Novel symmetric amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) with high plasmid DNA binding affinity as a biodegradable gene carrier

This study communicates the molecular design, preparation, and biological application of novel symmetric amphiphilic polycationic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) D2-LLA15-D2 bearing two two-generation poly(L-lysine) PLL dendrons D2 and a central hydrophobic biodegradable poly(L-lactide) block LLA15. First, an amino-protected precursor of L1-OH was designed and synthesized and was further employed to prepare L1-LLA15 with an organic 4-(dimethylamino)-pyridine-mediated living-ring-opening polymerization of l-lactide. Subsequently, the hydroxy end-capped L1-LLA15 was coupled to synthesize a new triblock L1-LLA15-L1 with two one-generation amino-protected PLL dendrons L1. Furthermore, with a repeated trifluoroacetic-acid-mediated amino deprotection-protection cycle, new amphiphilic triblock D2-LLA15-D2 was successfully prepared. By means of NMR, mass spectrometry, and gel permeation chromatography, these synthetic precursors and final amphiphilic product were characterized to bear well-defined triblock structures. In addition, this synthesized amphiphilic triblock polycationic macromolecule was applied as a new polycationic plasmid DNA carrier, and its DNA binding affinity was examined via an agarose electrophoresis and a fluorescence titration assay along with two important references of hydrophilic dendritic D2-HEX-D2 and double-hydrophilic D2-PEG-4K-D2 bearing the same two D2 dendrons; much enhanced DNA binding affinity was interestingly revealed for the new amphiphilic structural D2-LLA15-D2. Moreover, the assembled polyplex microparticles of plasmid DNA/polycationic carrier were further analyzed by dynamic light scattering and transmission electron microscopy, indicating their averaged nanoparticle size around 150-200 nm. As for the cytotoxicity of the new D2-LLA15-D2, MTT assays were conducted with a human hepatocellular carcinoma cell line (SMMC-7721), indicating a very low cytotoxicity as compared with commercial linear PLL-23K and PEI-2K, and a DNase I degradation of the assembled polyplex particles was also done in the HBS buffer solution to evaluate their stabilities. Finally, employing the new amphiphilic D2-LLA15-D2 as gene carrier, in vitro gene transfection experiments were conducted with the SMMC-7721 cell line, indicating a transfection efficiency increase of at least 10 times higher than that of the naked plasmid DNA under a N/P charge ratio of 10. Therefore, these interesting results may provide a new possible way to construct efficient polycationic macromolecular gene carriers with low toxicity and less expensive low-generation PLL dendrons.     

Characterization of a bacteriocin-producing strain of Enterococcus faecalis from cow’s milk used in the production of Moroccan traditional dairy foods

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.     

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall¹. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation². In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental³ and aging studies⁴ can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression⁵ and disease treatment⁶. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process⁷. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.     

Synthesis, structural characterization and in vitro cytotoxicity and anti-bacterial activity of some copper(I) complexes with N,N′-disubstituted thioureas

A series of copper(I) complexes of N,N′-disubstituted thioureas, [C₆H₅CONHCSNHR]Cu(I)Cl where R = C₆H₅ (1a), 2-ClC₆H₄ (2a), 3-ClC₆H₄ (3a), 4-ClC₆H₄ (4a), 2,3-Cl₂C₆H₃ (5a), 2,4-Cl₂C₆H₃ (6a), 2,5-Cl₂C₆H₃ (7a), 2,6-Cl₂C₆H₃ (8a), 3,4-Cl₂C₆H₃ (9a) and 3,5-Cl₂C₆H₃ (10a) have been synthesized. These complexes (1a–10a) have been characterized by elemental analyses, IR, ¹H and ¹³C NMR spectroscopy, cyclic voltammetry and single crystal XRD for 1a and 8a, and for ligand 7. The X-ray crystal structures reveal that the complexes 1a and 8a are mononuclear in the solid state in which the copper atoms adopt a distorted tetrahedral geometry. In both the cases, the neutral N,N′-disubstituted thiourea ligands have been coordinated to the Cu(I) through the sulphur atom in a terminal mode. The complexes have been screened for their in vitro cytotoxic activity in human cell lines carcinomas A498 (Renal), EVSA-T (Breast), H226 (Lung), IGROV (Ovarian), M19 (Melanoma-Skin), MCF-7 (Breast) and WIDR (Colon). They show a moderate cytotoxicity against these seven human cancer cell lines comparable to that of the less active standard chemotherapeutic drugs used for comparison. They were also screened for their anti-bacterial activity and were found less active than the standard drug Imipenem.     

Lack of association between interleukin-13 gene polymorphisms (−1055 C/T and +2044 G/A) in Iranian patients with lung cancer

Lung cancer is one of the leading causes of death from cancer. Both immune cells and tumor cells play a key role in lung cancer immunity by secretion of cytokines and developing type-2 cell-mediated immune response. IL-13 is an immunoregulatory cytokine affecting tumor immunosurveillance by deviation of immune response from Th1 to Th2. In the present study we sought to determine the association of single nucleotide polymorphisms (SNPs) of IL-13 gene at positions +2044 (G/A) and −1055 (C/T) and lung cancer. One hundred forty one patients and 113 controls were recruited; control group was subdivided into smoker and nonsmoker individuals for serum detection. Genotyping was carried out by PCR-RFLP assay and IL-13 detection by ELISA method. No statistically significant difference was found in the frequency of genotypes, alleles, and haplotypes at positions +2044 (G/A) and −1055 (C/T) of IL-13 gene between lung cancer patients and controls. Serum level of IL-13 was not detectable in both groups. The results of this study reveal that although +2044 (G/A) and −1055 (C/T) SNPs in IL-13 are implicated in some pulmonary processes, they do not confer susceptibility to lung cancer in Iranian population.     

Interleukin-18 gene promoter and serum level in women with ovarian cancer

IL-18, initially defined as a potent inducer of IFN- γ production, is a systemic, multifunctional cytokine with both pro-cancerous and anti-cancer activities. The contribution of the IL-18 promoter polymorphisms at positions −607 (C/A) and −137 (G/C) to cancer development has been reported. We sought to examine IL-18 serum level and its polymorphisms in Iranian women with ovarian cancer. Single nucleotide polymorphisms (SNPs) at positions −607 (C/A) and −137 (G/C) were analyzed by allele-specific polymerase chain reaction in 85 women with ovarian cancer and 158 healthy controls. IL-18 serum level was determined using ELISA method. No significant association was found between the allele, genotype, and haplotype distributions of the SNPs and ovarian cancer. Mean IL-18 serum level was significantly higher in patients than in controls (P = 0.008). Comparing IL-18 serum levels with genotypes at positions −607 and −137 revealed no significant difference. No association was also found between IL-18 levels and the disease stage. In conclusion, our results indicate that IL-18 promoter polymorphisms at positions −607 (C/A) and −137 (G/C) appear not to confer susceptibility to ovarian cancer in Iranian population; however, IL-18 serum level increases in ovarian cancer patients.     

VEGF gene mRNA expression in patients with coronary artery disease

To assess the expression of vascular endothelium growth factor (VEGF) mRNA in unstimulated peripheral blood mononuclear cells of patients with and without coronary artery disease (CAD). We also studied whether the functional VEGF -2,578C/A polymorphism may influence the level of VEGF mRNA expression in individuals undergoing coronary angiography because chest pain. We assessed 50 consecutive patients with angiographically confirmed CAD (CAD+). Also, 50 consecutive individuals with normal coronary studies were included in the study for comparison. VEGF mRNA expression was examined using quantitative real-time PCR and genotyping for VEGF -2,578C/A was performed using ARMS-PCR technique. VEGF mRNA expression was significantly decreased in CAD+ patients when compared to CAD- individuals (p = 0.01). The frequency of VEGF -2578 allele C and genotype CC was increased in CAD+ patients. In this regard, homozygosity for the CC genotype was more commonly observed in CAD+ (30 %) than in those without CAD disease (18 %). However, the difference was slightly out of the range of significance (p = 0.1). In addition, a trend for reduction in the expression of VEGF mRNA was observed when patients carrying the VEGF -2,578AA genotype were compared with those VEGF -2,578AC heterozygous or those homozygous for the VEGF -2,578CC genotype. VEGF gene expression is decreased in individuals with CAD+ disease. The VEGF -2,578C/A polymorphism may influences the expression of VEGF.     

Immediate and one-year outcome of patients presenting with Acute Coronary Syndrome complicated by stroke: Findings from the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2)

ABSTRACT: BACKGROUND: Stroke is a potential complication of acute coronary syndrome (ACS). The aim of this study was to identify the prevalence, risk factors predisposing to stroke, in-hospital and 1-year mortality among patients presenting with ACS in the Middle East. METHODS: For a period of 9 months in 2008 to 2009, 7,930 consecutive ACS patients were enrolled from 65 hospitals in 6 Middle East countries. RESULTS: The prevalence of in-hospital stroke following ACS was 0.70%. Most cases were ST segment elevation MI-related (STEMI) and ischemic stroke in nature. Patients with in-hospital stroke were 5 years older than patients without stroke and were more likely to have hypertension (66% vs. 47.6%, P = 0.001). There were no differences between the two groups in regards to gender, other cardiovascular risk factors, or prior cardiovascular disease. Patients with stroke were more likely to present with atypical symptoms, advanced Killip class and less likely to be treated with evidence-based therapies. Independent predictors of stroke were hypertension, advanced killip class, ACS type --STEMI and cardiogenic shock. Stroke was associated with increased risk of in-hospital (39.3% vs. 4.3%) and one-year mortality (52% vs. 12.3%). CONCLUSION: There is low incidence of in-hospital stroke in Middle-Eastern patients presenting with ACS but with very high in-hospital and one-year mortality rates. Stroke patients were less likely to be appropriately treated with evidence-based therapy. Future work should be focused on reducing the risk and improving the outcome of this devastating complication.     

Position-specific suppression and enhancement of HIV-1 integrase reactions by minor groove benzo[a]pyrene diol epoxide deoxyguanine adducts: implications for molecular interactions between integrase and substrates

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.