Structures of the human CST-Polα-primase complex bound to telomere templates.

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.

Personalized neoantigen vaccine enhances the therapeutic efficacy of bevacizumab and anti-PD-1 antibody in advanced non-small cell lung cancer.

Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.

Priority of Mixed Diamine Ligands in Cobalt Dithiolene Complex-Catalyzed H2 Evolution: A Theoretical Study.

Redox non-innocent metal dithiolene or diamine complexes are potential alternative catalysts in hydrogen evolution reaction and have been incorporated into 2D metal-organic frameworks to obtain unexpected electrocatalytic activity. According to an experimental study, Co-bis(dithiolene), Co-bis(diamine), and Co-dithiolene-diamine portions are considered as active sites where the generation of H2 occurs and a diamine ligand is necessary for high catalytic efficiency. We are interested in the difference between these catalytic active sites, and mechanistic studies on extracted Co-bis(dithiolene), Co-bis(diamine), and Co-dithiolene-diamine complex-catalyzed hydrogen evolution reactions are carried out by using density functional methods. Our calculated results indicate that the priority of ligand mixed complexes resulted from the readily occurring protonation of diamine ligands and large electron affinity of dithiolene ligands as well as the lowest overall barrier for H2 evolution.

Astragaloside IV sensitizes non-small cell lung cancer cells to gefitinib potentially via regulation of SIRT6.

Astragaloside IV, the active component of Astragalus membranaceus, exhibits diverse biological roles including the anti-tumor activity. In this study, we evaluated the chemosensitive role of astragaloside IV in non-small cell lung cancer cells. Cell Counting Kit-8 analysis was performed to determine cell viability. Real-time polymerase chain reaction and western blot were used to measure the messenger RNA and protein expression. Results showed that astragaloside IV treatment could suppress the proliferation of non-small cell lung cancer cells. In addition, combined treatment with astragaloside IV remarkably enhanced the chemosensitivity to gefitinib in three non-small cell lung cancer cell lines including NCI-H1299, HCC827, and A549. Furthermore, compared with gefitinib-treated cells, the messenger RNA expression of SIRT6 was obviously increased in non-small cell lung cancer cells treated with gefitinib combined with astragaloside IV. In addition, downregulation of SIRT6 was accomplished using small interference RNA technology. As a result, SIRT6 inhibition abolished the sensitization role of astragaloside IV in non-small cell lung cancer cells. Taken together, these data demonstrated that astragaloside IV sensitized tumor cells to gefitinib via regulation of SIRT6, suggesting that astragaloside IV may serve as potential therapeutic approach for lung cancer.

α­thalassemia deletion [­SEA (Southeast Asian)] and a compound heterozygote for the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia mutation: A case report.

In the present study, an α­thalassemia deletion [­SEA (Southeast Asian)] and a compound heterozygote for the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia mutation in a 15­year­old girl was identified by gap­PCR, PCR­reverse dot­blot hybridization and multiplex ligation­dependent probe amplification. Molecular analysis indicated that the proband's father carried a hemoglobin subunit ß (HBB) heterozygous mutation in codon 17 (CD17; c.52A>T), the mother was a double heterozygous carrier of the Chinese Gγ+(Aγδß)0­thalassemia mutation combined with an ­SEA deletion, and the proband inherited both mutations from her mother and father, thus carrying the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia combined with the­SEA deletion in a compound heterozygous state. The proband was diagnosed as severe thalassemia intermedia and experienced a clinical phenotype aggravation (severe anemia and splenomegaly) from no obvious clinical symptoms to being dependent on monthly blood transfusions.

Mild depolymerization of the sinocalamus oldhami alkali lignin to phenolic monomer with base and activated carbon supported nickel-tungsten carbide catalyst composite system.

In this study, the sinocalamus oldhami alkali lignin was depolymerized into phenolic products in a combined system by using the composite alkali and Ni-W2C/activated carbon (AC) as catalysts. FT-IR, GPC, TG, 2D-HSQC and GC-MS were used to analyze the composition, structure and distribution of degradation products, and the synergistic effect of metal and alkali catalysts on the depolymerization of lignin was also studied. The results showed that Ni-W2C/AC and composite alkali could effectively improve the catalytic degradation efficiency of lignin under mild conditions, 94.4% of lignin was converted and 17.18% of phenolic monomers were obtained under 260 °C for 5 h. In this composite system, the synergism of the basic sites, the metal active sites and the Lewis acid sites could promote the cleavage of C-O bonds in the lignin molecule and lower the char formation during the base-catalyzed solvolysis. Phenolic monomers were mainly composed of phenol, 2-methyl-phenol and p-cresol etc.

Characteristics of Antibiotic Resistance Genes and Antibiotic-Resistant Bacteria in Full-Scale Drinking Water Treatment System Using Metagenomics and Culturing.

The contamination of antibiotic resistance genes (ARGs) may directly threaten human health. This study used a metagenomic approach to investigate the ARG profile in a drinking water treatment system (DWTS) in south China. In total, 317 ARG subtypes were detected; specifically, genes encoding bacitracin, multidrug, and sulfonamide were widely detected in the DWTS. Putative ARG hosts included Acidovorax (6.0%), Polynucleobacter (4.3%), Pseudomonas (3.4%), Escherichia (1.7%), and Klebsiella (1.5%) as the enriched biomarkers in the DWTS, which mainly carried bacitracin, beta-lactam, and aminoglycoside ARGs. From a further analysis of ARG-carrying contigs (ACCs), Stenotrophomonas maltophilia and Pseudomonas aeruginosa were the most common pathogens among the 49 ACC pathogens in the DWTS. The metagenomic binning results demonstrated that 33 high-quality metagenome-assembled genomes (MAGs) were discovered in the DWTS; particularly, the MAG identified as S. maltophilia-like (bin.195) harbored the greatest number of ARG subtypes (n = 8), namely, multidrug (n = 6; smeD, semE, multidrug_transporter, mexE, semB, and smeC), beta-lactam (n = 1; metallo-beta-lactamase), and aminoglycoside [n = 1; aph(3')-IIb]. The strong positive correlation between MGEs and ARG subtypes revealed a high ARG dissemination risk in the DWTS. Based on the pure-culture method, 93 isolates that belong to 30 genera were recovered from the DWTS. Specifically, multidrug-resistant pathogens and opportunistic pathogens such as P. aeruginosa, Bacillus cereus, and S. maltophilia were detected in the DWTS. These insights into the DWTS's antibiotic resistome indicated the need for more comprehensive ARG monitoring and management in the DWTS. Furthermore, more effective disinfection methods need to be developed to remove ARGs in DWTSs, and these findings could assist governing bodies in the surveillance of antibiotic resistance in DWTSs.

Application of the transosseous approach for computed tomography-guided radioactive 125-iodine seed implantation for the treatment of thoracic and abdominal lymph node metastases.

AIM: This study aimed to investigate the technical procedure, safety, and clinical value of the transosseous approach for computed tomography (CT)-guided radioactive 125-iodine (125I) seed implantation for the treatment of thoracic and abdominal lymph node metastases. SUBJECTS AND METHODS: This was a retrospective study that Nine lymph node metastases in nine patients were treated in our hospital between January 2010 and August 2018. Under CT guidance, at least one puncture path was made through the transosseous approach. The seeds were planted according to the TPS. CT/MRI scans were performed every 2 months after the treatment to evaluate local therapeutic efficacy according to the Response Evaluation Criteria in Solid Tumors. RESULTS: The transosseous approach was successfully established in all patients. The median follow-up time was 11 months (6-36 months). At 2, 4, 6, 8, 10 and 12 months after operation, the objective effective rate and clinical benefit rate were 66.67%, 77.78%, 77.78%, 71.43%, 66.67% and 50.00%; and 88.89%, 88.89%, 88.89%, 71.43%, 66.67% and 50.00%, respectively. The survival rate of the patients at 6, 12, 18, 24, 30 and 36 months after operation was 53.00%, 26.00%, 26.00%, 13.00%, 13.00% and 13.00%, respectively. CONCLUSIONS: The transosseous approach for CT-guided radioactive 125I seed implantation was safe, effective, and minimally invasive for the treatment of thoracic and abdominal lymph node metastases.

Integrated microRNA and protein expression analysis reveals novel microRNA regulation of targets in fetal down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 and is associated with a number of deleterious phenotypes. To investigate the role of microRNA (miRNA) in the regulation of DS, high­throughput Illumina sequencing technology and isobaric tagging for relative and absolute protein quantification analysis were utilized for simultaneous expression profiling of miRNA and protein in fetuses with DS and normal fetuses. A total of 344 miRNAs were associated with DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to investigate the proteins found to be differentially expressed. Functionally important miRNAs were determined by identifying enriched or depleted targets in the transcript and the protein expression levels were consistent with miRNA regulation. The results indicated that GRB2, TMSB10, RUVBL2, the hsa­miR­329 and hsa­miR­27b, hsa­miR­27a targets, and MAPK1, PTPN11, ACTA2 and PTK2 or other differentially expressed proteins were connected with each other directly or indirectly. Integrative analysis of miRNAs and proteins provided an expansive view of the molecular signaling pathways in DS.

Quantitative proteomic analysis of Down syndrome in the umbilical cord blood using iTRAQ.

Down sydrome (DS) is a relatively frequent chromosomal disorder, which has no safe and effective method of prenatal diagnosis to date. The present study was designed to identify DS biomarkers. We quantified the changes in the umbilical cord blood protein levels between DS-affected and healthy (control) pregnant females using isobaric tags for relative and absolute quantification (iTRAQ) and Gene Ontology (GO) analysis. A total of 505 proteins were identified, and of these, five proteins showed significantly different concentrations between the DS and the control group. These proteins may thus be relevant to DS and constitute potential DS biomarkers.

[Efficacy observation on Jin's three-needle therapy for allergic rhinitis of lung qi deficiency and cold syndrome].

OBJECTIVE: To compare the clinical efficacy in the treatment of allergic rhinitis (AR) of lung qi deficiency and cold syndrome between Jin's three-needle therapy and western medication. METHODS: Sixty-six patients were randomized into an acupuncture group and a western medication group, 33 cases in each one. In the acupuncture group, acupuncture was applied at three-nose points [Yingxiang (LI 20), Shangyingxiang (EX-HN 8) and Yintang (GV 29); Cuanzhu (BL 2) was added for frontal headache] and three-back points [Dazhu (BL 11), Fengmen (BL 12) and Feishu (BL 13)], once every day. Ten treatments made one session. Two sessions of treatment were required. In the western medication group, desloratadine oral suspension was prescribed, 5 mg each time, once a day, for 20 days. The scores of the symptoms and physical signs in AR patients as well as the clinical efficacy were observed between the two groups. RESULTS: The total effective rate was 93.9% (31/33) in the acupuncture group, which was better than 72.7% (24/33) in the western medication group (P < 0.05). After treatment, the scores of AR symptoms and physical signs as well as the total score were all reduced compared with those before treatment in the two groups (all P < 0.01). The score of every item in the acupuncture group was lower than that in the western medication group after treatment (score of symptoms: 4.70 +/- 2.07 vs 6.55 +/- 2. 69, score of physical signs: 0.85 +/- 0.67 vs 1.45 +/- +0.62, total score: 5.36 +/- 2.70 vs 8.00 +/- 2.91, all P < 0.01). CONCLUSION: Jin's three-needle therapy achieves superior efficacy on AR of lung-qi deficiency and cold syndrome, which is better than desloratadine oral suspension.

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

[Influence of needle-pricking bleeding combined with pulling-rotating manipulation on blood rheology in patients with vertebral artery type cervical spondylosis].

OBJECTIVE: To observe the effect of needle-pricking bleeding combined with pulling-rotating manipulation and simple manipulation on blood rheology in vertebral artery type cervical spondylosis (VATCS) patients, so as to analyze their mechanisms in relieving VATCS. METHODS: A total of 198 VATCS patients were randomly divided into treatment group (n=101) which was treated with needle-pricking plus pulling-rotating manipulation, and control group (n=97) which was treated with simple pulling-rotating manipulation, according to the random number table. The treatment was given once every 7 days, 9 times altogether. The peak systolic blood flow velocity (Vpeak), end-diastolic blood velocity (Vmin), pulsatility index (PI) and resistent index (RI) of bilateral vertebral arteries (VA) and basilar artery (BA) were detected by transcranial doppler sonography (TCD). Whole blood apparent viscosity and the plasma viscosity in the treatment group were determined by using a blood viscosimeter. RESULTS: Of the 101 and 97 VATCS cases in the treatment and control groups, 62 (61.38%) and 12 (12.37%) were cured basically, 23 (22.77%) and 26 (26.80%) experienced marked improvement, 14 (13.86%) and 41 (42.27%) were improved. 2 (1.98%) and 18 (18.55%) failed in the treatment, with the total effective rates being 98.01% and 81.44% separately. The effective rate of the treatment group was significantly higher than that of control group (P<0.05). Compared with pretreatment, Vpeak and Vmin of the bilateral VA and BA in the treatment group, and Vpeak of the right VA in the control group increased significantly (P<0.01, P<0.05), PI and RI of the bilateral VA and BA in the treatment group, and PI and RI of the right VA in the control group decreased significantly (P<0.01, P<0.05), suggesting a marked reduction of the vascular resistance and an apparent increase of the cerebral blood supply after the treatment. The therapeutic effects of the above-mentioned indexes of treatment group were significantly superior to those of the control group (P<0.05). In comparison with pre-treatment, the whole blood apparent viscosity (high, medium and low shear rates) and plasma viscosity of the treatment group post-treatment were obviously reduced (P<0.01, P<0.05). CONCLUSION: Needle-pricking therapy combined with pulling-rotating manipulation can significantly improve VATCS patients' clinical symptoms, which may be closely related to its effects in lowering vascular blood resistance and blood viscosity and increasing cerebral blood supply.