NaOAc-Assisted Aerobic Oxidation Protocol for the Synthesis of Pentacoordinate Chalcogenyl Spirophosphoranes with P-Se/P-S Bonds under Open Air.

The NaOAc-assisted aerobic oxidation reaction of pentacoordinate hydrospirophosphoranes and dichalcogenyl compounds with open air as a green oxidant has been developed under mild conditions. A series of novel pentacoordinate spirophosphoranes with P-Se/P-S bonds were synthesized in excellent yields. The reaction mechanism was determined by 31P nuclear magnetic resonance tracing experiments, high-resolution mass spectrometry tracing experiments, and X-ray diffraction analysis. The method features a broad substrate scope, good functional group tolerance, and a high degree of atomic utilization and is meaningful for the synthesis of bioactive chalcogenphosphate compounds with chalcogen and phosphorus moieties.

Investigation of the Asymmetric Addition Reactions Induced by Pentacoordinated Hydrospirophosphorane Substrate.

Pentacoordinated bisaminoacyl hydrospirophosphoranes were first found to induce the asymmetric addition reactions as a novel chiral organic framework. Asymmetric addition reactions of bisaminoacyl hydrospirophosphoranes with aromatic aldehyde and in situ generated imine were investigated, and the corresponding α-hydroxyspirophosphonates and α-amino spirophosphonates were obtained. The addition reaction of hydrospirophosphoranes with ΔP configuration showed better stereoselectivity than that with ΛP configuration, not only for the addition reaction to aromatic aldehyde but also to in situ generated imine. Furthermore, the stereochemical mechanisms of asymmetric addition reactions induced by pentacoordinated hydrospirophosphorane were proposed by 31P NMR tracing experiment and X-ray diffraction analysis.

Toward more potent imidazopyridine inhibitors of Candida albicans Bdf1: Modeling the role of structural waters in selective ligand binding.

Novel agents to treat invasive fungal infections are urgently needed because the small number of established targets in pathogenic fungi makes the existing drug repertoire particularly vulnerable to the emergence of resistant strains. Recently, we reported that Candida albicans Bdf1, a bromodomain and extra-terminal domain (BET) bromodomain with paired acetyl-lysine (AcK) binding sites (BD1 and BD2) is essential for fungal cell growth and that an imidazopyridine (1) binds to BD2 with selectivity versus both BD1 and human BET bromodomains. Bromodomain binding pockets contain a conserved array of structural waters. Molecular dynamics simulations now reveal that one water molecule is less tightly bound to BD2 than to BD1, explaining the site selectivity of 1. This insight is useful in the performance of ligand docking studies to guide design of more effective Bdf1 inhibitors, as illustrated by the design of 10 new imidazopyridine BD2 ligands 1a-j, for which experimental binding and site selectivity data are presented.

Investigation of the Stereochemical Mechanism of the Nucleophilic Substitution Reaction at Pentacoordinate Phosphorus of Spirophosphorane.

The stereochemical mechanism of the nucleophilic substitution reaction at pentacoordinate phosphorus (P-V) atom is rarely studied. Here, we report the Atherton-Todd-type reaction of pentacoordinate hydrospirophosphorane with phenolic compounds in detail. The stereochemical mechanism of nucleophilic substitution at P-V atom was proposed by 31P NMR tracing experiment, X-ray diffraction analysis, and density functional theory calculations. The first step of the Atherton-Todd-type reaction is the formation of halogenated spirophosphorane intermediate with retention of configuration at phosphorus definitely. The second step is a nucleophilic substitution reaction at P-V atom of halogenated spirophosphorane. When using CCl4 as a halogenating agent, the reaction of chlorinated spirophosphorane proceeds via SN2(P-V) mechanism, and the backside attack of P-Cl bond is the main pathway. For chlorinated spirophosphorane with ΔP configuration, the completely P-inverted product is normally obtained. As for chlorinated spirophosphorane with ΛP configuration, which has larger steric hindrance behind P-Cl bond, the proportion of P-retained products apparently increases and a pair of diastereoisomers is acquired. Furthermore, if CBr4 is used as a halogenating agent, the nucleophilic substitution reaction of brominated spirophosphorane may go through a SN1(P-V) mechanism to afford a pair of diastereoisomers.

The Wnt inhibitor Dkk1 is required for maintaining the normal cardiac differentiation program in Xenopus laevis.

Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/ß-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.

Thioredoxin-interacting protein promotes high-glucose-induced macrovascular endothelial dysfunction.

Thioredoxin-interacting protein (TXNIP) emerges as a central regulator for glucose homeostasis, which goes awry in diabetic subjects. Endothelial dysfunction is considered the earliest detectable stage of cardiovascular disease (CVD), a major complication of diabetes. Here, we hypothesize that TXNIP may promote endothelial dysfunction seen in Type 1 diabetes mellitus (T1D). Using a T1D-like rat model, we found that diabetic rats showed significantly higher TXNIP mRNA and protein levels in peripheral blood, compared to their non-diabetic counterparts. Those changes were accompanied by decreased production of nitric oxide (NO) and vascular endothelial growth factor (VEGF), concurrent with increased expression of reactive oxygen species (ROS) and vascular cell adhesion molecule 1 (VCAM-1) in the aortic endothelium. In addition, TXNIP overexpression in primary human aortic endothelial cells (HAECs) induced by either high glucose or overexpression of carbohydrate response element binding protein (ChREBP), a major transcriptional activator of TXNIP, promoted early apoptosis and impaired NO bioactivity. The correlation between TXNIP expression levels and endothelial dysfunction suggests that TXNIP may be a potential biomarker for vascular complications in T1D patients.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

The expression of cytoglobin as a prognostic factor in gliomas: a retrospective analysis of 88 patients.

BACKGROUND: Evidence suggests that cytoglobin (Cygb) may function as a tumor suppressor gene. METHODS: We immunohistochemically evaluated the expression of Cygb, phosphatidylinositol-3 kinase (PI-3K), phosphorylated (p)-Akt, Interleukin-6 (IL-6), tumor necrosis factor-α (TNFα) and vascular endothelial growth factor (VEGF) in 88 patients with 41 high-grade gliomas and 47 low-grade gliomas. Intratumoral microvessel density (IMD) was also determined and associated with clinicopathological factors. RESULTS: Low expression of Cygb was significantly associated with the higher histological grading and tumor recurrence. A significant negative correlation emerged between Cygb expression and PI3K, p-Akt, IL-6, TNFα or VEGF expression. Cygb expression was negatively correlated with IMD. There was a positive correlation between PI3K, p-Akt, IL-6, TNFα and VEGF expression with IMD.High histologic grade, tumor recurrence, decreased Cygb expression, increased PI3K expression, increased p-Akt expression and increased VEGF expression correlated with patients' overall survival in univariate analysis. However, only histological grading and Cygb expression exhibited a relationship with survival of patients as independent prognostic factors of glioma by multivariate analysis. CONCLUSIONS: Cygb loss may contribute to tumor recurrence and a worse prognosis in gliomas. Cygb may serve as an independent predictive factor for prognosis of glioma patients.

Unexpected insertion of CO2 into the pentacoordinate P-N bond: Atherton-Todd-type reaction of hydrospirophosphorane with amines.

The Atherton-Todd-type reaction of pentacoordinate hydrospirophosphoranes with amines was investigated, and a novel CO2 insertion reaction into the pentacoordinate P-N bond under mild conditions was developed. The mechanism and stereochemistry of the CO2 insertion reaction between hydrospirophosphoranes and secondary amines were proposed via a carbon-13 labeling experiment, a (31)P NMR tracing experiment, and X-ray diffraction analysis. The chlorinated spirophosphorane intermediate was first generated with stereoretention of the configuration at phosphorus and subsequently was attacked by a carbamate anion formed from CO2 and a secondary amine. It was found that rear attack of nucleophilic substitution with stereoinversion at pentacoordinate phosphorus was the preferred route, although front attack happened for sterically hindered reactants. The configuration of the CO2 insertion product depended mainly upon the original phosphorus configuration of the hydrospirophosphoranes.

Comparison of manually shaped and computer-shaped titanium mesh for repairing large frontotemporoparietal skull defects after traumatic brain injury.

OBJECT: The object of this study was to compare the effects and complications of manual and computer-aided shaping of titanium meshes for repairing large frontotemporoparietal skull defects following traumatic brain injury. METHODS: From March 2005 to June 2011, 161 patients with frontotemporoparietal skull defects were observed. Patients were divided into 2 groups according to the repair materials used for cranioplasty: 83 cases used computer-aided shaping for the titanium mesh, whereas the remaining 78 cases used a manually shaped titanium mesh. The advantages and disadvantages of the 2 methods were compared. RESULTS: No case of titanium mesh loosening occurred in either group. Subcutaneous fluid collection, titanium mesh tilt, and temporal muscle pain were the most common complications. In the manually shaped group, there were 14 cases of effusion, 10 cases of titanium mesh tilt, and 15 cases of temporal muscle pain. In the computer-aided group, there were 6 cases of effusion, 3 cases of titanium mesh tilt, and 6 cases of temporal muscle pain. The differences were significant between the 2 groups (p < 0.05). Other common complications were scalp infection, exposure of titanium mesh, epidural hematoma, and seizures. In the computer-aided group, the operative time decreased (p < 0.01), the number of screws used was reduced (p < 0.01), and the satisfaction of patients was significantly increased (p < 0.05). CONCLUSIONS: Computer-aided shaping of titanium mesh for repairing large frontotemporoparietal skull defects decreases postoperative complications and the operative duration, reduces the number of screws used, increases the satisfaction of patients, and restores the appearance of the patient's head, making it an ideal choice for cranioplasty.

Different administration methods of endostar combined with second-line chemotherapy in advanced malignancies.

Background: This study aimed to compare the therapeutic efficacy and the side effects of different endostar administration methods in patients with advanced malignancy who underwent second-line chemotherapy. Methods: 98 patients with advanced malignancies were divided into 2 groups based on the delivery methods of endostar, including drip intravenous administration of endostar (DE) group and continuous intravenous administration of endostar (CE) group. Response rate (RR), disease control rate (DCR), and quality of life (QOL) of the patients were examined to evaluate the therapeutic efficacy, and toxicity reactions were analyzed to evaluate the adverse effects. Results: Compared with the DE group, the therapeutic efficacy of CE has been slightly improved, but the difference did not reach statistical significance (P > 0.05). Additionally, no different incidence rate was observed in toxic reactions, including leukopenia, thrombocytopenia, nausea and vomiting, diarrhea, and hepatic function damage, between the DE and CE groups (P > 0.05). Conclusion: In conclusion, no significant difference was observed between the traditional intravenous drip of endostar group and the intravenous drip followed by continuous pumping of endostar group in the patients with advanced malignancies.

Expression analysis of epb41l4a during Xenopus laevis embryogenesis.

Epbl41l4a (erythrocyte protein band 4.1-like 4a, also named Nbl4) is a member of the band 4.1/Nbl4 (novel band 4.1-like protein 4) group of the FERM (4.1, ezrin, radixin, moesin) protein superfamily. Proteins encoded by this gene family are involved in many cellular processes such as organization of epithelial cells and signal transduction. On a molecular level, band 4.1/Nbl4 proteins have been shown to link membrane-associated proteins and lipids to the actin cytoskeleton. Epbl41l4a has also recently been identified as a target gene of the Wnt/ß-catenin pathway. Here, we describe for the first time the spatio-temporal expression of epbl41l4a using Xenopus laevis as a model system. We observed a strong and specific expression of epb41l4a in the developing somites, in particular during segmentation as well as in the nasal and cranial placodes, pronephros, and neural tube. Thus, epbl41l4a is expressed in tissues undergoing morphogenetic movements, suggesting a functional role of epbl41l4a during these processes.

Gas-phase fragmentation of protonated 3-phenoxy imidazo[1,2-a] pyridines using tandem mass spectrometry and computational chemistry.

Imidazo[1,2-a] pyridine is one of the pharmaceutically important scaffolds and has been widely studied due to its extensive biological activities. In this work, electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive mode was used to study the gas-phase fragmentation behavior of a series of 3-phenoxy imidazo[1,2-a] pyridines. Proposed fragmentation pathways were supported by ESI-MS/MS data and computational thermochemistry. Homolytic cleavage of the 3-phenoxy C-O bond was the characteristic fragmentation of 3-phenoxy imidazo [1,2-a] pyridines. The eliminations of the one substituted phenoxy radical and CO produced other diagnostic ions for 3-phenoxy imidazo [1,2-a] pyridines, which were useful to identify the 3-phenoxy group and imidazo [1,2-a] pyridine scaffold. The results contribute to the further understanding of the gas-phase fragmentation of 3-phenoxy imidazo [1,2-a] pyridines and the identification of other analogs using tandem mass spectrometry techniques.

Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement.

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

MicroRNA-425 promotes the development of lung adenocarcinoma via targeting A disintegrin and metalloproteinases 9 (ADAM9).

PURPOSE: We aimed to investigate the roles of microRNA-425 (miR-425) in lung adenocarcinoma, as well as its possible regulatory mechanism. MATERIALS AND METHODS: The miR-425 expression in lung adenocarcinoma tissues and cells was determined. The regulatory relationship between miR-425 and IL-6/STAT3 signaling was investigated. In addition, miR-425 was downexpressed in H1299 cells, and its effects on cell proliferation and apoptosis were determined. Furthermore, the target relationship between miR-425 and A disintegrin and metalloproteinases 9 (ADAM9) in lung adenocarcinoma cells was explored. RESULTS: The miR-425 was significantly downregulated in lung adenocarcinoma tissues and cells and was markedly inhibited by IL-6/STAT3 signaling. In addition, miR-425 expression was successfully overexpressed by transfection with pre-miR-425. Overexpression of miR-425 decreased the proliferation and colony formation of H1299 cells and promoted cell apoptosis markedly. Moreover, ADAM9 was revealed as a target of miR-425, and ADAM9 expression was negatively regulated by miR-425. CONCLUSION: Our findings indicate that downregulation of miR-425 caused by IL-6/STAT3 signaling leads to loss of ADAM9 targeting, results in enhanced ADAM9 expression, and contributes to the development of lung adenocarcinoma. Thus, increasing miR-425 may be a promising therapeutic strategy for this disease.

The investigation of substituent effects on the fragmentation pathways of pentacoordinated phenoxyspirophosphoranes by ESI-MSn.

The fragmentation pathways of pentacoordinated phenoxyspirophosphoranes were investigated in the positive mode by electrospray ionization multistage mass spectrometry. The results demonstrate that the sodium adducts of the title compounds undergo two competitive fragmentation pathways, and the fragmentation patterns are heavily dependent on the various substituent patterns at the phenolic group. An electron-withdrawing substituent at the ortho-position always results in the removal of a corresponding phenol analogue, while cleavage by spiroring opening becomes the predominant fragmentation pathway if an electron-donating substituent is at the phenolic group. The substituent effects on the competitive fragmentation pathways were further elucidated by theoretical calculations, single crystal structure analysis, and high-resolution mass spectrometry. The results contribute to the understanding of the gas-phase fragmentation reactions and the structure identification of spirophosphorane analogues by electrospray ionization multistage mass spectrometry.

Downregulation of microRNA­574 in cancer stem cells causes recurrence of prostate cancer via targeting REL.

miR­574­5p has been reported involved in the pathogenesis of numerous human malignancies such as colorectal and lung cancer. In this study, we aimed to explore the roles of REL and miR­574 in the recurrence of prostate cancer (PCa) and to identify the underlying molecular mechanisms. Our literature search found that miR­574 is regulated in cancer stem cells (CSCs), and next we used the microRNA (miRNA) database (www.mirdb.org) to find REL as a target of miR­574. Luciferase assay was performed to verify the miRNA/target relationship. Oligo-transfection, real­time PCR and western blot analysis were used to support the conclusions. We validated REL to be the direct gene via luciferase reporter assay system, and real­time PCR and western blot analysis were also conducted to study the mRNA and protein expression level of REL between different groups (recurrence and non­recurrence) or cells treated with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors, indicating the negative regulatory relationship between miR­574 and REL. We also investigated the relative viability of prostate CSCs when transfected with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors to validate miR­574 to be positively interfering with the viability of prostate CSCs. We then investigated the relative apoptosis of prostate CSCs when transfected with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors. The results showed miR­574 inhibited apoptosis. In conclusion, miR­574 might be a novel prognostic and therapeutic target in the management of PCa recurrence.

Comparative analysis reveals distinct and overlapping functions of Mef2c and Mef2d during cardiogenesis in Xenopus laevis.

The family of vertebrate Mef2 transcription factors is comprised of four members named Mef2a, Mef2b, Mef2c, and Mef2d. These transcription factors are regulators of the myogenic programs with crucial roles in development of skeletal, cardiac and smooth muscle cells. Mef2a and Mef2c are essential for cardiac development in mice. In Xenopus, mef2c and mef2d but not mef2a were recently shown to be expressed during cardiogenesis. We here investigated the function of Mef2c and Mef2d during Xenopus laevis cardiogenesis. Knocking down either gene by corresponding antisense morpholino oligonucleotides led to profound heart defects including morphological abnormalities, pericardial edema, and brachycardia. Marker gene expression analyses and rescue experiments revealed that (i) both genes are required for proper cardiac gene expression, (ii) Mef2d can compensate for the loss of Mef2c but not vice versa, and (iii) the γ domain of Mef2c is required for early cardiac development. Taken together, our data provide novel insights into the function of Mef2 during cardiogenesis, highlight evolutionary differences between species and might have an impact on attempts of direct reprogramming.

TBX3 Directs Cell-Fate Decision toward Mesendoderm.

Cell-fate decisions and pluripotency are dependent on networks of key transcriptional regulators. Recent reports demonstrated additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor TBX3 has been implicated in regulating embryonic stem cell self-renewal and cardiogenesis. Here, we show that TBX3 is dynamically expressed during specification of the mesendoderm lineages in differentiating embryonic stem cells (ESCs) in vitro and in developing mouse and Xenopus embryos in vivo. Forced TBX3 expression in ESCs promotes mesendoderm specification by directly activating key lineage specification factors and indirectly by enhancing paracrine Nodal/Smad2 signaling. TBX3 loss-of-function analyses in the Xenopus underline its requirement for mesendoderm lineage commitment. Moreover, we uncovered a functional redundancy between TBX3 and Tbx2 during Xenopus gastrulation. Taken together, we define further facets of TBX3 actions and map TBX3 as an upstream regulator of the mesendoderm transcriptional program during gastrulation.

Fragmentation studies of pentacoordinated bisaminoacylspirophosphoranes by negative electrospray ionization mass spectrometry.

Fragmentation pathways of a series of pentacoordinated bisaminoacylspirophosphoranes were elucidated by electrospray ionization multistage mass spectrometry (ESI-MS(n)) in negative mode. The deprotonated ions of pentacoordinated bisaminoacylspirophosphoranes tend to eliminate a corresponding amino acid to form base peak. The hydrogen/deuterium exchange experiment, the high-resolution mass spectrometry, (13)C stable isotope labeling experiment and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The results indicate that the negative molecular ions of pentacoordinated bisaminoacylspirophosphoranes dissociate through its open-chain tricoordinated tautomers. The relative Gibbs free energies (ΔG) of the product ions and proposed fragmentation pathways were estimated using the B3LYP/6-31 + + G(d, p) model. The results have some potential applications in the identification structures of similar spirophosphorane compounds by ESI-MS(n).