The modern large-scale pyrosequencing technology is a revolution of DNA sequencing.One of the key points in this technology is to get an ATP sulfurylase immobilized on the surface of magnetic beads and with a high activity.Biotinylated ATP sulfurylase can be immobilized on magnetic beads coated with streptavidin through the specific conjunction between biotin and streptavidin, but using chemical modification method to biotinylate ATPS will affect the activity of the enzyme.ATP sulfurylase fused with the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein(BCCP87) was expressed in E.coli using fusion expression strategy.Results from Western blot analysis and SDS-PAGE analysis showed that the fusion protein could be biotinylated in vivo, and the molecular mass of the fusion protein was about 64 ku.The biotinylated ATP sulfurylase could be immobilized on the surface of magnetic beads coated with strepavidin, and the immobilized ATPS could be used for quantification of PPi and pyrosequencing.An effective enzyme for the large-scale chip-based pyrosequencing system was supplied.

Background and purpose: Paclitaxel is believed to be efficient in treating malignant ascites in gastric cancer. However, researches are still needed to get more evidence. The aim of this study was to discuss the efficacy and safety of the treatment of malignant ascites in gastric cancer with paclitaxel. Methods: Six cases of late phase gastric cancer patients were enrolled into the study, paclitaxel 60 mg/m~2 and 1 500-2 000 mL natural solution were administered via intraperitoneal injection, qw, for a of total 2-4 weeks. Efficacy and toxicity were determined according to WHO criteria. Results: Five (5/6) had complete response, and one (1/6) with partial response. The malignant ascites recession time was 0.5-10 months, overall survival time 2-10 months, 4 cases suffered grade Ⅰ-Ⅲabdominal pain, 4 cases grade Ⅰ leucopenia, 3 cases grade Ⅰ hair loss, 1 case grade Ⅰ liver injury (with past history of hepatitis). Conclusion: Paclitaxel is effective and relatively safe to treat malignant ascites of gastric cancer.

Background and purpose:Paclitaxel is believed to be efficient in treating malignant ascites in gastric cancer. However, researches are still needed to get more evidence. The aim of this study was to discuss the efficacy and safety of the treatment of malignant ascites in gastric cancer with paclitaxel. Methods:Six cases of late phase gastric cancer patients were enrolled into the study, paclitaxel 60 mg/m2 and 1 500-2 000 mL natural solution were administered via intraperitoneal injection, qw, for a of total 2-4 weeks. Efficacy and toxicity were determined according to WHO criteria. Results:Five (5/6) had complete response, and one (1/6) with partial response. The malignant ascites recession time was 0.5-10 months, overall survival time 2-10 months, 4 cases suffered grade Ⅰ-Ⅲ abdominal pain, 4 cases grade Ⅰ leucopenia, 3 cases grade Ⅰ hair loss, 1 case gradeⅠ liver injury (with past history of hepatitis). Conclusion:Paclitaxel is effective and relatively safe to treat malignant ascites of gastric cancer.

Objective To investigate the role of vitamin D3 in anti-pancreatic cancer.Methods After treatment of different concentrations of vitamin D3 on PANC1 cells (25,50,75,100μmol/L),MTT assay was used to detect the growth inhibition rates of PANC1 cells,the early apoptotic rates of the cell were detected by flow cytometry,PTCH and Gli-1 mRNA expression were detected by RT-PCR method,and cells without treatment were used as control.Results The vitamin D3 inhibited the proliferation of PANC1 cells in a dose-dependent manner,the highest inhibition rate was at 48 hours.After 48 hours,the control group,25,50,75,100 μmol/L vitamin D3 groups' inhibition rates were 0,16.1％,18.8％,31.8％ and 39.4％,the differences among these groups were statistically significance (P ＜ 0.05).After 24 hours,the control group,25,50,75,100μmol/L vitamin D3 groups' early apoptotic rates were (5.89 ±0.57)％,(6.06 ±0.44)％,(16.21 ± 1.62)％,(16.94± 0.91)％ and (20.96 ± 0.98)％,early apoptotic rates were inhibited in a dose-dependent manner,and the differences was statistically significance (P ＜ 0.05).After 24 hours,the control group,25,50,75,100μmol/L vitamin D3 groups' PTCH mRNA expression were 0.117 ± 0.009,0.104 ± 0.011,0.069 ± 0.011,0.052 ± 0.009 and 0.056 ± 0.007,meanwhile the Gli-1 mRNA expressions were 0.323 ± 0.007,0.312 ± 0.015,0.299 ± 0.015,0.233 ± 0.007 and 0.175 ± 0.014,all in a declining trend with the increase of concentration,and the difference was statistically significant (P ＜ 0.05).After 75 μmol/L vitamin D3's intervention in 0,12,24,36 and 48 hours,the expression of PTCH mRNA were 0.142±0.008,0.127± 0.009,0.111± 0.010,0.115± 0.003 and 0.102± 0.007,meanwhile the expression of Gli-1 mRNA were 0.341 ± 0.011,0.317 ± 0.017,0.320 ± 0.018,0.226 ± 0.011 and 0.191 ±0.010,all in a declining trend with time,and the difference was statistically significance (p＜0.05).Conclusions Vitamin D3 can effectively inhibit the proliferation of PANC1 cells and promote its apoptosis,and these effects may be related to blocking of hedgehog signaling pathway.

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Animals ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fireflies ; enzymology ; Luciferases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

The corona virus disease 2019 (COVID-19) caused by the new coronavirus (SARS-CoV-2) has spread rapidly around the world,posing a serious threat to the public"s health. As of September 30,2020,the number of infected people in the world has reached 33 million,causing more than 1 million deaths. Normalized nucleic acid detection methods based on lab have long turnaround time and high cost. Therefore,there is an urgent need to develop a convenient method to detect SARS-CoV-2,so as to achieve rapid testing and timely control of the epidemic when resources are limited.This review summarizes the point-of-care testing (POCT) methods developed for SARS-CoV-2 in terms of extraction,amplification and detection,and briefly introduces commercial POCT instruments that integrate these three steps,in order to provide references for emergency response and rapid deployment of COVID-19 and other emerging infectious diseases.

Objective:To propose a new grading system-the posterior ligament-bone injury classification and severity (PLICS) score for subaxial cervical spine injury, and evaluate its value in guiding the approach selection for subaxial cervical fracture and dislocation.Methods:All of 394 cases of subaxial cervical fracture and dislocation who received single anterior reduction and fixation in our hospital from January 2002 to December 2015 were retrospectively analyzed. According to the inclusion and exclusion criteria, 354 cases were finally included in this study. The Patients experienced internal fixation failure, postoperative kyphosis or interspinal process space dilation during follow-up were included into the failure group. Other patients were included into the successful group. The difference of visual analogue scale (VAS), neck disability index (NDI), ASIA score and PLICS score before and after surgery between the two groups was compared respectively. The PLICS score is composed of left, right and posterior columns. The injury of the posterior column was classified into mild, moderate and severe degrees, with 1-3 points respectively. The evaluation of lateral column injury included ligament and bone structure. The evaluation of ligament injury included: 1 point for the subluxation of the facet joint, 2 points for the dislocation or the facet joint; Fractures of the lateral column was classified into mild, moderate and severe degrees, with 1-3 points respectively. For the evaluation of the lateral column, the highest score of ligament or bone structure injury was reflected as the score of posterior column injury and was taken into the calculation of the final PLICS score.Results:All 354 patients had complete follow-up data, and the average follow-up time was 18.0±4.0 months, including 339 patients of successful group and 15 patients of failure group. At the 12 month follow-up, the average VAS score of the patients in the successful group decreased from 6.9±0.6 before the operation to 1.9±0.6 ( t=22.481, P<0.0001), and the average VAS score of the patients in the failure group decreased from 5.6±1.0 to 1.1±0.3 ( t=77.252, P<0.0001). The difference between the two groups was statistically significant. The NDI score of the successful group was significantly lower than that of the failure group (7.1%±1.2% vs 15.7%±2.7%, t=24.993, P<0.0001). PLICS score in two groups of patients were analyzed. There was a significant difference in PLICS scores between the two groups ( t=8.777, P<0.0001). According to the PLICS score, the ROC operating curve of the failure of internal fixation after anterior-only surgery was determined. The area under the curve was 0.981, and the 95% confidence interval was 0.943, 1.000. When the PLICS score was 6.5, the maximum value of the Jordan index was 0.927, the sensitivity was 0.994, and the specificity was 0.067. Considering the clinical practicality of PLICS score and the different clinical outcomes of the sub-classification groups of the PLICS score=7, we finally set the threshold as PLICS score=7 with the unilateral severe lateral mass fracture. Conclusion:The PLICS score is based on the anatomy of the posterior three-column structure of the subaxial cervical spine. It gives consideration to the bony structures, including bilateral lateral mass, spinous process, lamina, and the ligament structures. What's more, the overall damage severity of the posterior three columns of the subaxial cervical spine was quantified according to the evaluation of the injury characteristics of each column of ligament-bone structure. In this study, PLICS score and clinical efficacy were compared between two groups of patients, and it was finally determined that when PLICS score ≥7 with the unilateral severe lateral mass fracture, the risk of internal fixation failure is higher for anterior-only approach surgery alone. For these patients, anterior and posterior approach surgery may be considered.

The development of nanomedicine has recently achieved several breakthroughs in the field of cancer treatment; however, biocompatibility and targeted penetration of these nanomaterials remain as limitations, which lead to serious side effects and significantly narrow the scope of their application. The self-assembly of intermediate filaments with arginine-glycine-aspartate (RGD) peptide (RGD-IFP) was triggered by the hydrophobic cationic molecule 7-amino actinomycin D (7-AAD) to synthesize a bifunctional nanoparticle that could serve as a fluorescent imaging probe to visualize tumor treatment. The designed RGD-IFP peptide possessed the ability to encapsulate 7-AAD molecules through the formation of hydrogen bonds and hydrophobic interactions by a one-step method. This fluorescent nanoprobe with RGD peptide could be targeted for delivery into tumor cells and released in acidic environments such as endosomes/lysosomes, ultimately inducing cytotoxicity by arresting tumor cell cycling with inserted DNA. It is noteworthy that the RGD-IFP/7-AAD nanoprobe tail-vein injection approach demonstrated not only high tumor-targeted imaging potential, but also potent antitumor therapeutic effects in vivo. The proposed strategy may be used in peptide-driven bifunctional nanoparticles for precise imaging and cancer therapy.

OBJECTIVETo understand the prevalence of myopia in primary and middle school students in 6 provinces and the possible influencing factors.

METHODSPrimary and middle school students were selected through multistage cluster sampling in 60 primary and middle schools in 6 provinces in China. The questionnaire survey and eyesight test were conducted among all the students selected according to the national student's physique and health survey protocol. Pearson chi-square test and binary multivariate logistic regression analysis were done to identify the influencing factors for myopia in students.

RESULTSThe prevalence of myopia among primary and middle school students surveyed was 55.7%, the gender specific difference was statistically significant (59.7% for girls, 51.9% for boys) (P<0.01). The prevalence of myopia increased with age obviously. The prevalence was 35.8% in age group 6-8 years, 58.9% in age group 10-12 years, 73.4% in age group 13-15 years and 81.2% in age group 16-18 years, the differences were statistically significant (P<0.001). Single factor and multivariate analysis showed that parents' myopia, distance between computer screen and eyes, distance less than 30 cm between eyes and book while reading, distance less than 10 cm between chest and the table edge while studying, distance less than 3 cm between fingers and pen tip, sleep time, average outdoor activity time during last week, school sport activities in the afternoon, the size of television set at home, time spent on watching TV and playing computer were the influencing factors for myopia.

CONCLUSIONSThe prevalence of myopia is till high in primary and middle school students. Myopia is associated with both genetic factors and individual eye health related behaviors.

Adolescent ; Child ; China ; epidemiology ; Female ; Health Behavior ; Health Surveys ; Humans ; Logistic Models ; Male ; Multivariate Analysis ; Myopia ; epidemiology ; Parents ; Prevalence ; Schools ; Sleep ; Students ; Surveys and Questionnaires

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD⁺), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD⁺ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD⁺ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.
Humans ; NAD/metabolism* ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Nicotinamide Phosphoribosyltransferase/metabolism* ; Cytokines/metabolism* ; Quinones ; Oxidoreductases