Insights on the structural perturbations in human MTHFR Ala222Val mutant by protein modeling and molecular dynamics

Methylenetetrahydrofolate reductase (MTHFR) protein catalyzes the only biochemical reaction which produces methyltetrahydrofolate, the active form of folic acid essential for several molecular functions. The Ala222Val polymorphism of human MTHFR encodes a thermolabile protein associated with increased risk of neural tube defects and cardiovascular disease. Experimental studies have shown that the mutation does not affect the kinetic properties of MTHFR, but inactivates the protein by increasing flavin adenine dinucleotide (FAD) loss. The lack of completely solved crystal structure of MTHFR is an impediment in understanding the structural perturbations caused by the Ala222Val mutation; computational modeling provides a suitable alternative. The three-dimensional structure of human MTHFR protein was obtained through homology modeling, by taking the MTHFR structures from Escherichia coli and Thermus thermophilus as templates. Subsequently, the modeled structure was docked with FAD using Glide, which revealed a very good binding affinity, authenticated by a Glide XP score of ?10.3983 (kcal mol^?1). The MTHFR was mutated by changing Alanine 222 to Valine. The wild-type MTHFR-FAD complex and the Ala222Val mutant MTHFR-FAD complex were subjected to molecular dynamics simulation over 50 ns period. The average difference in backbone root mean square deviation (RMSD) between wild and mutant variant was found to be ~.11 Å. The greater degree of fluctuations in the mutant protein translates to increased conformational stability as a result of mutation. The FAD-binding ability of the mutant MTHFR was also found to be significantly lowered as a result of decreased protein grip caused by increased conformational flexibility. The study provides insights into the Ala222Val mutation of human MTHFR that induces major conformational changes in the tertiary structure, causing a significant reduction in the FAD-binding affinity.     

Big Data: Astronomical or Genomical?

Genomics is a Big Data science and is going to get much bigger, very soon, but it is not known whether the needs of genomics will exceed other Big Data domains. Projecting to the year 2025, we compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Our estimates show that genomics is a “four-headed beast”—it is either on par with or the most demanding of the domains analyzed here in terms of data acquisition, storage, distribution, and analysis. We discuss aspects of new technologies that will need to be developed to rise up and meet the computational challenges that genomics poses for the near future. Now is the time for concerted, community-wide planning for the “genomical” challenges of the next decade.We compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Astronomy has faced the challenges of Big Data for over 20 years and continues with ever-more ambitious studies of the universe. YouTube burst on the scene in 2005 and has sparked extraordinary worldwide interest in creating and sharing huge numbers of videos. Twitter, created in 2006, has become the poster child of the burgeoning movement in computational social science [6], with unprecedented opportunities for new insights by mining the enormous and ever-growing amount of textual data [7]. Particle physics also produces massive quantities of raw data, although the footprint is surprisingly limited since the vast majority of data are discarded soon after acquisition using the processing power that is coupled to the sensors [8]. Consequently, we do not include the domain in full detail here, although that model of rapid filtering and analysis will surely play an increasingly important role in genomics as the field matures.To compare these four disparate domains, we considered the four components that comprise the “life cycle” of a dataset: acquisition, storage, distribution, and analysis (

Transcellular secretion of group V phospholipase A2 from epithelium induces beta 2-integrin-mediated adhesion and synthesis of leukotriene C4 in eosinophils

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.     

Nbr1 is a novel inhibitor of ligand-mediated receptor tyrosine kinase degradation

Neighbor of BRCA1 (Nbr1) is a highly conserved multidomain scaffold protein with proposed roles in endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic α-helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function.     

Chemical composition of the fixed and volatile oils of Nigella sativa L. from Iran

The chemical composition of the extracted fixed oil (total fatty acid composition) and volatile oil of Nigella sativa L. seeds grown in Iran were determined by GC and GC/MS. Eight fatty acids (99.5%) and thirty-two compounds (86.7%) have been identified in the fixed and volatile oils, respectively. The main fatty acids of the fixed oil were linoleic acid (55.6%), oleic acid (23.4%), and palmitic acid (12.5%). The major compounds of the volatile oil were trans-anethole (38.3%), p-cymene (14.8%), limonene (4.3%), and carvone (4.0%).     

Characterization of a series of far-red-absorbing thiobarbituric acid oxonol derivatives as fluorescent probes for biological applications

Chromophores that absorb in the far-red region of the spectrum are increasingly being utilized for applications in the biosciences. We have synthesized and evaluated a novel series of fluorescent oxonols based on thiobarbituric acids containing aryl and heteroaryl substituents. The novel chromophores possess narrow absorption spectra ( approximately 40-nm bandwidths), reasonable Stokes shifts ( approximately 25 nm), and quantum yields of up to 0.67 in organic solvents and 0.3 in aqueous solvents, with absorption wavelength maxima at 620-640 nm. The spectral properties of the compounds are sensitive to base and exhibit a loss of far-red absorbance that is concentration and time dependent. Derivatives have been synthesized that can be used for the labeling of macromolecules such as proteins and DNA. The probes show environment sensitivity and the oligonucleotide conjugates sense the formation of duplex DNA. These novel far-red fluorophores have potential applications in diagnostic and research applications.     

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.     

VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models

Background

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA.

Methods and results

Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models.

Conclusion

Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.     

What Are the Reasons for Poor Uptake of HIV Testing among Patients with TB in an Eastern India District?

Background

National policy in India recommends HIV testing of all patients with TB. In West Bengal state, only 28% of patients with TB were tested for HIV between April-June, 2010. We conducted a cross-sectional survey to understand patient, provider and health system related factors associated with low uptake of HIV testing among patients with TB.

Methods

We reviewed TB and HIV program records to assess the HIV testing status of patients registered for anti-TB treatment from July-September 2010 in South-24-Parganas district, West Bengal, assessed availability of HIV testing kits and interviewed a random sample of patients with TB and providers.

Results

Among 1633 patients with TB with unknown HIV status at the time of diagnosis, 435 (26%) were tested for HIV within the intensive phase of TB treatment. Patients diagnosed with and treated for TB at facilities with co-located HIV testing services were more likely to get tested for HIV than at facilities without [RR = 1.27, (95% CI 1.20–3.35)]. Among 169 patients interviewed, 67 reported they were referred for HIV testing, among whom 47 were tested. During interviews, providers attributed the low proportion of patients with TB being referred and tested for HIV to inadequate knowledge among providers about the national policy, belief that patients will not test for HIV even if they are referred, shortage of HIV testing kits, and inadequate supervision by both programs.

Discussion

In West Bengal, poor uptake of HIV testing among patients with TB was associated with absence of HIV testing services at sites providing TB care services and to poor referral practices among providers. Comprehensive strategies to change providers’ beliefs and practices, decentralization of HIV testing to all TB care centers, and improved HIV test kit supply chain management may increase the proportion of patients with TB who are tested for HIV.