Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Gene expression profiling demonstrates a novel role for foetal fibrocytes and the umbilical vessels in human fetoplacental development

There is a difference in the susceptibility to inflammation between the umbilical vein (UV) and the umbilical arteries (UAs). This led us to hypothesize that there is an intrinsic difference in the pro-inflammatory response between UA and UV. Real-time quantitative RT-PCR and microarray analysis revealed higher expression of interleukin (IL)-1β and IL-8 mRNA in the UV and differential expression of 567 genes between the UA and UV associated with distinct biological processes, including the immune response. Differential expression of human leukocyte antigen (HLA)-DRA mRNA between the UA and UV was due to unexpected HLA-DR⁺ cells migrating via the umbilical vessels into Wharton's jelly, more frequently in the UV. A significant proportion of these cells co-expressed CD45 and type I pro-collagen, and acquired CD163 or α-smooth muscle actin immunoreactivity in Wharton's jelly. Migrating cells were also found in the chorionic and stem villous vessels. Furthermore, the extent of migration increased with progression of gestation, but diminished in intrauterine growth restriction (IUGR). The observations herein strongly suggest that circulating foetal fibrocytes, routing via umbilical and placental vessels, are a reservoir for key cellular subsets in the placenta. This study reports fibrocytes in the human umbilical cord and placenta for the first time, and a novel role for both circulating foetal cells and the umbilical vessels in placental development, which is deranged in IUGR.     

Design of Benzene-1,2-diamines as selective inducible nitric oxide synthase inhibitors: a combined de novo design and docking analysis

Selective inhibition of inducible nitric oxide synthases (iNOS) has been a challenging problem for researchers pursuing work in finding methods to treat inflammatory disorders, shock, etc. Though many inhibitors have been studied to date, all are associated with selectivity or potency problems. Additionally, most of the reported compounds have several similarities and fewer number of novel structures are being tried. There is an increasing need to design novel molecules for this target. In this work, de novo design using LUDI, combined with docking analysis using FlexX has been employed in an attempt to identify novel scaffolds. Benzene-1,2-diamines were identified which could mimic the interactions of the substrate analogs and other inhibitors. Comparative docking scores in each of the isoforms of nitric oxide synthase were employed to recognize hits for iNOS selectivity. Figure Figure shows the docked poses of the ligand M226 along with that of the reference GW274150. (FlexX analysis)     

A Time-Series-Based Feature Extraction Approach for Prediction of Protein Structural Class

This paper presents a novel feature vector based on physicochemical property of amino acids for prediction protein structural classes. The proposed method is divided into three different stages. First, a discrete time series representation to protein sequences using physicochemical scale is provided. Later on, a wavelet-based time-series technique is proposed for extracting features from mapped amino acid sequence and a fixed length feature vector for classification is constructed. The proposed feature space summarizes the variance information of ten different biological properties of amino acids. Finally, an optimized support vector machine model is constructed for prediction of each protein structural class. The proposed approach is evaluated using leave-one-out cross-validation tests on two standard datasets. Comparison of our result with existing approaches shows that overall accuracy achieved by our approach is better than exiting methods.     

A computational study of the aerodynamic performance of a dragonfly wing section in gliding flight

A comprehensive computational fluid-dynamics-based study of a pleated wing section based on the wing of Aeshna cyanea has been performed at ultra-low Reynolds numbers corresponding to the gliding flight of these dragonflies. In addition to the pleated wing, simulations have also been carried out for its smoothed counterpart (called the 'profiled' airfoil) and a flat plate in order to better understand the aerodynamic performance of the pleated wing. The simulations employ a sharp interface Cartesian-grid-based immersed boundary method, and a detailed critical assessment of the computed results was performed giving a high measure of confidence in the fidelity of the current simulations. The simulations demonstrate that the pleated airfoil produces comparable and at times higher lift than the profiled airfoil, with a drag comparable to that of its profiled counterpart. The higher lift and moderate drag associated with the pleated airfoil lead to an aerodynamic performance that is at least equivalent to and sometimes better than the profiled airfoil. The primary cause for the reduction in the overall drag of the pleated airfoil is the negative shear drag produced by the recirculation zones which form within the pleats. The current numerical simulations therefore clearly demonstrate that the pleated wing is an ingenious design of nature, which at times surpasses the aerodynamic performance of a more conventional smooth airfoil as well as that of a flat plate. For this reason, the pleated airfoil is an excellent candidate for a fixed wing micro-aerial vehicle design.     

Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components

Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-β-cyclodextrin (MβCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MβCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.     

NMR identification of local structural preferences in HIV-1 protease tethered heterodimer in 6 M guanidine hydrochloride.

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus-1 protease in the 'unfolded state'. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native-like, and there are also some non-native structural elements. Three-bond H(N)-H(alpha) coupling constants that could be measured for some of the N-terminal and C-terminal residues are consistent with the native-like beta-structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.     

Protein and carbohydrate histochemistry in relation to the keratinization in the epidermis of Barbus sophor (Cyprinidae, Pisces)

The distribution of protein and carbohydrate constituents in the epidermis of Barbus sophor is described in order to give a better understanding of its cellular organization and physiology.
Various cytochemical techniques show the keratinized nature of the outer free margins of the polygonal cells in the most-superficial layer. These contain appreciable amounts of cysteine bound sulphydryl groups, basic proteins, protein bound NH₂ groups, ribonucleic acid and calcium and give a strong Papanicolaou's reaction. Absence of cystine bound disulphide groups suggests that the cornified layer in B. sophor is probably mechanically weak as adjacent keratin chains remain unbonded. The polygonal cells showing keratinization at the outer free margins remain metabolically active and are not sloughed off at the surface. This is in contrast to the keratinized epidermis of other teleosts so far reported in which the keratinized cells are dead and are sloughed off at the surface.
In addition to keratinization the polygonal cells undergo mucogenesis synthesizing sulphated acid mucopolysaccharides.
The presence of eosinophilic granular cells in the epidermis is interesting. The possible role of these cells in the protection of the epidermis has been discussed. The epidermis on the inner surface of the scale is very thin so it may not have much protective significance in these areas.     

The merrit alloantigenic system of human B lymphocytes: evidence for thirteen possible factors including one six-member segregant series.

An analysis of the typing results of a 70-member chronic lymphatic leukemia B cell panel revealed evidence for 13 possible groups of the Merrit alloantigenic system. Six of these appeared possibly allelic and may represent a segregant series. The CLL panel was also fully typed for HLA and some degree of linkage dysequilibrium between Merrit and HLA seemed apparent from the data. Merrit antibodies can be absorbed out with selected surface membrane immunoglobulin (SMIG)-positive normal lymphocytes and less so or not at all with E rosette-forming T cells or Fc-positive SMIG-negative lymphocytes.