Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos.

The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3' untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features.

A meta-analysis of association between serum iron levels and lung cancer risk.

Many studies conducted on the relationship between serum iron levels and lung cancer risk had produced inconsistent results. We therefore conducted a meta-analysis to determine whether serum iron levels were lower in lung cancer patients compared to those in controls.A literature survey was conducted by searching the PubMed, WanFang, CNKI, and SinoMed databases for articles published as of Mar 1, 2018. Standard mean differences (SMD) with the corresponding 95% confidence intervals (CI) were executed by Stata 12.0 software. A total of 13 publications involving 1118 lung cancer patients and 832 controls were included in our study. The combined results showed that serum iron levels in lung cancer cases had no significantly lower when compared to those in controls [summary SMD = -0.125, 95%CI= -0.439, 0.189, Z = 0.78, p for Z test= 0.435], with high heterogeneity (I2= 89.9%, P< 0.001) found. In the stratified analysis by geographic locations, consistent results were found for serum iron levels between lung cancer patients and controls both in Asian populations [summary SMD = -0.113, 95%CI= -0.471, 0.245] and European populations [summary SMD = -0.215, 95%CI= -0.835, 0.404]. Publication bias was not found when evaluated by Begg's funnel plot and Egger's regression asymmetry test.In summary, the current study showed that serum iron levels had no significant association on lung cancer risk.

Combined detection of CEA and CA125 for the diagnosis for lung cancer: A meta-analysis.

This study aimed to systematically evaluate the value of combined detection of serum CEA and CA125 concentrations for the diagnosis of lung cancer. Related studies regarding the diagnosis of lung cancer were searched in PubMed, Embase, CNKI, and Wanfang using a computer. The number of patients who were true-positive, false-positive, false-negative, and true-negative were extracted from each study. Meta-analysis was performed using the Meta-Disc l.4, RevMan 5.3. Seven studies involving 2,216 cases were finally included. Regarding the diagnosis of lung cancer, the sensitivity, specificity, and diagnostic odds ratio of combined CEA and CA125 detection were higher than those of CEA detection alone. The area under the curve (AUC) of combined detection was 0.90, whereas the independently detected AUC was 0.73. Combined CEA and CA125 detection has higher diagnostic efficiency for lung cancer than CEA detection alone. The significance of combined serum CEA and CA125 detection in lung cancer is confirmed.

Association between BIM polymorphism and lung cancer outcomes: a meta-analysis.

Accumulating evidences have indicated that BIM expression largely decides the development of lung cancer and outcome of EGFR-mutant lung cancers after TKI treatments. BIM polymorphism is a 2,903-bp deletion in the second exon. To clarify the relationship between this BIM polymorphism and clinical outcomes of lung cancers, we conducted this meta-analysis and observed the survival and responses to TKIs. Sixteen cohort studies, covering 4393 WT and 916 BIM deletion patients were included. Overall, BIM deletion polymorphism was associated with significantly shorter progression-free survival (PFS) and slightly shorter overall survival (OS), compared to the WT group. Moreover, patients with BIM deletion polymorphism showed significantly inferior response to EGFR TKIs. In conclusion, our analysis confirmed that lung cancer patients harboring the BIM deletion have inferior survival and TKI responses. Examination of the novel biomarker BIM deletion in lung cancer patients, especially for the EGFR mutant cohort, could provide some prognostic utility.

Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t1/2 ) at 40 °C and a 4.6 and 5.1 °C increase in T5010 . In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40 °C and a 5.5 °C increase in T5010 . Molecular dynamics simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C-D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121-136.

Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations.

Amine transaminases have recently gained a lot of attention for the synthesis of chiral amines. Using (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) as a transaminase model, in silico design was applied employing B-factor and folding free energy (ΔΔGfold) calculations. Mutation sites were selected by targeting flexible regions with the greatest B-factors, and were substituted with amino acids that were determined by folding free energy calculations (ΔΔGfold < 0) to be more rigid than the original ones. By site-directed mutagenesis, we obtained four stabilized mutants (T130M, T130F, E133F and D134L) with improved stability from 19 candidates. Compared to the wild type, the best single mutant (T130M) showed an increase in thermal stability with a nearly 2.2-fold improvement of half-life (t1/2) at 40 °C and a 3.5 °C higher T1/210 min. The optimum catalytic temperature of T130F was increased by 10 °C. In addition, the T130M/E133F double mutant displayed the largest shift in thermostability with 3.3-fold improvement of t1/2 at 40 °C and a 5.0 °C higher T1/210 min. Modeling analysis showed that new hydrophobic interactions and hydrogen bonds might contribute to the observed thermostability improvement.

[Isolation, characterization and real-time RT-PCR for relative quantification of gene expression in H+-ATPase-defective mutants from Lactobacillus plantarum].

Objective: The aim of this study was to isolate Lactobacillus plantarum acid-sensitive mutants with lower H+-ATPase activity, and to study the mechanism of H+-ATPase regulation in Lactobacillus plantarum. Methods: We used neomycin to isolate acid-sensitive mutants of L. plantarum, and measured H+-ATPase activity and lactic acid production of wild-type and mutants. Genomic DNA was extracted from the wild-type ZUST and two mutants (ZUST-1, ZUST-2), and used as PCR templates. H+-ATPase genes of the strain were amplified, and the PCR products were sequenced. Sequence similarity of H+-ATPase was analyzed. Real-time RT-PCR was used to evaluate the relative quantification of the H+-ATPase genes expression. Results: The growth of the mutants was characterized in MRS broth, which revealed that their cell biomass and acid production were lower than that of the wild-type. H+-ATPase activity of the mutants ZUST-1 and ZUST-2 was 10.1% and 28.8% lower than that of the wild-type. Results showed that atpA gene of the mutants ZUST-1 and ZUST-2 existed 22 mutations by alignment of the wild-type sequence, and atpC gene of ZUST-2 existed 6 mutations. Mutants ZUST-1 and ZUST-2 atpA gene expression were 41.1% and 35.7% lower than that of the wild-type in exponential phase, 43.6% and 14.2% in stationary phase, respectively. The atpC gene expression of ZUST-1 was similar to that of the wild-type in exponential phase, and was 30% higher than that of the wild-type in stationary phase, and ZUST-2 atpC gene was not expressed. Conclusion: The mutants with lower H+-ATPase activity were found to up-regulate the expression of H+-ATPase genes in stationary phase, except ZUST-2 atpC gene was not expressed. H+-ATPase activity has an important connection with the difference in gene expression of atpA and atpC. The results of this study will pave the way for gaining further insights into the mechanism of the H+-ATPase-defective mutants.

Sleep duration, snoring habits and risk of acute myocardial infarction in China population: results of the INTERHEART study.

BACKGROUND: Less sleep time and snoring have been associated with cardiovascular disease (CVD) risk in Western populations; however, few studies have evaluated the different aspects of sleep duration and snoring frequency in relation to CVD, and this association has not been examined in China. The present study aimed to address the relation between sleep duration, snoring frequency and risk of acute myocardial infarction (AMI) in China population. METHODS: We conducted a hospital-based case-control study. Cases were first AMI (n = 2909). Controls were matched to cases on age and sex. 2947 controls who did not report previous angina or physical disability completed a questionnaire on sleep duration and snoring frequency. We used logistic regression to control for other risk factors. RESULTS: We observed an inverse association between serious snoring frequency and AMI risk. After adjustment for all the risk factors, and the OR for everyday group and 3-5 times per week group was 1.45 (95% CI: 1.01 to 1.91) and 1.93 (95% CI: 1.52-2.46) compared to no snoring group. The OR for serious level group and moderate group was 1.77 (95% CI: 1.29 to 2.43) and 1.37 (95% CI: 1.10 to 1.69) compared to no snoring group. People having serious snoring increased 77% risk of AMI. 15.2% people in control group have ≤ 6 hours sleeping, compared with 17.4% in AMI group. CONCLUSIONS: Snoring frequency, including as much as everyday and 3-5 times per week, was positively associated with AMI risk and less sleep duration was associated with risk of AMI. Less sleep time could increase AMI risk in China population.

Policy design and implementation: a quantitative evaluation and empirical study of China's rural sports policy based on the PMC index model.

The formulation and implementation of a rural sports policy is an important means of promoting rural sports, improving the physical wellbeing of farmers, and enhancing the cohesion of rural communities. However, introducing such a policy faces problems in the process of specific policy practices, such as poor effective implementation, a lagging implementation effect, and goal cognitive bias. How to look at the current rural sports policy implementation blockage problem and the governance of the blockage, in order to improve the level of rural sports public service, is the focus of this paper's research. On this basis, this paper selects 56 policy texts, issued from 2002 to 2023, that are highly relevant to rural sports and have high timeliness and authority from the sports policies issued in China. Also, ROST CM6 software is used to count high-frequency words; this study then draws keyword social network mapping for the visual analysis of policy preferences and selects 20 rural sports policy texts as typical samples. Finally, a policy modeling research consistency (PMC) index model is used to evaluate the texts comprehensively and quantitatively. The results show that the overall design of China's rural sports policies is relatively reasonable. However, the consistency and effectiveness of their implementation need to be improved. Twenty representative policy texts have an average PMC index score of 5.96, with a concave index of 3.04 (which is good overall), with the highest mean value for rural sports policies at the national level. This is followed by the second highest value at the municipal and county levels, and the smallest at the provincial level. Therefore, in the future formulation and implementation of rural sports policies, a multi-dimensional rural sports policy system should be constructed. This would help to strengthen the consistency and effectiveness of the implementation of the policy system and promote the high-quality development of rural sports.

Identification of a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine as a novel inhibitor of the transcription factor p53.

AIM: To identify novel small compound inhibitor of p53 protein. METHODS: Mouse embryonic fibroblasts (MEF) and mouse embryonic stem (ES) cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips. RESULTS: Treatment of MEF cells with a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine (G5) at 10 µmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 (10 µmol/L) up to 48 h neither affected the proliferation rate nor caused morphological alterations. CONCLUSION: G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.

A multi-axis robot-based bioprinting system supporting natural cell function preservation and cardiac tissue fabrication.

Despite the recent advances in artificial tissue and organ engineering, how to generate large size viable and functional complex organs still remains as a grand challenge for regenerative medicine. Three-dimensional bioprinting has demonstrated its advantages as one of the major methods in fabricating simple tissues, yet it still faces difficulties to generate vasculatures and preserve cell functions in complex organ production. Here, we overcome the limitations of conventional bioprinting systems by converting a six degree-of-freedom robotic arm into a bioprinter, therefore enables cell printing on 3D complex-shaped vascular scaffolds from all directions. We also developed an oil bath-based cell printing method to better preserve cell natural functions after printing. Together with a self-designed bioreactor and a repeated print-and-culture strategy, our bioprinting system is capable to generate vascularized, contractible, and long-term survived cardiac tissues. Such bioprinting strategy mimics the in vivo organ development process and presents a promising solution for in vitro fabrication of complex organs.

[Cloning, sequencing of G3PD gene from Brugia malayi and prediction of B cell epitopes in its amino acid sequence].

Specific primers were designed and synthesized based on the reported glyceraldehyde-3-phosphate dehydrogenase (BmG3PD) gene of Brugia malayi (GenBank Accession No. U18137). Total RNA was extracted from Brugia malayi and its BmG3PD gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was purified and cloned into plasmid pGEM-T, then transformed into Escherichia coli DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pGEM-T-BmG3PD was confirmed by sequencing and homology comparison. Five parameters and methods were used to predict B-cell epitopes in amino acid sequence of BmG3PD. The amplified DNA fragment (1,020 bp) had a high identity of 99% with the BmG3PD gene sequence of Brugia malayi. B-cell epitopes of BmG3PD were probably at or adjacent to 22-36, 242-255, 303-318 and 326-336 in its amino acid sequence.

Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2) at 40 °C, and a 4.6 °C increase in T5010 among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T5010. Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T5010.

[Cloning and eukaryotic expression of the gene encoding myosin from Brugia malayi].

Total RNA was extracted from periodic microfilariae of Brugia malayi and its myosin partial gene (Bm-M55) was amplified by RT-PCR. The PCR product was cloned and then subcloned into pcDNA3.1 (+)vector. The recombinant eukaryotic plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and was transfected into COS-7 cells subsequently. The expressed protein was identified by SDS-PAGE. Bm-M55 mRNA was highly expressed in transfected COS-7 cells. The deduced amino acid sequence showed to be identical with that of Bm-M55, and the recombinant protein was about Mr 55000.

METTL3-mediated N6-methyladenosine mRNA modification enhances long-term memory consolidation.

The formation of long-term memory is critical for learning ability and social behaviors of humans and animals, yet its underlying mechanisms are largely unknown. We found that the efficacy of hippocampus-dependent memory consolidation is regulated by METTL3, an RNA N6-methyladenosine (m6A) methyltransferase, through promoting the translation of neuronal early-response genes. Such effect is exquisitely dependent on the m6A methyltransferase function of METTL3. Depleting METTL3 in mouse hippocampus reduces memory consolidation ability, yet unimpaired learning outcomes can be achieved if adequate training was given or the m6A methyltransferase function of METTL3 was restored. The abundance of METTL3 in wild-type mouse hippocampus is positively correlated with learning efficacy, and overexpression of METTL3 significantly enhances long-term memory consolidation. These findings uncover a direct role of RNA m6A modification in regulating long-term memory formation, and also indicate that memory efficacy difference among individuals could be compensated by repeated learning.

Modified physiological and operative score for the enumeration of mortality and morbidity risk assessment model in general surgery.

AIM: To establish a scoring system for predicting the incidence of postoperative complications and mortality in general surgery based on the physiological and operative severity score for the enumeration of mortality and morbidity (POSSUM), and to evaluate its efficacy. METHODS: Eighty-four patients with postoperative complications or death and 172 patients without postoperative complications, who underwent surgery in our department during the previous 2 years, were retrospectively analyzed by logistic regression. Fifteen indexes were investigated including age, cardiovascular function, respiratory function, blood test results, endocrine function, central nervous system function, hepatic function, renal function, nutritional status, extent of operative trauma, and course of anesthesia. Modified POSSUM (M-POSSUM) was developed using significant risk factors with its efficacy evaluated. RESULTS: The significant risk factors were found to be age, cardiovascular function, respiratory function, hepatic function, renal function, blood test results, endocrine function, nutritional status, duration of operation, intraoperative blood loss, and course of anesthesia. These factors were all included in the scoring system. There were significant differences in the scores between the patients with and without postoperative complications, between the patients died and survived with complications, and between the patients died and survived without complications. The receiver operating characteristic curves showed that the M-POSSUM could accurately predict postoperative complications and mortality. CONCLUSION: M-POSSUM correlates well with postoperative complications and mortality, and is more accurate than POSSUM.

[Construction of eukaryotic expression plasmids with paramyosin gene of periodic Brugia malayi].

Total RNA was extracted from periodic Brugia malayi Specific primers were designed on the basis of known sequences of paramyosin gene from B. malayi (BmPmy). The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli (E. coli) strain DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The right gene fragments encoding BmPmy in positive clones for prokaryotic and eukaryotic expression plasmids were digested with restrictive endonuclease, and were subcloned into pcDNA3.1(+). The recombinant eukaryotic plasmid (pcDNA3.1-BmPmy) was then transfected into COS-7 cells. The transient expression of BmPmy was examined with RT-PCR. BmPmy mRNA was highly expressed in transfected COS-7 cells.

[Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus].

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T50¹° (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

Preparation and copper ions adsorption properties of thiosemicarbazide chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of ß-chitin from squid pens. Thiosemicarbazide group was introduced to chitosan via formaldehyde-derived linkages, and thiosemicarbazide chitosan (TSFCS) with different degrees of substitution (DS) was synthesized. The DS values of TSFCS calculated by elemental analysis were 0.19, 0.36 and 0.63. The structure of the TSFCS was confirmed by elemental analysis, FTIR, XRD, TGA and SEM. The adsorption capacity of Cu(II) ions by TSFCS showed good correlation with the DS and pH (pH range 2.2-5.8). The maximum Cu(II) ions adsorption capacity of all three TSFCS samples reached 134.0mgg-1 at pH 3.6, but chitosan showed no adsorption at this pH. The adsorption equilibrium process of Cu(II) ions onto TSFCS was better described by the Langmuir model than the Freundlich isotherm model. Cu(II) ions adsorbed by TSFCS could be released using 0.01M Na2EDTA and the adsorption capacity could retain above 80% after five adsorption-desorption cycles. TSFCS exhibited good potential for heavy metal removal because of its high adsorption capacity at the low pH.