Unveiling the Selectivity of Ru(II)-Catalyzed C-H Activation for Defluorinative Cyclization of 2-Arylbenzimidazole and Trifluoromethyl Diazo: A DFT Study.

The synthesis of monofluorinated heterocyclic compounds by C-H activation combined with defluorination is useful. Studies on the reaction mechanism and selectivity have shown that these processes play a positive role in promoting the development of monofluorinated reactions. Density functional theory (DFT) calculations were performed to investigate the mechanism and selectivity of Ru(II)-catalyzed 2-arylbenzimidazole with trifluoromethyl diazo. DFT calculations showed that C-H activation occurs through a concerted metalation/deprotonation (CMD) mechanism. After that, deprotonation and defluorinative cyclization are assisted by acetate and trifluoroethanol (TFE). Further mechanistic insights through noncovalent interaction (NCI) analysis were also obtained to elucidate the origin of the selectivity in the defluorination process.

Numerical Simulation Analysis of the Hydrogen-Blended Natural Gas Leakage and Ventilation Processes in a Domestic House.

The blending of hydrogen in natural gas may have effects on the safety of its usage in a domestic house. In this work, the leakage accident of hydrogen-blended natural gas (HBNG) in the kitchen of a domestic house is analyzed by CFD with a hydrogen blending ratio (HBR) ≤ 30%. The whole process is divided into the gas accumulation process and the ventilation process. In the initial leakage stage, the influence of heights and the HBR on the gas distribution is analyzed. HBNG concentration increases with increasing height. Based on the exit Froude number, the formation of a gas cloud in the kitchen is significantly influenced by the initial momentum and buoyancy, while it is more driven by the concentration gradient beyond the kitchen. In contrast to height, the variation of HBR on the HBNG distribution is not significant. In the ventilation process, the evolution of the hazardous gas cloud volume is analyzed. With windows and doors closed, the hazardous gas cloud fills the house in approximately 3600 s after the leakage occurs. When windows and doors are open for ventilation, the volume of the hazardous gas cloud first declines rapidly and then slowly. The reasons for the variation rate of hazardous gas cloud volume are analyzed according to ventilation conditions. The difference during the decline stage for different HBRs is analyzed according to the gas layering properties. Under a lack of convection condition, the ventilation process finally reaches a stagnant stage. In addition, another ventilation process has been investigated after extending the gas accumulation time. After extending the gas accumulation time, the effect of different HBRs on the ventilation process remains the same as before. However, it postpones the time point to enter the stagnation stage. As gas accumulation time extends from 3600 to 5400 and 7200 s, the ventilation time into the stagnation stage increases from about 4800 to 5400 and 6000 s, respectively. This study has implications for the establishment of a risk assessment system based on hazardous gas cloud volume.

Overexpression of MBD3 Improves Reprogramming of Cloned Pig Embryos.

Methyl-CpG-binding domain protein 3 (MBD3) is a core component of the nucleosome remodeling and deacetylase (NuRD) complex, which is crucial for pluripotent stem cell differentiation and embryonic development. MBD3 was shown to play important roles in transcription factor-induced somatic cell reprogramming. Expression level of MBD3 was demonstrated to be higher in somatic cell nuclear transfer-generated cloned pig embryos than in fertilization-derived porcine embryos. However, the functions of MBD3 in nuclear transfer-mediated somatic cell reprogramming are unknown. In this study, MBD3 was overexpressed in cloned pig embryos, and the effects of MBD3 overexpression on gene transcription, DNA methylation, and in vitro developmental competence of cloned pig embryos were analyzed. Results indicated that overexpression of MBD3 in cloned pig embryos not only increased blastocyst rate and number of cells per blastocyst but also upregulated mRNA expression levels and decreased the DNA methylation of NANOG, OCT4, and LINE1 genes to the levels close to those in in vivo fertilization-produced pig embryos. These findings suggest that overexpression of MBD3 improves reprogramming of cloned pig embryos.

Genome-wide association study reveals genetic loci and candidate genes for average daily gain in Duroc pigs.

OBJECTIVE: Average daily gain (ADG) is an important target trait of pig breeding programs. We aimed to identify single nucleotide polymorphisms (SNPs) and genomic regions that are associated with ADG in the Duroc pig population. METHODS: We performed a genome-wide association study involving 390 Duroc boars and by using the PorcineSNP60K Beadchip and two linear models. RESULTS: After quality control, we detected 3,5971 SNPs, which included seven SNPs that are significantly associated with the ADG of pigs. We identified six quantitative trait loci (QTL) regions for ADG. These QTLs included four previously reported QTLs on Sus scrofa chromosome (SSC) 1, SSC5, SSC9, and SSC13, as well as two novel QTLs on SSC6 and SSC16. In addition, we selected six candidate genes (general transcription factor 3C polypeptide 5, high mobility group AT-hook 2, nicotinamide phosphoribosyltransferase, oligodendrocyte transcription factor 1, pleckstrin homology and RhoGEF domain containing G4B, and ENSSSCG00000031548) associated with ADG on the basis of their physiological roles and positional information. These candidate genes are involved in skeletal muscle cell differentiation, diet-induced obesity, and nervous system development. CONCLUSION: This study contributes to the identification of the casual mutation that underlies QTLs associated with ADG and to future pig breeding programs based on marker-assisted selection. Further studies are needed to elucidate the role of the identified candidate genes in the physiological processes involved in ADG regulation.

Organo-Catalyzed Asymmetric Michael-Hemiketalization-Oxa-Pictet-Spengler Cyclization for Bridged and Spiro Heterocyclic Skeletons: Oxocarbenium Ion as a Key Intermediate.

A Michael-hemiketalization-oxa-Pictet-Spengler cyclization has been developed for the construction of chiral bridged and spiro heterocyclic skeletons with one spiro stereogenic carbon center and two bridgehead carbon centers, utilizing cooperative catalysts of a Takemoto thiourea catalyst and a triflimide. In particular, an oxocarbenium ion acts as a key intermediate for this cyclization reaction. Additionally, biological evaluation of this type of novel structure has revealed obvious antiproliferative activity against some cancer cell lines.

Telomerase inhibition and telomere loss in BEL-7404 human hepatoma cells treated with doxorubicin.

AIM: To study the effects of doxorubicin on telomerase activity and telomere length in hepatocellular carcinoma. METHODS: Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplification protocol-based method. The effect of doxorubicin (DOX) on the growth of BEL-7404 human hepatoma cells was determined by microculture tetrazolium assay. Mean telomere length (terminal restriction fragment) was detected by Southern blot method. The expression of telomerase subunits genes was investigated by RT-PCR. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. RESULTS: Telomerase activity was inhibited in a dose and time-dependent manner in BEL-7404 human hepatoma cells treated with DOX for 24, 48 or 72 h in concentrations from 0.156 to 2.5 microM which was correlated with the inhibition of cell growth. No changes were found in the mRNA expression of three telomerase subunits (hTERT, hTR and TP1) after drug exposure for 72 h with indicated concentrations. The cells treated with DOX showed shortened mean telomere length and accumulated at the G(2)/M phase. However, there was almost no effects on cell apoptosis by DOX. CONCLUSION: The telomerase inhibition and the telomere shortening by DOX may contribute to its efficiency in the treatment in hepatocellular carcinoma.

Growth inhibition of BEL-7404 human hepatoma cells by expression of mutant telomerase reverse transcriptase.

Human hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa. Human telomerase reverse transcriptase (hTERT) is expressed in HCC but absent in normal human liver cells, which is consistent with the expression pattern of telomerase. In the present study, expression of a dominant-negative form of hTERT (DN-hTERT) resulted in inhibition of telomerase activity and decreased mean telomeric length of BEL-7404 human hepatoma cells, whereas expression of wild-type hTERT (WT-hTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes in telomerase level. Flattened large cells were found in late generations with the DN-hTERT treatment. When mean telomeric length of DN-hTERT-transfected cells reached a critical length (about 1.7 kb), apoptosis was induced. Tumorigenicity of DN-hTERT-expressing cells was eliminated in vivo. These data indicated that hTERT was essential for the growth of hepatoma cells. hTERT can also be used as an important target for anti-HCC drug screening.

Growth-inhibiting effects of taxol on human liver cancer in vitro and in nude mice.

AIM:To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms.METHODS:In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA, RNA and protein were analyzed by incorporation of (3)H-thymidine, (3)H-uridine and (3)H-leucine respec-tively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis.RESULTS:Taxol was effective against SMMC-7721 human hepatoma cell growth in the ranges of 2.5nmol/L-10nmol/L with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72h. Taxol at 2.5nmol/L reduced (3)H-thymidine uptake to about 34% of the control value (P<0.05). Increasing the dose of taxol to 20nmol/L resulted in a greater decrease in (3)H-thymidine incorporation to 60% of the control value (P < 0.01). At a concentration of 20nmol/L, the (3)H-uridine and (3)H-leucine uptakes were reduced to 52% (P<0.05) and 63% (P<0.01), respectively. In vivo,taxol significantly inhibited SMMC-7721 tumor growth at 10mg/kg, i.p., once daily for 10d. A more than 90% decrease in tumor volume was observed by day 11 (P < 0.01) similarly with mitotic arrest and cell apoptosis.CONCLUSION:Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently, apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol.

Expression of alpha fetoprotein messenger RNA in BEL-7404 human hepatoma cells and effect of L-4 oxalysine on the expression.

AIM:To investigate alpha-fetoprotein (AFP) mRNA expression in BEL-7404 human hepatoma cells and the effect of L-4-oxalysine (OXL) on the expression.METHODS: Bel-7404 human hepatoma cells were maintained in RPMI 1640 media.Human AFP cDNA probe was labelled with digoxigenin-11-dUTP by the random primer labelling method. The expression of AFP mRNA in Bel-7404 cells was determined by an in situ hybridization technique with digoxigenin-labelled human AFP cDNA probe. The positive intensities of AFP mRNA in cells were analyzed by microspectrophotometer and expressed as absorbance at 470nm. For the experiment with OXL, cells were incubated with various concentrations of the agent for 72h.RESULTS:Essentially all the hepatoma cells contained AFP mRNA in the cytoplasm,although in various amounts. The specificity of the hybridization reaction was confirmed by control experiments in which the use of Rnase-treated BEL-7404 cells, non-AFP producing cells (HL-60 human leukemia cells) or a nonspecific cDNA probe resulted in negative hybridization. When the cells were treated with OXL (25, 50mg/L), the content of AFP mRNA in the cytoplasm was decreased with the inhibition percentages of 34.3% and 70.1%, respectively (P< 0.05).CONCLUSION:AFP mRNA was expressed in BEL-7404 human hepatoma cells and OXL suppressed AFP mRNA expression in the cells.

Presence of Fas and Bcl-2 proteins in BEL-7404 human hepatoma cells.

AIM:To study the expression of Fas and Bcl-2 proteins in BEL-7404 human hepatoma cells in order to analyze the possible relationship between cell growth regulation by alpha-fetoprotein(AFP) and Fas/Bcl-2 proteins.METHODS:BEL-7404 human hepatoma cells were maintained in RPMI 1640 medium supplemented with 10% new-born calf serum. Cells adhered to coverslips were used to detect Fas and Bcl-2 protein expression by the avidin-biotin complex (ABC) immunocytochemical assay.RESULTS: Immunocytochemical study showed that essentially all the BEL-7404 human hepatoma cells could express Fas and Bcl-2 proteins, although in various amount. No positive staining for Fas and Bcl-2 proteins was observed when cells were incubated with non-relevant sera, to establish the specificity.CONCLUSION:Fas apoptosis signals and Bcl-2 rescue/survival signals from apoptosis are expressed in BEL-7404 human hepatoma cells. The finding strongly implys that AFP-mediated cell apoptosis and growth enhancement are potentially associated with Fas and Bcl-2 proteins present in those cells.