In vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in human acute promyelocytic leukemia HL-60 cells.

OBJECTIVE: Our study aims to explore the in vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in HL-60 cells. METHODS: According to the construction of lentiviral vector LV-OSCK of reprogramming factors (Oct-4, Sox2, Klf4, c-Myc), 293T cells were transfected to detect virus titer. The endogenous pluripotent genes (Oct4, SOX2, c-Myc and Klf4) and CD34 mRNA and protein expressions were detected by AP staining, immunofluorescence staining, qRT-PCR and flow cytometry. RESULTS: Expressions of Oct4, SOX2, c-Myc and Klf4 were 0.220±0.013, 0.186±0.009, 0.287±0.015 and 0.153±0.007. These levels were significantly higher in the experimental group than the control and blank groups. CD34 protein expression in the experimental group was also discovered to be significantly higher than the other two groups. CONCLUSION: The reprogramming factors could increase the expressions of pluripotent genes and CD34 gene in HL-60 cells.

A new chromone and a new aliphatic ester isolated from Daldinia eschscholtzii.

A new chromone and a new aliphatic ester were isolated from the EtOAc extract of myceliums of Daldinia eschscholtzii. Their structures were elucidated as (R)-5-hydroxy-8-methoxy-2-methylchroman-4-one (1) and (E)-6-(non-3-en-1-yl) -2H-pyran-2-one (2) by interpretation of the spectroscopic evidence.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Nitric oxide production inhibition and mechanism of phenanthrene analogs in lipopolysaccharide-stimulated RAW264.7 macrophages.

Natural phenanthrene derivatives are considered to be important resource for the anti-inflammatory therapeutics, but their structure-activity relationship and mechanisms are still unknown. In this study we evaluated 20 synthesized phenanthrene analogs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compounds 10, 11 and 17 were found to inhibit the production of nitric oxide (NO) with IC50 values of 37.26µM, 5.05µM and 20.31µM, respectively. Compound 11 decreased LPS-induced expression of inducible NO synthase (iNOS), inhibited phosphorylation of p38 mitogen-activated protein kinase (MAPK) and serine/threonine kinase Akt. It also suppressed the phosphorylation and degradation of inhibitory kappa B-α (IκBα). Data obtained suggest that compound 11 exerts anti-inflammatory effects by inhibiting p38 MAPK and nuclear factor κB (NF-κB) pathways, which warrants further investigation as a new anti-inflammatory pharmaceutical tool.

[Chemical Constituents from Fruiting Bodies of Amanita caesarea].

Objective: To study the chemical constituents from fruiting bodies of Amanita caesarea. Methods: Silica gel column chromatography was applied for the isolation and purification of the constituents. The structures were established by means of spectroscopic and chemical data. Results: Eight compounds were obtained and identified as ergosterol( 1),ergosterol peroxide( 2),9,11-dehydroergosterol peroxide( 3),glycerol-1-monooleate( 4),ethyl oleate( 5),( 2S,3R,4E,8E)-1-( ß-D-glucopyranosyl)-3-hydroxy-2-[( R)-2'-hydroxyoctadecanoyl]amino-9-methyl-4,8-octadecadiene( 6),tetradecy lamine( 7) and berberine( 8). Conclusion: All the compounds are isolated from Amanita caesarea for the first time.

[Optimization of submerged culture conditions for Xylaria striata mycelium].

OBJECTIVE: To establish the optimum submerged culture condition of Xylaria striata mycelium. METHODS: One-factor-at-a-time method and orthogonal experiment design were applied and the dry weight of mycelium was tested for evaluation the biomass of the growth of Xylaria striata mycelium. RESULTS: The results from one-factor-at-a-time experiments showed that maltose, glucose or corn powder could be used as the best carbon source and the optimum nitrogen source was soybean powder. The best ratio of carbon source to nitrogen source was 5:1. In addition, the higher dry weight of mycelium was found at pH 6 when 2 mycelial discs were inoculated and fermented for 7 days. Otherwise, the growth of mycelium was observed to be promoted significantly by addition with K,Mg,P and VB1. The consequence of orthogonal experiment showed that the optimum carbon source and nitrogen source were maltose 4% and soybean powder 0.8%, respectively,and the highest mycelium biomass could be obtained at pH 7 and 25 °C shaking for 13 days. CONCLUSION: A large amount of mycelium will be obtained under the optimum condition of liquid culture for Xylaria striata.

[Multi Scenario Carbon Peak Prediction and Emission Reduction Path Analysis of Xi'an Hi-tech Zone].

The fifth session of the 13th National People's Congress proposed to be committed to promoting carbon peaking and carbon neutrality, promoting the comprehensive green and low-carbon transformation of the economy and society and achieving high-quality development. As an important scientific and technological innovation and industrial cluster in Shaanxi Province, the economic development of the Xi'an Hi-tech Zone largely relies on energy consumption, making the task of carbon reduction particularly challenging. Firstly, taking the Xi'an Hi-tech Zone as the research object, through systematic accounting of carbon emissions within the park, we analyzed the current carbon emission status of enterprises in different energy types and industries. Then, using the Kaya model, multiple independent carbon peak scenarios were set up to predict the total carbon emissions and peak time under different scenarios. Finally, based on the development characteristics of the Xi'an Hi-tech Zone, we scientifically selected corresponding carbon emission reduction paths and provided reasonable emission reduction suggestions. The results showed that the proportion of carbon emissions consumed by electricity was currently the highest, and the share was increasing yearly. Industrial carbon emissions had always been dominant, and the development of the tertiary industry was becoming increasingly prosperous. In the scenario prediction, the carbon emission factor scenario, energy intensity scenario, and economic level scenario could reach the carbon peak by 2030. Among them, the economic development level had the greatest impact on the peak and time of the future carbon peak in the Xi'an Hi-tech Zone, whereas the industrial structure scenario, energy source structure scenario, and population size scenario had no peak before 2030. The future emission reduction path mainly started from decarbonization of the power sector, stable and high-quality economic development, green upgrading of energy and industrial structure, and building a green transportation system. This can reserve more preparation time for achieving carbon neutrality and provide decision-making reference for the low-carbon development of industrial parks in China.

Adverse drug interactions as a high-risk factor for lethal post-transplant complications in Chinese population.

Metabolism of triazole antifungal agents is highly competitive to conventional post-transplant immunosuppressants like cyclosporine A (CsA) via the cytochrome P450-dependent pathway. We present the first report on lethal complications that may arise due to this type of drug interaction. A retrospective survey identified 10 of 104 cases (9.62%) that suffered life-threatening complications associated with the interaction between CsA and itraconazole or voriconazole following allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center. According to the close drug monitoring, all 10 patients experienced supratherapeutic levels of CsA even with a preemptive CsA dosage reduction and prompt dose adjustment. Six patients developed grade I to III acute graft-versus-host disease (aGVHD) and eventually died from either idiopathic pneumonia syndrome or diffuse alveolar hemorrhage; another four patients died from CSA-associated neurological complications. Impaired hepatic and renal function was noted in only one of these 10 cases. The high frequency as well as the unpredictability of severe complications lead us to suggest that triazole should always be replaced by another antifungal medication (e.g., amphotericin B or Echincandins) while patients receive CsA after HSCT, especially in the Chinese population.

[Extraction technology of diester-type alkaloids from Radix Aconiti lateralis].

OBJECTIVE: To determine the suitable extraction technology of diester-type alkaloids, such as mesaconitine, aconitine and hypaconitine from the roots of Aconitum carmichaeli (Radix Aconiti lateralis). METHODS: The contents of mesaconitine, aconitine and hypaconitine were determined by HPLC. Single-factor experiment was used to study the extraction factors. RESULTS: The suitable extraction technology for diester-type alkaloids was as follows: coarse sizings of Radix Aconiti lateralis was extracted by 10 times ethanol for 3 times (each time for 1 day) at 15 degrees C. The total yield of diester-type alkaloids was 0.57%, including 0.16% aconitine, 0.032% mesaconitine and 0.38% hypaconitine, and the purity was 18.81%. CONCLUSION: The extraction technology is efficient, harmfulless, economical, convenient and can be used for industrial production.

Effects of acute normovolemic hemodilution on perioperative coagulation and fibrinolysis in elderly patients undergoing hepatic carcinectomy.

OBJECTIVE: To observe the effects of acute normovolemic hemodilution (ANH) on coagulation function and fibrinolysis in elderly patients undergoing hepatic carcinectomy. METHODS: Thirty elderly patients (aged 60-70 years) with liver cancer (American Society of Anesthesiologists physical status I-II) scheduled for hepatic carcinectomy from February 2007 to February 2008 were randomly divided into ANH group (n = 15) and control group (n = 15). After tracheal intubation, patients in ANH group and control group were infused with 6% hydroxyethyl starch (HES) (130/0.4), and basic liquid containing 6% HES and routine Ringer's solution, respectively. In all the studied patients, blood samples were drawn at five different time points: before anesthesia induction (T1), 30 minutes after ANH (T2), 1 hour after start of operation (T3), immediately after operation (T4), and 24 hours after operation (T5). Then coagulation function, soluble fibrin monomer complex (SFMC), prothrombin fragment (F1+2), and platelet membrane glycoprotein (activated GPIIb/GPIIIa and P-selectin) were measured. RESULTS: The perioperative blood loss was not significantly different between the two groups (P > 0.05). The volume of allogeneic blood transfusion in ANH group was significantly smaller than that in control group (350.5 +/- 70.7 mL vs. 457.8 +/- 181.3 mL, P < 0.01). Compared with the data of T1, prothrombin time (PT) and activated partial thromboplastin time in both groups prolonged significantly after T3 (P < 0.05), but still within normal range. There were no significant changes in thrombin time and D-dimer between the two groups and between different time points in each group (all P > 0.05). SFMC and F1 + 2 increased in both groups, but without statistical significance. P-selectin expression on the platelet surface of ANH group was significantly lowered at T2 and T3 compared with the level at T1 (P < 0.05). Compared with control group, P-selectin was significantly lower in ANH group at T2-T5 (all P < 0.05). CONCLUSIONS: In elderly patients undergoing resection of liver cancer, ANH may not hamper fibrinolysis and coagulation function. It could therefore be safe to largely reduce allogeneic blood transfusion.

Antishock Characteristics of Erythrocyte-mediated Endoplasmic Reticulum Stress in Macrophages in Severe Hemorrhagic Shock Environment Based on TLR9-cGAS-STING-IFN Signal Axis.

This study aimed to investigate the protective effects of erythrocyte-mediated endoplasmic reticulum (ER) stress in macrophages in hemorrhagic shock. An hemorrhagic shock model was established in male BALB/c mice. Animals were randomly divided into three groups (n = 8): control group (A), erythrocyte reinfusion group (B), and TLR9 inhibition group (C). Eight healthy BALB/c mice were also included as group N (n = 8). Mice in group A were not treated, while mice in groups B and C were transfused with red blood cells separated from the blood of mice in group N. Flow cytometry was used to detect the expression of erythrocyte surface protein TLR9 in each group. Immunofluorescence assay was used to analyze the distribution and relative expression of protein STING in macrophages. Flow cytometry was used to analyze the expression of STING, ATF6, and IRE1 in macrophages. Enzyme-linked immunosorbent assay was used to analyze the levels of inflammatory signal molecules, including IFN-α, IFN-ß, IL-6, CCL4, CCL5, and IL-6. FITC-Annexin V was used to analyze the apoptosis of immune cells (macrophages) in mouse blood samples and to detect the concentration of calcium ions in erythrocyte cytoplasm. The results showed that the expression of erythrocyte surface protein TLR9; the distribution of STING-positive cells in macrophages; the expressions of STING, ATF6, and IRE1 in macrophages; the levels of inflammatory signal molecules; the apoptosis rate of macrophages; and the intracellular calcium concentration in erythrocytes in group B were higher than those in group A, followed by group C. These results suggest that TLR9 regulates ER stress in macrophages of mice with hemorrhagic shock through the TLR9-cGAS-STING-IFN signaling pathway. Increased expression of TLR9 enhanced macrophage activity, reduced apoptosis, enhanced inflammatory response and immune response, and restored electrolyte level, which might be a therapeutic option for the treatment of hemorrhagic shock.

[Analysis of volatile oil from Ardisia brevicaulis].

OBJECTIVE: To analyze and compare the compounds in the essential oil from the leaves and roots of Ardisia brevicaulis. METHODS: The essential oil were obtained by steam distillation. The chemical components were separated and identified by gas chromatography-mass spectrometry (GC-MS). RESULTS: 38 compounds were identified from the leaves (65.952% of the total essential oil) and 46 compounds were identified from the roots (54.890% of the total essential oil). The main constituents of the leaves essential oil were Palmitic acid (43.329%), Fitone (2.430%), Phytol (3.142%), and so on. The main constituents of the roots essential oil were Calamenene (2.913%), cis-alpha-Bisabolene (5.222%), gamma-Muurolene (14.227%), Caryophyllene (11.592%), and so on. CONCLUSION: The constituents of volatile oil extracted from the leaves and roots of Ardisia brevicaulis were different, so the leaves and roots of Ardisia brevicaulis should be utilized differently in clinical application.

[Relationship between Ghrelin polymorphism and serum lipoprotein levels in Han Chinese with or without coronary heart disease risk factors].

OBJECTIVE: To investigate the relationship between polymorphism of Ghrelin gene and serum levels of lipoprotein in Han Chinese with or without coronary heart disease (CHD) risk factors. METHODS: PCR restriction fragment length polymorphism assay was used to detect the distribution of genotypes of Ghrelin gene in 225 Han Chinese (40 to 69 years-old) with CHD risk factors, 78 subjects without CHD risk factors served as normal controls. Serum levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) were measured to analyze the relationship with the polymorphism of Ghrelin gene. RESULTS: Ghrelin genotype frequencies of AA, AG, GG (0.975, 0.025, 0.00 in control group and 0.956, 0.040, 0.004 in the high-risk group, all P > 0.05) as well as the allele frequencies of A, G (0.987, 0.013 in control group and 0.976, 0.024 in the high-risk group, all P > 0.05) were similar between the groups. HDL-C levels of the Arg/Gln carriers were significantly lower than those of Arg/Arg carriers in control group and in the high-risk group (all P < 0.05). CONCLUSION: Arg/Gln carriers were associated lower HDL-C levels in Han Chinese.

Suppression of cortical TRPM7 protein attenuates oxidative damage after traumatic brain injury via Akt/endothelial nitric oxide synthase pathway.

Neuronal death after traumatic brain injury (TBI) is a complex process resulting from a combination of factors, many of which are still unknown. Transient receptor potential melastatin 7 (TRPM7) is a transient receptor potential channel that has been demonstrated to mediate ischemic and traumatic neuronal injury in vitro. In the present study, TRPM7 was suppressed in the rat cerebral cortex by intracortical injections of viral vectors bearing shRNA specific for TRPM7 to investigate its potential role in an in vivo TBI model. We found that TRPM7 suppression significantly reduced brain edema, brain contusion volume and motor functional deficits, which was sustained for at least 2 weeks after the insult. These protective effects were accompanied by inhibited apoptosis in injured cortex. Also, TRPM7 suppression attenuated lipid peroxidation, decreased the expression of protein carbonyl, and preserved the endogenous antioxidant enzyme activities. The results of western blot analysis showed that TRPM7 suppression markedly increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). In addition, blocking Akt/eNOS pathway activation by the specific inhibitor LY294002 (LY, 10 µL, 10 mmol/L) or L-NIO (0.5 mg/kg) partially reversed the protective effects of TRPM7 suppression and its anti-oxidative activities. Taken together, these findings demonstrated that regional inhibition of TRPM7 in cerebral cortex exerts neuroprotective effects against TBI through activation of Akt/eNOS pathway. Thus, TRPM7 might represent a potential drug development target for the treatment of TBI.

[Effects of Triptolide Combined with Homoharringtonine on Proliferation and Apoptosis of KG-1α Cells].

OBJECTIVE: To investigate the effect and possible mechanism of low concentration of triptolide (TPL) combined with homoharringtonine (HHT) on the proliferation and apoptosis of KG-1α cells. METHODS: CCK-8 method was used to detect the antiproliferating effects of different concentrations of TPL and HHT single-use and combined use on KG-1α cells, and the combined index (CI) was calculated. The colony formation ability was also determined by methylcellulose colony formation assay, cell surface molecules, apoptosis rate and cell cycle changes were detected by flow cytometry. Westerrn blot was used to detect the expression of Akt signaling pathway related proteins before and after low dose TPL combined with HHT using. RESULTS: High expression of CD34 and CD123 were on KG-1a cells, which being lack expression of CD38. TPL and HHT dose-dependently inhibited the proliferation of KG-1α cells. Compared with low dosage TPL and HHT single-use groups, the cell proliferation and colony formation efficiency were lower, and the cell apoptosis rate was higher in the combined group. CI values also indicated that low concentration TPL combined with HHT possessed highly synergistic effect. After the combination of the 2 drugs, the expressions of P-Aktser473, P-Aktthr308, BCL-2, PARP and survivin protein were down-regulated and the cleavage of PARP protein was increased. CONCLUSION: Low concentration of TPL combined with HHT can synergistically inhibit KG-1α cell proliferation and induce its apoptosis through the PI3K/Akt signaling pathway and downstream protein.

Xylastriasan A, a new cytochalasan from the fungus Xylaria striata.

Xylastriasan A (1), a new cytochalasan alkaloid with a rare 5/6/6/5/6 pentacyclic skeleton, and ergosterol (2) were isolated from the ethanol extract of fruiting bodies of the fungus Xylaria striata. Their structures were determined by analysis of their spectroscopic data. Compound 1 exhibited weak cytotoxic activity against HEPG2, B16 and A549 cell lines with IC50 values of 93.61, 85.61 and 91.58 µM, respectively. Ergosterol (2) potentiated pentobarbital-induced sleep by not only increasing the number of falling asleep and prolonging sleeping time but also reducing sleep latency at a dosage of 5 mg/kg.

[Synthesis and molecule recognition capability of corilagin-molecularly imprinted polymer].

To study the molecule recognition capability of corilagin-molecularly imprinted polymer (MIP) by the high performance liquid chromatography (HPLC), the molecularly imprinted polymer was synthesized by using corilagin as the template. Chromatographic performance of corilagin was investigated in different mobile phases. The MIP was investigated for the recognition of corilagin and its derivatives and other compounds in the same mobile phase. The MIP exhibited very high affinity for corilagin in the mobile phase of acetonitrile. The K' value will be reduced when the content of polar solvent increased in the mobile phase. The MIP has good selectivity in the mobile phase of acetonitrile-methanol (95:5), but it has no affinity for corilagin's derivatives. The corilagin-MIP has good selectivity for corilagin and it can be used in extracting corilagin and its analogs from herbs.

Bone marrow-derived mesenchymal stem cells overexpressing MiR-21 efficiently repair myocardial damage in rats.

OBJECTIVE: We investigated the ability of bone marrow derived mesenchymal stem cells (BMSCs) overexpressing microRNA-21 (miR-21) to repair cardiac damage induced by anthracyclines in rats. METHODS: Sprague-Dawley (SD) rats of 2~3 weeks old were selected to isolate and culture BMSCs. A lentivirus harboring pLVX-miR-21 was generated and transfected into rat BMSCs. The rats were assigned into an untreated negative control group, and groups injected with adriamycin alone or with adriamycin followed by BMSCs, pLVX-BMSCs or pLVX-miR-21-BMSCs (n = 10 each). Proliferation and migration of cells were detected by cholecystokinin-8 (CCK- 8) and transwell. MiR-21 expression, mRNA expressions of B cell lymphoma 2 (Bcl2), BAX (BCL-2-associated X protein) and vascular endothelial growth factor (VEGF) were tested by qRT-PCR. Western blotting was applied to detect protein expressions of Bcl-2, Bax and VEGF. RESULTS: Using CCK- 8 and transwell assays, we found that pLVX-miR-21-BMSCs, which overexpressed miR-21, exhibited greater proliferation and migration than untransfected BMSCs or pLVX-BMSCs. Ultrasonic cardiograms and immunohistochemical analysis demonstrated that among the five groups, the pLVX-miR-21-BMSC group exhibited the most improved heart function and enhanced angiogenesis. Moreover, the pLVX-miR-21-BMSC group showed enhanced expression of Bcl-2, VEGF and Cx43 and reduced expression of Bax, BNP and troponin T. CONCLUSION: These findings suggest miR-21 overexpression enhanced the proliferation, invasiveness and differentiation of BMSCs as well as expression of key factors (Bcl-2, VEGF and Bax) essential for repairing the cardiac damage induced by anthracyclines and restoring heart function.

Cipadesins A-C: novel tetranortriterpenoids from Cipadessa cinerascens.

[structure: see text] Three novel tetranortriterpenoids, cipadesins A-C (1-3), were isolated from the aerial parts of Cipadessa cinerascens. They possess a novel carbon skeleton, in which rings A and C were joined via C-10 and C-11. Their structures were elucidated by spectral evidence. X-ray crystallographic analysis confirmed the structure of 1.

[Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn].

Balsampear (M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method. By joining the two amplified fragments, the full-length cDNA of M. charatica homologous gene of plant class V chitinase (McChi5) was obtained. The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mochi5 gene were discussed.