Use of in silico tools for classification of novel missense mutations identified in dystrophin gene in developing countries

DMD gene which is composed of 79 exons is the largest known gene located on X chromosome (Xp21). Point mutations in the dystrophin gene are responsible for 30–35% of cases with DMD/BMD. Mutation analysis of all the exons of the DMD gene is costly in developing countries, therefore, a few of the exons are selected to be analyzed routinely in clinical laboratories. In this study, direct sequencing was used for detection of point mutations in 10 exons of dystrophin gene in patients affected with DMD without detectable large rearrangements. Freely available programs were used to predict the damaging effects of the mutations. Point mutations were successfully detected in three patients. Three novel mutations, two missense mutations located on nonconservative domains and a single nucleotide deletion, were detected. Missense mutations were predicted to change splicing efficiency. Detection of point mutations by DNA analysis followed by prediction of the pathogenecity by using bioinformatic tool might be an asset to provide proper diagnosis or genetic counseling to patients and their family.     

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0420-4) contains supplementary material, which is available to authorized users.     

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris

Chondroitinase ABC I (cABC I) from Proteus vulgaris cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The clinical utilization of this enzyme is mainly limited by its thermal instability. This study has been undertaken to determine the effects of glycerol, sorbitol and trehalose on cABC I activity and thermal stability. The results indicated that the enzyme catalytic activity and intrinsic fluorescence intensity increased in the presence of these cosolvents whereas no considerable conformational changes observed in far-UV CD spectra. Thermal CD experiment revealed an increase in T(m) of cABC I in the presence of cosolvents which was significant for trehalose. Our results support the idea that cABC I has stabilized in the presence of glycerol, sorbitol and trehalose. Therefore, the use of these cosolvents seems to be promising for improvement in shelf-life and clinical applications of this drug enzyme.     

Nitrotyrosine as a marker for peroxynitrite‐induced neurotoxicity: the beginning or the end of the end of dopamine neurons?

This review examines the involvement of nitrotyrosine as a marker for peroxynitrite-mediated damage in the dopamine neuronal system. We propose that the dopamine neuronal phenotype can influence the cytotoxic signature of peroxynitrite. Dopamine and tetrahydrobiopterin are concentrated in dopamine neurons, and both are essential for their proper neurochemical function. It is not well appreciated that dopamine and tetrahydrobiopterin are also powerful blockers of peroxynitrite-induced tyrosine nitration. What is more, the reaction of peroxynitrite with either dopamine or tetrahydrobiopterin forms chemical species (i.e. o-quinones and pterin radicals, respectively) whose cytotoxic effects may be manifested far earlier than nitrotyrosine formation in the course of dopamine neuronal damage. A better understanding of how the dopamine neuronal phenotype modulates the effects of reactive nitrogen species could reveal early steps in drug- and disease-induced damage to the dopamine neuron and form the basis for rational, protective therapies.     

A theoretical study on the coordination behavior of some phosphoryl,carbonyl and sulfoxide derivatives in lanthanide complexation

The selectivity of phosphoryl P(O)R₃, sulfoxide S(O)R₂, and carbonyl C(O)R₂ (R?=?NH₂, CH₃, OH, and F) derivatives with lanthanide cations (La³⁺, Eu³⁺, Lu³⁺) was studied by density functional theory calculations. Theoretical approaches were also used to investigate energy and the nature of metal–ligand interaction in the model complexes. Atoms in molecules and natural bond orbital (NBO) analyses were accomplished to understand the electronic structure of ligands, L, and the related complexes, L–Ln³⁺. NBO analysis demonstrated that the negative charge on phosphoryl, carbonyl, and sulfoxide oxygen (O_P, O_C, and O_S) has maximum and minimum values when the connected –R groups are –NH₂ and –F. The metal–ligand distance declines as, –F?>?–OH?>?–CH₃?>?–NH₂. Charge density at the bond critical point and on the lanthanide cation in the L–Ln³⁺ complexes varies in the order –F?<?–OH?<?–CH₃?<?–NH₂, due to greater ligand to metal charge transfer, which is well explained by energy decomposition analysis. It was also illustrated that E⁽²⁾ values of Lp(N)?→?σ*(Y–N) vary in the order P=O ? S=O ? C=O and the related values of Lp(N)?→?σ*(Y=O) change as C=O ? S=O ? P=O in (NH₂)_nYO ligands (Y?=?P, C, and S). Trends in the L–Ln³⁺ CP–corrected bond energies are in good accordance with the optimized O_Y?Ln distances. It seems that, comparing the three types of ligands studied, NH₂–substituted are the better coordination ligands.

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Graphical Abstract Density functional theory (B3LYP) calculations were used to compare structural, electronic and energy aspects of lanthanide (La, Eu, Lu) complexes of phosphine derivatives with those of carbonyls and sulfoxides in which the R– groups connected to the P=O, C=O and S=O are –NH₂, –CH₃, –OH and –F.

     

A novel flexible model for lot sizing and scheduling with non-triangular,period overlapping and carryover setups in different machine configurations

This paper develops and tests an efficient mixed integer programming model for capacitated lot sizing and scheduling with non-triangular and sequence-dependent setup times and costs incorporating all necessary features of setup carryover and overlapping on different machine configurations. The model’s formulation is based on the asymmetric travelling salesman problem and allows multiple lots of a product within a period. The model conserves the setup state when no product is being processed over successive periods, allows starting a setup in a period and ending it in the next period, permits ending a setup in a period and starting production in the next period(s), and enforces a minimum lot size over multiple periods. This new comprehensive model thus relaxes all limitations of physical separation between the periods. The model is first developed for a single machine and then extended to other machine configurations, including parallel machines and flexible flow lines. Computational tests demonstrate the flexibility and comprehensiveness of the proposed models.     

Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.     

Bioactivity of endophytic Trichoderma fungal species from the plant family Cupressaceae

Trichoderma fungal species are universal soil residents that are also isolated from decaying wood, vegetables, infected mushroom and immunocompromised patients. Trichoderma species usually biosynthesize a plethora of secondary metabolites. In an attempt to explore endophytic fungi from healthy foliar tissues of the plant family Cuppressaceae, we explored Cupressus arizonica, C. sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp. and Thuja orientalis plants and recovered several endophytic Trichoderma fungal strains from Trichoderma atroviride and Trichoderma koningii species. We found that the host plant species and biogeographical location of sampling affected the biodiversity and bioactivity of endophytic Trichoderma species. Furthermore, the bioactivity of Trichoderma isolates and the methanol extracts of their intra- and extra-cellular metabolites were assessed against a panel of pathogenic fungi and bacteria. Fungal growth inhibition, conidial cytotoxicity, minimum inhibitory concentration and minimum bactericidal concentration were evaluated and analyzed by statistical methods. Our data showed that both intra- and extracellular secondary metabolites from all endophytic isolates had significant cytotoxic and antifungal effects against the model target fungus Pyricularia oryzae and the cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. Further research indicated their significant antimicrobial bioactivity against the model phytopathogenic bacteria Pseudomonas syringae, Erwinia amylovora and Bacillus sp., as well. Altogether, the above findings show for the first time the presence of T. atroviride and T. koningii as endophytic fungi in Cupressaceae plants and more importantly, the Trichoderma isolates demonstrate significant bioactivity that could be used in future for agrochemical/drug discovery and pathogen biocontrol.