Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Synthesis of 4-thia-[6-13C]lysine from [2-13C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine

4-Thialysine (S-(2-aminoethyl)-l-cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-¹³C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-¹³C]glycine via formation of five intermediates including 2-amino[2-¹³C]ethanol and 2-bromo[1-¹³C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-¹³C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.     

Fish oil increases raft size and membrane order of B cells accompanied by differential effects on function

Fish oil (FO) targets lipid microdomain organization to suppress T-cell and macrophage function; however, little is known about this relationship with B cells, especially at the animal level. We previously established that a high FO dose diminished mouse B-cell lipid raft microdomain clustering induced by cross-linking GM1. To establish relevance, here we tested a FO dose modeling human intake on B-cell raft organization relative to a control. Biochemical analysis revealed more docosahexaenoic acid (DHA) incorporated into phosphatidylcholines than phosphatidylethanolamines of detergent-resistant membranes, consistent with supporting studies with model membranes. Subsequent imaging experiments demonstrated that FO increased raft size, GM1 expression, and membrane order upon cross-linking GM1 relative to no cross-linking. Comparative in vitro studies showed some biochemical differences from in vivo measurements but overall revealed that DHA, but not eicosapentaenoic acid (EPA), increased membrane order. Finally, we tested the hypothesis that disrupting rafts with FO would suppress B-cell responses ex vivo. FO enhanced LPS-induced B-cell activation but suppressed B-cell stimulation of transgenic naive CD4(+) T cells. Altogether, our studies with B cells support an emerging model that FO increases raft size and membrane order accompanied by functional changes; furthermore, the results highlight differences in EPA and DHA bioactivity.     

Controlled synthesis of O-glycopolypeptide polymers and their molecular recognition by lectins

The facile synthesis of high molecular weight water-soluble O-glycopolypeptide polymers by the ring-opening polymerization of their corresponding N-carboxyanhydride (NCA) in very high yield (overall yield > 70%) is reported. The per-acetylated-O-glycosylated lysine-NCA monomers, synthesized using stable glycosyl donors and a commercially available protected amino acid in very high yield, was polymerized using commercially available amine initiators. The synthesized water-soluble glycopolypeptides were found to be α-helical in aqueous solution. However, we were able to control the secondary conformation of the glycopolypeptides (α-helix vs nonhelical structures) by polymerizing racemic amino acid glyco NCAs. We have also investigated the binding of the glycopolypeptide poly(α-manno-O-lys) with the lectin Con-A using precipitation and hemagglutination assays as well as by isothermal titration calorimetry (ITC). The ITC results clearly show that the binding process is enthalpy driven for both α-helical and nonhelical structures, with negative entropic contribution. Binding stoichiometry for the glycopolypeptide poly(α-manno-O-lys) having a nonhelical structure was slightly higher as compared to the corresponding polypeptide which adopted an α-helical structure.     

Frequency distribution and association of Fat-mass and obesity (FTO) gene SNP rs-9939609 variant with Diabetes Mellitus Type-II population of Hyderabad,Sindh, Pakistan

Background/AimDiabetes Mellitus (DM) is one of the important public health issues worldwide. The Fat mass obesity (FTO) gene rs-9939609 variant identified single nucleotide polymorphism (SNP) with the T to A missense mutation, and has a strong association with T2DM. FTO gene is present on chromosome “16q12.2” comprising of nine exons. FTO gene rs-9939609 a variant is commonly found in the Pakistani Population. The purpose of the study was to alert the population about the rs-9939609 variant SNP, having a strong association with T2DM.Material and MethodsTotal of 190 participants were included in the present cross-sectional study. To collect the samples non-probability convenience technique was used. subjects were recruited and divided into three groups, normal healthy subjects, obese and T2DM. The patients were selected from the Medicine department Jamshoro/Hyderabad by filling the pre-designed proforma, as well as verbal and written consent taken from study participants. To analysed the data ANOVA Post hoc (Tukey-test) was applied for comparison among groups (P < 0.05) and “SNP-STAT” online software was used for frequencies.ResultsThe BMI, neck circumference, waist circumference and lipid profile, fasting blood sugar and HbA1c was found significant (p < 0.001) in both genders as compared to control. Homozygous and heterozygous distribution of allelic and genotyping frequency was found in study participants. 37.9 %T/A, 57.4% T/T, and A/A were 4.7%. The FTO gene rs-9939609 variant amplified and have an increased risk of developing T2DM in the Sindh population. Codominant model odd ratio of T/A showed 2.42 (CI)1.23–3.84, with significant p < 0.032.ConclusionThe present study concluded that the FTO gene SNP rs-9939609 variant was found in the population of Hyderabad, Sindh and having strong association with T2DM and obese individuals. Increase BMI, neck and waist circumference are the biomarkers of obesity and causative factors of T2DM.     

Abnormal Fixational Eye Movements in Amblyopia

Purpose

Fixational saccades shift the foveal image to counteract visual fading related to neural adaptation. Drifts are slow eye movements between two adjacent fixational saccades. We quantified fixational saccades and asked whether their changes could be attributed to pathologic drifts seen in amblyopia, one of the most common causes of blindness in childhood.

Methods

Thirty-six pediatric subjects with varying severity of amblyopia and eleven healthy age-matched controls held their gaze on a visual target. Eye movements were measured with high-resolution video-oculography during fellow eye-viewing and amblyopic eye-viewing conditions. Fixational saccades and drifts were analyzed in the amblyopic and fellow eye and compared with controls.

Results

We found an increase in the amplitude with decreased frequency of fixational saccades in children with amblyopia. These alterations in fixational eye movements correlated with the severity of their amblyopia. There was also an increase in eye position variance during drifts in amblyopes. There was no correlation between the eye position variance or the eye velocity during ocular drifts and the amplitude of subsequent fixational saccade. Our findings suggest that abnormalities in fixational saccades in amblyopia are independent of the ocular drift.

Discussion

This investigation of amblyopia in pediatric age group quantitatively characterizes the fixation instability. Impaired properties of fixational saccades could be the consequence of abnormal processing and reorganization of the visual system in amblyopia. Paucity in the visual feedback during amblyopic eye-viewing condition can attribute to the increased eye position variance and drift velocity.     

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.