Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

Cardioprotective Mechanism Of S-nitroso-n-acetylcysteine Via S-nitrosated Betadrenoceptor-2 In The Ldlr-/- Mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with β₂-AR signaling in mediating this protection. Ventricular superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O₂⁻ and H₂O₂ production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H₂O₂ and O₂⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in β₂-AR expression associated with coupling change to Gi; β₂-ARs-S-nitrosation (β₂-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with β₂-ARs overexpression and β₂-AR-SNO via an anti-apoptotic pathway.

Characterization Of Cumulative Joint Damage Patterns In Patients With Rheumatoid Arthritis: A Clinical, Serological, And Gene Polymorphism Perspective.

To characterize cumulative joint damage (CJD) patterns in rheumatoid arthritis (RA) and determine their associations with demographic/clinical features and HLA-DRB1 gene polymorphism. Hand and foot radiographs were obtained from 404 patients with RA. CJD patterns were determined by 3 derivations from Sharp/van der Heijde scores, obtained by the mathematical division of scores for hands/feet (Sharp-h/f score), fingers/wrists (Sharp-f/w score), and erosion/space narrowing (Sharp-e/sn score), respectively. DNA and serum were obtained for determination of HLA-DRB1 polymorphism, rheumatoid factor (RF), and anticitrullinated protein antibodies (ACPA). Patients with wrist-dominant CJD pattern were more likely to have severe RA than those with finger-dominant pattern (68.4% vs 46.0%; p = 0.036) as were those with foot-dominant vs hand-dominant CJD pattern (76.5% vs 56.4%; p = 0.044). HLA-DRB1 shared epitope (SE) alleles were associated with erosion-dominant CJD pattern (p = 0.021). Patients with erosion-dominant CJD pattern had higher levels of RF and ACPA than those with space-narrowing-dominant CJD pattern (median RF 71.35 U/ml vs 22.05 U/ml, respectively; p = 0.003; median ACPA 187.9 U/ml vs 143.2 U/ml, respectively; p < 0.001). The majority of triple-positive patients (SE+, RF+, ACPA+) had erosion-dominant CJD pattern (62.3%) while the majority of triple-negative patients (SE-, FR-, ACPA-) had space narrowing-dominant CJD pattern (75%; p = 0.017). ACPA was associated with HLA-DRB1 SE alleles (p < 0.05). Patients with foot-dominant CJD pattern were taller than those with hand-dominant CJD pattern (p = 0.002); those with erosion-dominant CJD pattern had higher weight and body mass index than those with space narrowing-dominant CJD pattern (p = 0.014, p = 0.001). CJD patterns were associated with disease severity, HLA-DRB1 SE status, presence and titer of ACPA and RF, and morphometric features.

Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia

Type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3β-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3β-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.