Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZ_EI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.     

Knowledge,Attitudes and Perceptions of Saudis towards Participating in Clinical Trials

Aim

To assess the knowledge, attitudes, and perceptions of Saudis towards participating in clinical trials (CTs).

Methods

A cross-sectional study was conducted on 232 Saudi adult patients and their companions visiting adult outpatient clinics at King Fahad Medical City, Riyadh, Saudi Arabia. Data were collected using a self-administered questionnaire based on information obtained from the literature. The questionnaire was divided into four sections, one covering the respondents’ demographics, and the other three assessing knowledge, attitudes, and perceptions towards participating in CTs.

Results

A total of 148 (63.8%) respondents were males, and 52 (22.4%) participants had been invited to participate in a CT previously. Of those, 39 (75%) participated. Knowledge about the essential elements of informed consent ranged from 55.7% (number of participants needed) to 85.7% (confidentiality of personal information). The majority (163, 73.8%) of respondents was willing to participate in a CT after consulting their family physician and 130 (58.0%) respondents would be motivated to participate in a CT if they were healthy. Only 36.8% of the respondents believed that patients who participated in a CT received the best care. Moreover, 110 (48.7%) respondents believed that research was conducted in a responsible and ethical manner.

Conclusions

The present study assessed the current understanding of CTs among Saudi participants. Although the majority of participants had an acceptable level of knowledge about CTs, they exhibited conditional attitudes and misperceptions towards participating in a CT. Increased patient awareness may improve patients’ attitudes towards ethical conduct of CTs.     

Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.     

Turbulence Model Selection for Low Reynolds Number Flows

One of the major flow phenomena associated with low Reynolds number flow is the formation of separation bubbles on an airfoil’s surface. NACA4415 airfoil is commonly used in wind turbines and UAV applications. The stall characteristics are gradual compared to thin airfoils. The primary criterion set for this work is the capture of laminar separation bubble. Flow is simulated for a Reynolds number of 120,000. The numerical analysis carried out shows the advantages and disadvantages of a few turbulence models. The turbulence models tested were: one equation Spallart Allmars (S-A), two equation SST K-ω, three equation Intermittency (γ) SST, k-kl-ω and finally, the four equation transition γ-Re_θ SST. However, the variation in flow physics differs between these turbulence models. Procedure to establish the accuracy of the simulation, in accord with previous experimental results, has been discussed in detail.     

Effects of fusaric acid treatment on the protocorm-like bodies of <Emphasis Type="Italic">Dendrobium</Emphasis> sonia-28

Dendrobium sonia-28 is a popular orchid hybrid due to its flowering recurrence and dense inflorescences. Unfortunately, it is being decimated by fungal diseases, especially those caused by Fusarium proliferatum. In this study, selection of F. proliferatum-tolerant protocorm-like bodies (PLBs) was carried out by assessing the effects of differing concentrations of fusaric acid (FA). PLBs were cultured on Murashige and Skoog (MS) medium supplemented with 0.05 to 0.2 millimolar (mM) concentrations of FA. Higher concentrations of FA increased mortality of PLBs and reduced their growth. The survival rate for 0.05 mM FA was 20 % but only 1 % at the highest dose of 0.2 mM. Additionally, two different size ranges of PLBs were investigated, and growth increased more at lower FA concentrations for larger PLBs, whilst the growth rate of smaller PLBs was inhibited at an FA concentration of 0.2 mM. Histological examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses disclosed severe cell wall and organelle damage, as well as stomatal closure in PLBs treated with the high FA concentrations. Reductions in plantlet growth were much greater at the highest concentrations of FA. Some randomly amplified polymorphic DNA (RAPD) markers clearly discriminated between selected and non-selected variants of Dendrobium sonia-28, showing different banding patterns for each FA concentration and specific bands for selected and control plants.     

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.     

Identification,cDNA Cloning,and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.