A draft human pangenome reference.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Association between air pollution and atopic dermatitis in Guangzhou, China: modification by age and season.

BACKGROUND: The short-term effect of ambient air pollution on atopic dermatitis (AD), along with its effect modifiers, has not been fully addressed. OBJECTIVES: To examine the short-term associations between air pollution and AD, and to identify effect modifications by age and season. METHODS: We used the generalized additive model to evaluate the short-term effect of ambient air pollution on daily hospital visits for AD, adjusting for potential confounders. Subgroup analyses were performed to identify potential effect modifications by season and age (< 18 years and ≥ 18 years). RESULTS: A total of 29 972 hospital visits for AD were recorded in Guangzhou, China, from 19 January 2013 to 31 December 2017. Among them, 72·8% were visits by children and 51·4% occurred in the cool season. Acute and delayed effects on AD hospital visits were significant for all air pollutants. Stronger effects were seen in the cool season (approximately 1·7-3·0 times higher than effects in the warm season). Stronger effects were also observed in children (approximately 1·3-1·8 times higher than effects in adults). Sensitivity analyses indicated the results were robust. CONCLUSIONS: Air pollution might be an important trigger for AD in subtropical Guangzhou, China. Children are more vulnerable than adults, and the effects are stronger in the cool season.

Effect of fasting blood glucose on risk of new-onset hypertension in rural Chinese population: a 15-year follow-up cohort.

BACKGROUND: The purpose of this study was to examine the correlation between fasting blood glucose and new-onset hypertension and examine any synergistically effect modification with multiple risk factors. METHODS: We conducted post-hoc analyses of repeated-measures data in the original Dongzhi osteoporosis cohort study. In total, 3985 participants without hypertension aged 25-64 years were included in the current analyses. Generalized estimating equation models were used to assess the relationship between fasting blood glucose and risk of new-onset hypertension after adjusting for pertinent covariates and autocorrelations among siblings. RESULTS: 393 men (19.4%) and 398 women (20.3%) without hypertension at the baseline developed hypertension by the end of the study period. Compared to lower baseline fasting blood glucose levels (Q1-Q3: < 5.74 mmol/L; clinical cut points: < 5.6 mmol/L), higher baseline fasting blood glucose levels (Q4: ≥ 5.74 mmol/L; clinical cut points: ≥ 5.6 mmol/L and < 7.0 mmol/L) increased the risk of new-onset hypertension significantly [(OR: 1.54, 95% CI 1.19-1.98, P < 0.001); (OR: 1.38, 95% CI 1.09-1.75, P = 0.008)] in women. Additionally, a stronger significant association was found in women with elevated fasting blood glucose on risk of new-onset of hypertension with higher total cholesterol (≥ 5.2 mmol/L) [(OR: 2.76; 95% CI: (1.54, 4.96), P < 0.001)]. However, no association was found between fasting blood glucose and risk of new-onset hypertension in men. CONCLUSIONS: High fasting blood glucose may be significantly associated with risk of new-onset hypertension in Chinese women, especially in women with higher total cholesterol. Further randomized studies are needed to confirm our findings.

Molecular tagging of biocontrol fungus Trichoderma asperellum and its colonization in soil.

AIMS: To conduct molecular tagging of the biocontrol fungus Trichoderma asperellum strain T4 and elucidate its colonization patterns in soil. METHODS AND RESULTS: We constructed an expression vector harbouring a hygromycin B-resistant gene (hph) and an efficient green fluorescent protein (egfp) gene. By applying Agrobacterium AGL-1-mediated genetic transformation technology, we conducted molecular tagging of T. asperellum and monitored the colonization dynamics of T. asperellum in soil. The results of tracking five independent transformants of T. asperellum indicated that its expansion rates ranged from 4·7 to 6·8 cm week-1 . After inoculation in soil, the quantities of T. asperellum could be maintained at over 10 × 104  CFU per gram soil in the first year. In the third year after inoculation, the quantities of T. asperellum in soil were still higher than 1 × 103  CFU per gram soil. In addition, molecularly tagged T. asperellum in soil in the second year (i.e. 12 months) after inoculation could still reach the biocontrol effect on cucumber Rhizoctonia rot by more than 74%. CONCLUSION: Trichoderma asperellum strain T4 is capable of effectively colonizing in soil and surviving for more than 1 year. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided the scientific basis for applying T. asperellum as the biocontrol fungus for prevention and control of plant diseases.

[NEK2 gene expression in mouse cryptorchidism model and its mechanism involved in apoptosis].

Objective: To observe the expression of NEK2 mRNA and protein in the cryptorchidism mice model, and to explore its role in apoptosis of testicular tissue. Methods: A mouse cryptorchid model was constructed, and the spermatids in the spermatic tubules were observed by HE staining. Apoptosis was detected by Tunel test, and expression of NEK2 mRNA and protein was detected by RT-PCR and immunohistochemistry, respectively. Results: After the mouse cryptorchidism model was successfully constructed, the HE staining results showed that the damage of spermatogonia cells, primary spermatocytes and sperm cells in the seminiferous tubules became more severe with time. The results of Tunel test showed that the number of apoptotic cells first increased and then decreased, 1, 3, 6, 9 and 15 d apoptotic cells were 3.67±2.08 (t=2, P=0.0412), 7.67±1.53 (t=6.325, P=0.003), 17.67±3.51 (t=7.906, P=0.001), 30.67±3.51 (t=14.072, P<0.001) and 14.33±3.21 (t=6.860, P=0.002). The results of immunohistochemistry showed that NEK2 protein was expressed in the nucleus and cytoplasm in normal testis and cryptorchidism. RT-PCR and immunohistochemistry showed that expression of NEK2 mRNA and protein gradually increased after modeling. After reaching the peak, the expression gradually decreased with time, and was significantly lower than the normal control group. Conclusion: The trend of NEK2 expression in cryptorchidism tissue is consistent with the trend of cell apoptosis in cryptorchidism tissue, suggesting that abnormal expression of NEK2 may affect the damage of sperm cells in the seminiferous tubules through apoptosis, leading to infertility in patients with cryptorchidism.

Examining the relationships between life satisfaction and alcohol, tobacco and marijuana use among school-aged children.

BACKGROUND: There are many known risk factors associated with youth substance use. Nonetheless, the impact of life satisfaction (LS) on the use of alcohol, tobacco and marijuana by adolescents still remains largely unknown. METHODS: The present analysis utilized data from the Health Behavior in School-Aged Children 2009-10 US study. Multilevel logistic regression models were used to assess the relationship between LS and individual substance use. Multilevel multinomial regression models examined the relationship with total number of substances used. RESULTS: After controlling for numerous variables associated with substance use, individuals reporting low LS were significantly more likely to ever use tobacco (OR = 1.34, 95% CI = [1.01, 1.78]), alcohol (OR = 1.45, 95% CI = [1.10, 1.92]) and marijuana (OR = 1.98, 95% CI = [1.39, 2.82]). Additionally, students with low LS were significantly more likely to use two substances (OR = 1.90, 95% CI = [1.15, 3.14]) and three substances concurrently (OR = 2.00, 95% CI = [1.27, 3.16]). CONCLUSIONS: The present study identified strong associations between LS and individual, as well as concurrent, substance use among adolescents. Interventions aiming to reduce adolescent substance use may benefit from incorporating components to improve LS.

[Tailored therapy in treatment of Helicobacter pylori infectionbasedon clarithromycinsensitivity].

Objective: To compare eradication rates and compliance of patients with Helicobacter pylori(H. pylori)infection based on clarithromycin sensitivity. Methods: From July 2015 to January 2018,patients with H. pylori infection in Peking university people's hospital were randomly assignedto a 14-day treatment with clarithromycin quadruple therapy versus tailored quadruple therapy for a prospective study. In the group of tailored therapy, medications were adjusted based on clarithromycin sensitivity. In the control group, all patients were given proton pump inhibitors (PPI), amoxicillin, clarithomycin and bismuth. Eradication status was assessed 4 weeks after treatment withurea breath test. Results: The H.pylori eradication rate were higher in the tailor therapy group than that in the control group in intention-to-treat[77.8% vs 65.3%,(P=0.001)] and per,protocol analyses [86.4% vs 70.2%,(P<0.001)], the differences between the two groups were statistically significant.The incidence of compliance between the two groups were also comparable. Conclusions: The tailored therapy basedon clarithromycinsensitivity has a better eradication efficacy and a higher eradication ratesin the patients with H. pylori infection.

Revisiting the low birthweight paradox using sibling data with implications for the classification of low birthweight.

Background: We examined the birthweight threshold for increased odds of neonatal death among second births based on their elder sibling's birthweight category. Methods: This population-based cohort study included 190 575 women who delivered their first two non-anomalous singleton live births in Missouri (1989-2005). We examined the birthweight distribution and neonatal mortality curves of second births whose elder sibling had low versus adequate/high birthweight. We determined the optimal cut-off point for the classification of low birthweight among infants in each group based on the Youden index. Results: Infants whose elder sibling had low birthweight had a lower mean birthweight and a higher percentage of low birthweight infants versus those whose elder sibling had adequate/high birthweight, but low birthweight infants in the former group had a lower rate of neonatal mortality. Upon standardizing the birthweight distribution to a Z-scale, neonatal mortality rates became comparable between the two groups at every rescaled birthweight for Z-scores ≥-3.7. The optimal cut-off point for low birthweight was 2500 and 3000 g among infants whose elder sibling had low and adequate/high birthweight, respectively. Conclusions: Using sibling data for the classification of LBW may enable the identification of average-sized infants who may be at increased risk of neonatal mortality.

Risk factors and clinical outcomes for carbapenem-resistant Enterobacteriaceae nosocomial infections.

This study was aimed to determine the risk factors of Carbapenem-resistant Enterobacteriaceae (CRE) nosocomial infections and assess the clinical outcomes. A case-case-control design was used to compare two groups of case patients with control patients from March 2010 to November 2014 in China. Risk factors for the acquisition of CRE infections and clinical outcomes were analyzed by univariable and multivariable analysis. A total of 94 patients with CRE infections, 93 patients with Carbapenem-susceptible Enterobacteriaceae (CSE) infections, and 93 patients with organisms other than Enterobacteriaceae infections were enrolled in this study. Fifty-five isolates were detected as the carbapenemase gene. KPC-2 was the most common carbapenemase (65.5 %, 36/55), followed by NDM-1 (16.4 %, 9/55), IMP-4 (14.5 %, 8/55), NDM-5 (1.8 %, 1/55), and NDM-7 (1.8 %, 1/55). Multivariable analysis implicated previous use of third or fourth generation cephalosporins (odds ratio [OR], 4.557; 95 % confidence interval [CI], 1.971-10.539; P < 0.001) and carbapenems (OR, 4.058; 95 % CI, 1.753-9.397; P = 0.001) as independent risk factors associated with CRE infection. The in-hospital mortality of the CRE group was 57.4 %. In the population of CRE infection, presence of central venous catheters (OR, 4.464; 95 % CI, 1.332-14.925; P = 0.015) and receipt of immunosuppressors (OR, 7.246; 95 % CI, 1.217-43.478; P = 0.030) were independent risk factors for mortality. Appropriate definitive treatment (OR, 0.339; 95 % CI, 0.120-0.954; P = 0.040) was a protective factor for in-hospital death of CRE infection. Kaplan-Meier curves of the CRE group had the shortest survival time compared with the other two groups. Survival time of patients infected with Enterobacteriaceae with a high meropenem MIC (≥8 mg/L) was shorter than that of patients with a low meropenem MIC (2,4, and ≤ 1 mg/L). In conclusion, CRE nosocomial infections are associated with prior exposure to third or fourth generation cephalosporins and carbapenems. Patients infected with CRE had poor outcome and high mortality, especially high meropenem MIC (≥8 mg/L). Appropriate definitive treatment to CRE infections in the patient is essential.

[Expression changes of aldehyde dehydrogenase 1A1 in rat cryptorchidism and normal testis].

Objective: To study the mRNA and protein expression of aldehyde dehydrogenase 1A1(ALDH1A1)in rat cryptorchidism and normal testis. Methods: We established the cryptorchidism model by flutamide and took normal testis as normal group.The testicular tissue samples were collected on 15 days, 45 days, and 90 days after birth respectively.The expression of ALDH1A1 in rat cryptorchidism and normal testis were investigated by real-time PCR, Western blot and immunohistochemisty intissue microarray. Results: The mRNA expression of ALDH1A1 in cryptorchidism group in infant, adolescent and adult period were 1.01±0.19, 1.60±0.32, 0.75±0.16, and 1.66±0.23, 0.52±0.08, 0.15±0.10 in normal group, respectively.The expression of ALDH1A1 in cryptorchidism group was significantly lower than that in normal group in infant period, but it was significantly higher than that in the normal group in adolescent and adult period(P<0.05). Conclusions: The expression of ALDH1A1 was different in different age period during the process of testicular development of rat. It showed an important relationship between ALDH1A1 and cryptorchidism.

Co-delivery of G-CSF and EPO released from fibrin gel for therapeutic neovascularization in rat hindlimb ischemia model.

OBJECTIVE: G-CSF and EPO have shown a notable capability in neovascularization. However, their use is limited because of untoward leucocytosis, erythrogenesis, and short half-life in the plasma. Herein, we examined whether G-CSF and EPO released from fibrin gel injected into ischemic tissues would synergistically promote neovascularization with limited systematic effects in a rat hindlimb ischemic model. METHODS AND RESULTS: In vivo study, group Gel received an intramuscular injection of fibrin gel; group Gel+G-CSF received fibrin gel containing human G-CSF; group Gel+EPO received fibrin gel containing human EPO; group Gel+G-CSF&EPO received fibrin gel containing G-CSF and EPO; group G-CSF&EPO received G-CSF and EPO. Through promoting the expression of SDF-1, local high concentration of EPO could traffic CXCR4+ cells mobilized by G-CSF to enhance neovascularization in ischemic muscle. The treatment with Gel+G-CSF&EPO was superior to the other treatments on blood flow reperfusion, capillary density, and α smooth muscle actin-positive vessel density. And this treatment induced a modest WBC count increase in peripheral blood. CONCLUSIONS: G-CSF and EPO released from fibrin gel had a combined effect on postischemia neovascularization. This treatment may be a novel therapeutic modality for ischemic peripheral artery disease.

Decolorization of methylene blue in aqueous suspensions of gold nanoparticles using parallel nanosecond pulsed laser.

Using 532 nm parallel nanosecond pulsed laser, the decolorization of methylene blue (MB) in aqueous suspensions of gold nanoparticles (GNPs) was studied. The effects of various experimental parameters, such as irradiation time, laser energy, and initial MB concentration on the decolorization rate were investigated. Experiments using real samples of textile dyeing wastewater were also carried out to examine the effectiveness of the method in more complex samples. From the results, the following conclusions may be drawn: (i) Under the optimum conditions (pH 7.19, 135 mJ laser energy, 4 mg/L MB concentration, and 11.6 mg/L GNP concentration), the rate of MB decolorization could reach 94% in 15 min. The decolorization follows pseudo-first-order kinetics; (ii) The amount of MB decreased rapidly during the decolorization. No intermediates of the decolorization could be detected by high-performance liquid chromatography. These observations indicate that MB was decolorized through a very rapid degradation mechanism; (iii) The rate of MB decolorization increased with the increase in laser energy (at laser energies of 0 to 135 mJ); and, (iv) The efficient decolorization of MB in real samples of textile dyeing wastewater was achieved at a decolorization rate of about 85% in 15 min.

[Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway].

OBJECTIVE: To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism. METHODS: The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 µmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 µmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed. RESULTS: The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 µmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01). CONCLUSION: Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.

Gene expression profile in human skeletal muscle cells infected with human adenovirus type 36.

Human adenovirus type-36 (HAdV-36) is a specific pathogen that may lead to increased adiposity and obesity. In order to evaluate the effects of HAdV-36 on gene transcription, a microarray analysis of muscle cells infected with HAdV-36 was performed. Gene expression profile was determined by microarray analysis in cultured human skeletal muscle cells with or without HAdV-36 infection. Quantitative real-time PCR (qPCR) assay was performed in selected 35 genes to verify the results of the microarray analysis. A total of 13,060 unique genes were detected in the HAdV-36 infected muscle cells infected with HAdV-36. Among them, 1,004 genes were significantly altered by using a cut-off point at fold change ≥1.5 and P value <0.05. Most of the principal 100 altered genes were involved in development, immune response, signal transduction, transcriptional regulation as well as carbohydrate, lipid and protein metabolism. Thirty-two genes (91.4%) from the 35 selected genes were confirmed by qPCR assay. In addition, HAdV-36 altered 252 genes that are associated with cancer. The study showed HAdV-36 infection upregulated host cell antiviral defense. HAdV-36 also induces changes in gene expression related to cellular signaling pathways of signal transduction, transcriptional regulation as well as carbohydrate, lipid and protein metabolism. However, it remains to be investigated if HAdV-36 infection could lead to oncogenesis.

DNA polymerase ß may be involved in protecting human bronchial epithelial cells from the toxic effects induced by methyl tert-butyl ether exposure.

Methyl tert-butyl ether (MTBE), a widely used gasoline additive and a ubiquitous environmental pollutant in many countries and regions, can cause various kinds of toxic effects on human health. However, the molecular mechanism underlying its toxic effects remains elusive. The present study aimed to explore the cytotoxicity, DNA damage and oxidative damage effects of MTBE on human bronchial epithelial cells (16HBE) and the possible role of DNA polymerase ß (pol-ß) in this process. RNA interference (RNAi) was used to obtain pol-ß gene knocked-down cells (pol-ß-). CCK-8 assay was adopted to analyze the cell viability. Alkaline single-cell gel electrophoresis (SCGE) was performed to detect the DNA damage effects of MTBE. The enzyme activity of GSH-Px, SOD, CAT and the level of MDA were assessed. The data indicated that when treated with MTBE at the concentration exceeding 50 µmol/L and for the time exceeding 24 h, the pol-ß- exhibited significantly decreased cell viability and increased DNA damage effects, as compared to the control (P < 0.05). Furthermore, there was significant difference in the levels of GSH-pX, SOD, CAT and MDA between the pol-ß- and the control (P < 0.05). Our investigation suggests that MTBE can cause obvious cytotoxicity, DNA damage and oxidative damage effects on 16HBE cells. DNA polymerase ß may be involved in protecting 16HBE cells from the toxic effects induced by MTBE exposure. These findings provide a novel insight into the molecular mechanism underlying the toxic effects of MTBE on human cells.

Prognostic factors in patients with complete response of the tumour (ypT0) after neoadjuvant chemoradiotherapy and radical resection of rectal cancer.

BACKGROUND: There are only a few studies on the prognosis of patients with complete response of the tumour (ypT0) after neoadjuvant chemoradiotherapy (NCRT) and radical resection of rectal cancer. The aim of the study was to identify prognostic factors with regard to oncological outcome in ypT0 patients after NCRT and radical resection. METHODS: All ypT0 patients with rectal cancer after NCRT and radical resection between January 2010 and June 2019 were included. Cox univariate and multivariate regression analyses were used to determine the prognostic factors of these patients. RESULTS: Seventy-six patients with ypT0 rectal cancer were included. In nine patients (11.8%), lymph node metastasis was identified. Age, gender, elevated carcinoembryonic antigen (CEA) and ypN+ were risk factors associated with a worse 5-year disease-free survival (DFS) rate in univariate analysis (P = 0.08, 0.14, 0.007 and 0.003, respectively). In multivariate analysis, ypN+ and elevated CEA before NCRT were independent risk factors for worse 5-year DFS (P = 0.005 and 0.021, respectively). Elevated CEA before NCRT, post-operative chemotherapy and ypN+ were risk factors associated with worse overall survival in univariate analysis (P = 0.14, 0.002 and 0.17, respectively). However, in multivariate analysis, none of these three factors were independent risk factors for worse overall survival (P = 0.20, 0.34 and 0.06, respectively). CONCLUSION: ypN+ and elevated CEA before NCRT were found to be independent risk factors for an unfavourable DFS in ypT0 patients with complete response of the tumour after neoadjuvant chemoradiotherapy for rectal cancer.

LncRNA NEAT1 alleviates sepsis-induced myocardial injury by regulating the TLR2/NF-κB signaling pathway.

OBJECTIVE: To investigate the effect of long non-coding ribonucleic acid nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) on lipopolysaccharide (LPS)-induced myocardial injury in mice and the underlying mechanism. This study aims to provide some references for the prevention and treatment of sepsis-induced myocardial injury. MATERIALS AND METHODS: According to the random number table, 60 male C57 mice were divided into the Sham group (n=20), LPS group (n=20) and LPS + NEAT1 small interfering ribonucleic acid (siRNA) group (n=20). Sepsis-induced myocardial injury model in mice was established by intraperitoneal injection of LPS (10 mg/kg), and the NEAT1 knockout model was established by tail vein injection of NEAT1 siRNAs. After 12 h, the cardiac function of mice in each group was detected via the two-dimensional ultrasound; ejection fraction [EF (%)] and fraction shortening [FS (%)] were recorded. Hematoxylin and eosin (H&E) staining was conducted to evaluate the pathological changes in the heart tissues in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect the apoptotic levels of myocardial cells and fibroblasts in each group. In addition, the expression level of the oxidative stress marker 4-hydroxynonena (4-HNE) and the positive proportions of cluster of differentiation 45 (CD45) and CD68 in the mouse heart of three groups were detected via immunohistochemical staining. Moreover, the messenger RNA (mRNA) expression levels of inflammatory indicators [interleukin-1 (IL-1), IL-6, monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor-alpha (TNF-α)] in mouse serum of the three groups were examined by enzyme-linked immunosorbent assay (ELISA). Finally, the effects of NEAT1 siRNAs on the Toll-like receptor 2 (TLR2)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway were detected by Western blotting. RESULTS: ENEAT1 knockdown could significantly improve ischemia/reperfusion (I/R)-induced cardiac insufficiency in rats, and increase EF (%) and FS (%) (p<0.05). Besides, NEAT1 knockdown remarkably inhibited the LPS-induced myocardial injury. Compared with the LPS group, LPS + NEAT 1 siRNA group has more orderly arranged cardiac myofilament, a lower degree of degradation and necrosis, and significantly reduced cell edema. TUNEL staining showed that NEAT1 knockdown markedly reduced LPS-induced apoptosis of cardiac cells (p<0.05). Immunohistochemical results revealed that NEAT1 knockdown could remarkably reverse LPS-induced elevation of the myocardial 4-HNE expression and decrease the oxidative stress in the heart (p<0.05). At the same time, CD45+ and CD68+ cells were reduced after NEAT1 knockdown in myocardial tissues (p<0.05). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that the mRNA levels of inflammatory indicators in LPS + NEAT1 siRNA group were lower than that in the LPS group (p<0.05). According to Western blotting results, NEAT1 siRNAs could significantly downregulate the protein expressions of TLR2 and p-p65. CONCLUSIONS: NEAT1 knockdown can improve LPS-induced myocardial injury in mice by inhibiting the TLR2/NF-κB signaling pathway. LncRNA NEAT1 is expected to be a potential target for clinical treatment of the sepsis-induced myocardial injury.

Design, synthesis, and biological activity evaluation of campthothecin-HAA-Norcantharidin conjugates as antitumor agents in vitro.

Three components of Camptothecin, hydroxyacetic acid, and functionalized norcantharidins were constructed together to form a novel series of camptothecin derivatives in a good yield. The synthesized campthothecin-HAA-norcantharidin conjugate pro-drugs could suppress cancer cell growth in vitro. These conjugated pro-drug molecules possess therapeutic potential as novel bi-functional conjugates platforms for cancer treatment.

Synthesis of Dual Target CPT-Ala-Nor Conjugates and Their Biological Activity Evaluation.

BACKGROUND: Target therapy has been one of the important strategies in new drug discovery and the resulting drug resistance has also been a serious problem for concern. At the same time, there are several cancer genes or pathways operating within a given cancer. Given these two things, the combination therapy will be needed for optimal therapeutic effect. OBJECTIVE: Camptothecin and norcantharidin were thus chosen to construct a dual anticancer drugs assemblies mainly because CPT was the DNA-topoisomerase I inhibitor and norcantharidin could also suppress the cancer cell growth by inhibiting protein phosphatase. The designed conjugate of camptothecin and norcantharidin linked by alanine was expected to have dual target drug properties. METHODS: EDCI/DMAP was chosen as a coupling agent for the coupling of CPT with substituted norcantharidin derivatives and CCK-8 method was used to test the cytotoxicity and intensity on human hepatoma cell line HepG2. Two kinds of enzymes, Top I and CDC 25B were selected to screen the binding affinity in molecular level. RESULTS: Nine of dual targets camptothecin derivatives were smoothly synthesized by twice coupling in the condition of EDCI/DMAP in moderate yield. All of the synthesized compounds were characterized by 1HNMR and 13CNMR spectrum and exhibited strong potent inhibition against Hep G2, SW480, BGC803, and PANC-1 cell line in vitro. The newly synthesized camptothecin compounds, such as 3j and 3i have strengthened inhibition activity compared to camptothecin and norcantharidin. CONCLUSION: We have successfully synthesized a series of novel camptothecin derivatives constructed from three components of camptothecin, alanine and norcantharidin. These compounds not only preserved strong activity against several cancer cell lines in vitro, but also exhibited potential binding affinity to target Top I and CDC 25B. Therefore, these conjugates linked by alanine could suppress cancer cell growth by inhibiting Top I and protein phosphatase simultaneously, which makes it much valuable as a novel bi-functional target drug candidate to develop in vivo.

Bioactives of Artemisia dracunculus L enhance cellular insulin signaling in primary human skeletal muscle culture.

An alcoholic extract of Artemisia dracunculus L (PMI 5011) has been shown to decrease glucose and improve insulin levels in animal models, suggesting an ability to enhance insulin sensitivity. We sought to assess the cellular mechanism by which this botanical affects carbohydrate metabolism in primary human skeletal muscle culture. We measured basal and insulin-stimulated glucose uptake, glycogen accumulation, phosphoinositide 3 (PI-3) kinase activity, and Akt phosphorylation in primary skeletal muscle culture from subjects with type 2 diabetes mellitus incubated with or without various concentrations of PMI 5011. We also analyzed the abundance of insulin receptor signaling proteins, for example, IRS-1, IRS-2, and PI-3 kinase. Glucose uptake was significantly increased in the presence of increasing concentrations of PMI 5011. In addition, glycogen accumulation, observed to be decreased with increasing free fatty acid levels, was partially restored with PMI 5011. PMI 5011 treatment did not appear to significantly affect protein abundance for IRS-1, IRS-2, PI-3 kinase, Akt, insulin receptor, or Glut-4. However, PMI 5011 significantly decreased levels of a specific protein tyrosine phosphatase, that is, PTP1B. Time course studies confirmed that protein abundance of PTP1B decreases in the presence of PMI 5011. The cellular mechanism of action to explain the effects by which an alcoholic extract of A dracunculus L improves carbohydrate metabolism on a clinical level may be secondary to enhancing insulin receptor signaling and modulating levels of a specific protein tyrosine phosphatase, that is, PTP1B.