Cooperative interaction of CST and RECQ4 resolves G-quadruplexes and maintains telomere stability.

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.

Diabetic oxidative stress-induced telomere damage aggravates periodontal bone loss in periodontitis.

Periodontitis, one of the most common oral complications of diabetes mellitus (DM), causes a reduction in alveolar bone height and loss of alveolar bone mass. It has been shown that DM aggravates the progression of periodontitis, but the mechanism remains inconclusive. The hyperglycemic environment of DM has been proven to generate reactive oxygen species (ROS). Since telomeres, guanine-rich repeats, are highly susceptible to oxidative attack, we speculate that the excessive accumulation of ROS in DM could induce telomere damage resulting in dysfunction of periodontal ligament cells, especially periodontal ligament stem cells (PDLSCs), which reduces the ability of tissue repair and reconstruction in diabetic periodontitis. In this study, our current data revealed that oxidative telomere damage occurred in the periodontal ligaments of diabetic mice. And Micro-CT scans showed reduced alveolar bone height and impaired alveolar bone mass in a diabetic periodontitis model. Next, cultured mouse PDLSCs (mPDLSCs) were treated with the oxidant tert-butyl hydroperoxide (t-BHP) in vitro, as we expected telomere damage was observed and resulted in cellular senescence and dysfunction. Taken together, oxidative stress in DM causes telomere dysfunction and PDLSCs senescence, which influences periodontal bone tissue regeneration and reconstruction and ultimately exacerbates bone loss in periodontitis.

PRIMPOL competes with RAD51 to resolve G-quadruplex-induced replication stress via its interaction with RPA.

PRIMPOL (primase-polymerase) is a recently discovered DNA primase-polymerase involved in DNA damage tolerance and replication stress response in eukaryotic cells. However, the detailed mechanism of the PRIMPOL response to replication stress remains elusive. Here, we demonstrate that replication-related factors, including replication protein A (RPA), regulate the accumulation of PRIMPOL in subnuclear foci in response to replication stress induced by replication inhibitors. Moreover, PRIMPOL works at G-quadruplexes (G4s) in human cells to resolve the replication stress induced by G4s. The formation of PRIMPOL foci persists throughout the cell cycle. We further demonstrate that PRIMPOL competes with RAD51 to resolve G4-induced replication stress. In conclusion, our results provide novel insight into the mechanism of PRIMPOL in G4s to resolve replication stress and competition between PRIMPOL (repriming)- and RAD51 (fork reversal)-mediated pathways, which indicates a new strategy to improve the tumor response to DNA-damaging chemotherapy by targeting the PRIMPOL pathway.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation.

Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.

Development of a reverse-transcription loop-mediated isothermal amplification assay for the detection of Tobacco mild green mosaic virus (TMGMV).

Tobacco mild green mosaic virus (TMGMV), a member species of the genus Tobamovirus, infects pepper (Capsicum annuum) and a number of other economically important species in the Solanaceae family. TMGMV infections had seriously impacted pepper production worldwide, including China. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect TMGMV in pepper field samples and seed. This assay was based on four primers that matched to six sequences in the C-terminal region of the TMGMV genome. RT-LAMP assay could detect the presence of the virus in 3.0 × 10-7 µg of total RNA extract from pepper leaves, which was ten times more sensitive than the corresponding reverse-transcription polymerase chain reaction (RT-PCR) assay. This method specifically detected TMGMV but not the closely related species of the same genus Pepper mild mottle virus, Cucumber green mottle mosaic virus and Tomato mosaic virus. In addition, the use of SYBR Green I facilitated the detection of the TMGMV RT-LAMP products by the naked eye. These results indicated that the RT-LAMP assay was a simple, sensitive, specific and affordable diagnostic tool that has the potential to detect and monitor TMGMV infection in field samples.

Ruthenium(II)-Catalyzed Regioselective [3 + 2] Spiroannulation of 2H-Imidazoles with 2-Alkynoates.

The C═N double bond of 2H-imidazole has been employed as a C-electrophile for the ruthenium(II)-catalyzed [3 + 2] spiroannulation reaction of 4-phenyl-2H-imidazoles and 2-alkynoates to synthesize spiroimidazole-4,1'-indenes. This strategy features high regioselectivity, broad functional group tolerance, and use of ruthenium as a catalyst, providing a new method to synthesize spirocycles with potential applications in pharmaceuticals.

Rhodium(III)-Catalyzed [4+3] Annulation of N-Aryl-pyrazolidinones and Propargylic Acetates: Access to Benzo[c][1,2]diazepines.

The generation of 2,3-dihydro-benzo[c][1,2]diazepine derivatives via rhodium(III)-catalyzed [4+3] annulation of pyrazolidinones and propargylic acetates is disclosed. The reaction proceeds smoothly under relatively mild conditions from propargylic acetates as novel C3 synthons. A range of dinitrogen-fused heterocyclic compounds are readily accessed by this approach.

Mammalian CST averts replication failure by preventing G-quadruplex accumulation.

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization, even when ATR/ATM pathways were inhibited. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

Rh(III)-Catalyzed One-Pot Synthesis of Benzimidazoquinazolines via C-H Amidation-Cyclization of N-LG-2-phenylbenzoimidazoles.

An efficient protocol to synthesize substituted benzo[4,5]imidazo[1,2- c]quinazolines starting from N-LG-2-phenylbenzoimidazole and dioxazolones catalyzed by Rh(III) or Ir(III) has been developed. Various substituted benzo[4,5]imidazo[1,2- c]quinazolines could be easily provided in up to 99% yield. A large range of substrates and functional groups are compatible for this transformation. This method features low catalyst loading, acid-free conditions, and low solvent consumption.

Plasma D-dimer Can Effectively Predict the Prospective Occurrence of Ascites in Advanced Schistosomiasis Japonica Patients.

China still has more than 30,000 patients of advanced schistosomiasis while new cases being reported consistently. D-dimer is a fibrin degradation product. As ascites being the dominating symptom in advanced schistosomiasis, the present study aimed to explore a prediction model of ascites with D-dimer and other clinical easy-achievable indicators. A case-control study nested in a prospective cohort was conducted in schistosomiasis-endemic area of southern China. A total of 291 patients of advanced schistosomiasis were first investigated in 2013 and further followed in 2014. Information on clinical history, physical examination, and abdominal ultrasonography, including the symptom of ascites was repeatedly collected. Result showed 44 patients having ascites. Most of the patients' ascites were confined in the kidney area with median area of 20 mm2. The level of plasma D-dimer and pertinent liver function indicators were measured at the initial investigation in 2013. Compared with those without ascites, cases with ascites had significantly higher levels of D-dimer (0.71±2.44 µg/L vs 0.48±2.12 µg/L, P=0.005), as well ALB (44.5 vs 46.2, g/L) and Type IV collagen (50.04 vs 44.50 µg/L). Receiver operating characteristic curve analyses indicated a moderate predictive value of D-dimer by its own area under curve (AUC) of 0.64 (95% CI: 0.54-0.73) and the cutoff value as 0.81 µg/L. Dichotomized by the cutoff level, D-dimer along with other categorical variables generated a prediction model with AUC of 0.76 (95% CI: 0.68-0.89). Risks of patients with specific characteristics in the prediction model were summarized. Our study suggests that the plasma D-dimer level is a reliable predictor for incident ascites in advanced schistosomiasis japonica patients.

Synthesis, structure-activity relationship and binding mode analysis of 4-thiazolidinone derivatives as novel inhibitors of human dihydroorotate dehydrogenase.

A series of 4-thiazolidinone derivatives were synthesized and evaluated as novel human dihydroorotate dehydrogenase (hDHODH) inhibitors. Compounds 26 and 31 displayed IC50 values of 1.75 and 1.12 µM, respectively. The structure-activity relationship was summarized. Further binding mode analysis revealed that compound 31 could form a hydrogen bond with Tyr38 and a water-mediated hydrogen bond with Ala55, which may be necessary for maintaining the bioactivities of the compounds in this series. Further structural optimization of the para- or meta-position of the phenyl group at R will lead to the identification of more potent hDHODH inhibitors.

[Investigation on the gene expression pattern in cervical squamous cell carcinoma of uygur].

OBJECTIVE: To investigate the differentially expressed gene in cervical squamous cell carcinoma of uygur and normal cervical tissue using gene chip. METHOD: Gene chips containing 2,048 human genes were used to investigate the gene expression pattern of 3 samples of cervical cancer. RESULT: By applying this gene chip we identified 64 differentially expressed genes in all 3 clinical cervical cancer samples, and we further identified 53 upregulated and 11 downregulated genes for functional analysis. CONCLUSION: The analysis of gene expression pattern of tumor based on gene chip can realize high-throughput screening of the genes associated with the cervical cancer.

[Study of the interactions between diorganotin (IV) complexes of 1,3-dimethyl-4-acetyl-5-pyrazolone and mononucleotides and DNA].

AIM: The interactions between diorganotin (IV) complexes of 1,3-dimethyl-4-acetyl-5-pyrazolone (HL1) and mono-nucleotides together with DNA near physiological condition were investigated. METHODS: The mode of action of the diorganotin (IV) complexes with mononucleotides and DNA under different conditions and different times were investigated by high resolution NMR technology and UV spectra. RESULTS: The interaction of [(L1)2SnEt2] with AMP was shown to result in significant change of chemical shift of H(8), H(2) and 31P of AMP. Hyperchromic effect of DNA could be observed due to the interaction of; [(L1)2SnEt2] with DNA, while interaction of [(L1)2SnMe2] with AMP and DNA could only cause obvious change of chemical shift of 31P and lead to hypochromic effect of DNA. CONCLUSION: The results indicate that [(L1)2SnEt2] can selectively bind to the N1 atom of the base and the phosphate oxygen atom of AMP and may further destroy the helical structure of DNA, while the dimethyltin (IV) compound of 1,3-dimethyl-4-acetyl-5-pyrazolone [(L1)2SnMe2] merely binds to the the phosphate oxygen atom of AMP and causes the contraction of DNA helical structure.