Insights into cardiovascular risk and nutritional status in subjects with wheat-related disorders

Objective: Wheat-related disorders are a spectrum of disorders associated with different autoimmune and non-autoimmune diseases. However, it is unclear whether these wheat-related disorders lead to adverse health effects such as cardiovascular risk, nutritional deficiencies etc. The objective of the study was to explore the lipid profiles and the nutritional status of subjects with wheat-related disorders to understand the potential threat of wheat on cardiovascular risk and nutritional deficiency.

Method: A total of 1041 subjects who showed wheat-related symptoms were initially tested for the wheat protein antibody panel (Wheat Zoomer (WZ) panel and Coeliac Disease (CD) panel), then for cardiovascular panel and the micronutrient panel at Vibrant America Clinical Laboratory.

Results: Subjects with both Wheat Zoomer positivity (WZ+) and Coeliac Disease positivity (CD+) had significantly low levels of high-density lipoproteins (HDL) (279/483(57.8%) and 29/47(61.7%) respectively), but only subjects with WZ?+?had low levels of Apo A1 (44/424(9.5%)), and high levels of Omega 6 fatty acids (53/334(15.9%)). None of the micronutrients tested showed a significant imbalance in WZ?+?subjects.

Conclusion: Subjects with positive serology for WZ have deranged blood lipid profiles but did not show any significant micronutrient deficiency. Hence, our results showcase a significant association of wheat-related disorders to cardiovascular risk.     

Adapting collagen/CNT matrix in directing hESC differentiation

The lineage selection in human embryonic stem cell (hESC) differentiation relies on both the growth factors and small molecules in the media and the physical characteristics of the micro-environment. In this work, we utilized various materials, including the collagen-carbon nanotube (collagen/CNT) composite material, as cell culture matrices to examine the impact of matrix properties on hESC differentiation. Our AFM analysis indicated that the collagen/CNT formed rigid fibril bundles, which polarized the growth and differentiation of hESCs, resulting in more than 90% of the cells to the ectodermal lineage in Day 3 in the media commonly used for spontaneous differentiation. We also observed the differentiated cells followed the coarse alignment of the collagen/CNT matrix. The research not only revealed the responsiveness of hESCs to matrix properties, but also provided a simple yet efficient way to direct the hESC differentiation, and imposed the potential of forming neural-cell based bio-devices for further applications.     

Nanoparticles of Polyethylene Sebacate: A New Biodegradable Polymer

The present study demonstrates feasibility of preparation of nanoparticles using a novel polymer, polyethylene sebacate (PES), and its application in the design of drug-loaded nanocarriers. Silymarin was selected as a model hydrophobic drug for the present study. Two methods of preparation, viz., nanoprecipitation and emulsion solvent diffusion, were evaluated for preparation of nanoparticles. Effect of surfactants polyvinyl alcohol (PVA), lutrol F 68, and Tween 80 on the preparation of blank and silymarin-loaded PES nanoparticles was evaluated. Nanoprecipitation resulted in the formation of nanoparticles with all the surfactants (<450 nm). Increase in surfactant concentration resulted in decrease in entrapment efficiency and particle size except with PVA. The type and concentration of surfactant was critical to achieve low size and adequate drug entrapment. While increase in concentration of PES resulted in larger nanoparticles, inclusion of acetone in the organic phase resulted in particles of smaller size. In case of emulsion solvent diffusion, nanoparticles were obtained only with lutrol F 68 as surfactant and high surfactant concentration. The study revealed nanoprecipitation as a more versatile method for preparation of PES nanoparticles. Scanning electron microscopy studies revealed spherical shape of nanoparticles. Freeze-dried nanoparticles exhibited ease of redispersion, with a marginal increase in size. Differential scanning calorimetry and X-ray diffraction analysis revealed amorphous nature of the drug. The study demonstrates successful design of PES nanoparticles as drug carriers.     

The role of the basal ganglia in exploration in a neural model based on reinforcement learning

We present a computational model of basal ganglia as a key player in exploratory behavior. The model describes exploration of a virtual rat in a simulated water pool experiment. The virtual rat is trained using a reward-based or reinforcement learning paradigm which requires units with stochastic behavior for exploration of the system's state space. We model the Subthalamic Nucleus-Globus Pallidus externa (STN-GPe) segment of the basal ganglia as a pair of neuronal layers with oscillatory dynamics, exhibiting a variety of dynamic regimes such as chaos, traveling waves and clustering. Invoking the property of chaotic systems to explore state-space, we suggest that the complex exploratory dynamics of STN-GPe system in conjunction with dopamine-based reward signaling from the Substantia Nigra pars compacta (SNc) present the two key ingredients of a reinforcement learning system.     

High-performance thin-layer chromatographic determination of diltiazem hydrochloride as bulk drug and in pharmaceutical preparations

A simple, rapid, selective and precise high-performance thin-layer chromatographic method for the analysis of diltiazem hydrochloride both as a bulk drug and in pharmaceuticals is reported. The mobile phase composition was ethyl acetate–methanol–strong ammonia solution (80:10:10, v/v). Densitometric analysis of diltiazem hydrochloride was carried out at 238 nm. The calibration curve of diltiazem hydrochloride in distilled water was linear in the range 40–400 ng. The mean value of correlation coefficient, slope and intercept were 0.997±0.0008, 0.0617±0.0012 and 7.16±0.2562, respectively. The limits of detection and quantitation were 20 ng and 40 ng, respectively. The recovery of diltiazem hydrochloride was about 99.5%. The method was utilized to analyse diltiazem hydrochloride from conventional and sustained release tablets in the presence of commonly used excipients.     

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).     

Effect of covalent links on the structure, spectra, and redox properties of myeloperoxidase--a density functional study

The enzyme myeloperoxidase shows several unusual properties compared to other peroxidases, e.g. a red-shifted absorption spectrum and a peroxidase activity towards chloride. It has been suggested that this is caused by the unusual covalent links between the heme group and the surrounding protein, but whether it is caused by the two ester links to Glu-242 and Asp-94 or the sulfonium ion linkage to Met-243 is unclear. To investigate these suggestions, we have used density functional theory to study the structure, spectra, and reduction potential of 25 models of myeloperoxidase in the reduced (Fe^II) and oxidized (Fe^III) states, as well as in the compound I (formally Fe^VO) and II (Fe^IVO or Fe^IVOH) states, using appropriate models of the linkages to the Asp, Glu, and Met residues (including the back-bone connection between Glu-242 and Met-243) in varying combinations. The calculated spectral shifts indicate that both the ester and sulfonium linkages play a role in the spectral shift. On the other hand, the sulfonium linkage seems to be mainly responsible for the high positive reduction potential for the both ferric/ferrous and compound I/II couples of myeloperoxidase.     

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.     

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.