM system (Sequenome, San Diego, CA). Results: Two SNPs at 1q22, rs4072037 and rs4460629, were significantly associated with a reduced risk of GC, best fitting the dominant genetic model. Logistic regression models adjusted for age and sex showed that rs4072037 AG and GG (OR=0.64, P=0.017, compared with AA) and rs4460629 CT and TT (OR=0.54, P=0.0016, compared with TT) significantly reduced the risk of GC. However, no significant results for the five SNPs at 10q23 were obtained in this study. Conclusion: These outcomes indicate that 1q22 is associated with GC susceptibility in this southern Chinese population, while an association for the locus at 10q23 was not confirmed.

Mitochondria were isolated from the hybrid rice Xiangzaoxian 31, then the effects of low and high concentrations of Ce (Ш) on metabolism of mitochondria fed by pyruvate were investigated respectively, by microcalorimetry and oxygen electrode method The thermogenic curve of mitochondria without Ce (Ш) could be divided into three parts: activity recovery phase, stationary phase and decline phase. And the thermokinetic parameters have been calculated through the metabolic thermogenic curves. With addition of different concentrations of Ce (Ш), the results demonstrated that low levels of cerium ion stimulated the metabolic activity of energized mitochondria and the inhibition was discovered with high concentrations of Ce (Ш). At the same time, it is shown that the effect in respiration correspond to the effect on mitochondrial metabolism on addition of different concentrations of Ce (Ш). Moreover, the addition of low and high concentrations of Ce (Ш) had no obvious effect on the total heat output (Q). The concentration-dependent effect of Ce (Ш) on metabolism of mitochondria is similar to plant growth response to rare earth elements (Hormesis effect).

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.

Recently, a genome-wide association study of gastric cancer (GC) reported the significant association of seven genetic variants (rs4072037 and rs4460629 on 1q22; rs753724, rs11187842, rs3765524, rs2274223, and rs3781264 on 10q23) with GC in a Chinese population. These findings were confirmed in a subsequent independent study. However, it remains unknown whether these loci are associated with an increased risk of colorectal cancer (CRC). This study was to test whether the seven single nucleotide polymorphisms (SNPs) associated with GC were also associated with CRC in a Chinese population. The seven SNPs were genotyped using MassARRAY system. Allelic, genotypic, and haplotypic associations of the SNPs with CRC were investigated using χ(2) tests and logistic regression analysis. The SNPs rs3765524 and rs2274223, located on 10q23, were found to have significant protective effects against CRC, with equal odds ratios per allele. The two SNPs located on 1q22 (rs4072037 and rs4460629) showed a weak association with CRC. No significant association was identified with CRC for the remaining three SNPs located on 10q23 (rs753724, rs11187842, and rs3781264). These results suggest that rs3765524 and rs2274223 on 10q23 are associated with a protective effect against CRC in a Chinese population.