Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2) in plasmid DNA extraction

Recent updates on Magnetic Nano-Particles (MNPs) based separation of nucleic acids have received more attention due to their easy manipulation, simplicity, ease of automation and cost-effectiveness. It has been indicated that DNA molecules absorb on solid surfaces via hydrogen-bonding, and hydrophobic and electrostatic interactions. These properties highly depend on the surface condition of the solid support. Therefore, surface modification of MNPs may enhance their functionality and specification. In the present study, we functionalized Fe₃O₄ nano-particle surface utilizing SiO₂ and TiO₂ layer as Fe₃O₄/SiO₂ and Fe₃O₄/SiO₂/TiO₂ and then compare their functionality in the adsorption of plasmid DNA molecules with the naked Fe₃O₄ nano-particles. The result obtained showed that the purity and amount of DNA extracted by Fe₃O₄ coated by SiO₂ or SiO₂/TiO₂ were higher than the naked Fe₃O₄ nano-particles. Furthermore, we obtained pH 8 and 1.5 M NaCl as an optimal condition for desorption of DNA from MNPs. The result further showed that, 0.2 mg nano-particle and 10 min at 55 °C are the optimal conditions for DNA desorption from nano-particles. In conclusion, we recommended Fe₃O₄/SiO₂/TiO₂ as a new MNP for separation of DNA molecules from biological sources.     

Solution properties and structure of brain proteolipids

Bovine white matter proteolipids have been studied by several physical methods and have been found to exist as micelles in 2 : 1 (v/v) chloroform–methanol solution. The data would indicate the existence of a critical micelle concentration at 0.017–0.022 g/100 ml. The curve appears linear in the range 0.017–0.2 g/100 ml, but from the data at higher concentrations it would appear that a change in slope is occurring in the region 0.2–0.3 g/100 ml. Light-scattering measurements on 2 : 1 (v/v) chloroform–methanol solutions containing more than 0.2 g/100 ml of proteolipid yielded a weight-average aggregate weight of 2.9 × 10⁶and a radius of gyration of 64.5 Å. The intrinsic viscosity of the solutions was 0.32 dl/g and the Huggins constant was 1.085. Light-scattering measurements in 88.5% formic acid–0.5M sodium formate yielded a weight-average aggregate weight of 7.1 × 10⁶ and a radius of gyration of 241 Å. The intrinsic viscosity observed for this solvent system is 0.14 dl/g and the Huggins constant is 1.005. Osmotic pressure measurements in 2 : 1 (v/v) chloroform–methanol containing less than 0.2 g/ 100 ml of proteolipid yielded a number-average aggregate weight of 7.2 × 10⁴ Ultracentrifugal analysis in 1.5:1 (v/v) methylene chloride–methanol showed two broad peaks with, s values of s_1.5% = 20.05 S, s_2% = 19.79 S for the minor peak and s_1.5%,2% = 1.86 S for the major peak. Optical rotatory dispersion studies revealed large changes in b₀ with change in solvent and proteolipid concentration. The present data suggest that the mode of attachment of protein to lipid is primarily of a noncovalent type. The results of this investigation also suggest that the proteolipid micelle above 0.2 g/100 ml is cylindrical (prolate ellipsoid) in 2:1 (v/v) chloroform-methanol and approaches a more spherical shape in 88.5% formic acid. A structure for the proteolipid micellar complex above concentrations of 0.2 g/100 ml is proposed.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Inhibitory role of cAMP on doxorubicin-induced apoptosis in pre-B ALL cells through dephosphorylation of p53 serine residues

Exposure of cells to chemotherapeutic drug doxorubicin, a DNA-damaging agent, induces an increase in the levels and activity of the wild-type p53 protein. Less well appreciated was the effect of cAMP levels on posttranslational modifications of p53 in response to doxorubicin. Here we show that elevation of cAMP in pre-B acute lymphoblastic leukemia NALM-6 cells significantly attenuated phosphorylation state of p53 at Ser6, Ser9, Ser15, Ser20, Ser37, Ser46 and Ser392 upon exposure to doxorubicin. Increased cAMP levels also shifted the ratio of the death promoter to death repressor genes via alteration of Bcl-2 and Bax proteins expression. In conclusion, our results suggest that activation of cAMP-signaling system may repress p53-dependent apoptosis in malignant cells exposed to doxorubicin.     

Fold-recognition and comparative modeling of human α2,3-sialyltransferases reveal their sequence and structural similarities to CstII from Campylobacter jejuni

Background  

The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.     

Variance components for survival of piglets at farrowing using a reduced animal model

Farrowing survival is usually analysed as a trait of the sow, but this precludes estimation of any direct genetic effects associated with individual piglets. In order to estimate these effects, which are particularly important for sire lines, it is necessary to fit an animal model. However this can be computationally very demanding. We show how direct and maternal genetic effects can be estimated with a simpler analysis based on the reduced animal model and we illustrate the method using farrowing survival information on 118 193 piglets in 10 314 litters. We achieve a 30% reduction in computing time and a 70% reduction in memory use, with no important loss of accuracy. This use of the reduced animal model is not only of interest for pig breeding but also for poultry and fish breeding where large full-sib families are performance tested.     

An age-dependent branching process model for the analysis of CFSE-labeling experiments

Background  

Over the past decade, flow cytometric CFSE-labeling experiments have gained considerable popularity among experimentalists, especially immunologists and hematologists, for studying the processes of cell proliferation and cell death. Several mathematical models have been presented in the literature to describe cell kinetics during these experiments.