Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

Membrane Bound GSK-3 Activates Wnt Signaling through Disheveled and Arrow

Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. Here we examine the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. We find that targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. Our results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol.     

The effects of chemokine, adhesion and extracellular matrix molecules on binding of mesenchymal stromal cells to poly(l-lactic acid)

Abstract Background aims. Mesenchymal stromal cells (MSC) are pluripotent adult stem cells capable of osteogenesis and chondrogenesis to form bone and cartilage. This characteristic gives them the potential for bone and cartilage regeneration. Synthetic polymers have been studied to examine whether they could be used as a scaffold for tissue engineering. In the current study a two-dimensional (2-D) poly(l-lactic acid) (PLLA) scaffold was treated with chemokine, adhesion and extracellular matrix molecules with the aim of using biologic molecules to improve the attachment of human MSC. Methods. MSC were isolated from human bone marrow and applied to a 2-D PLLA scaffold. Chemokines ligand (CXCL12 and CXCL13), adhesion molecules [P-selectin, vascular cell adhesion molecule (VCAM)-1 and heparin] and extracellular matrix molecules (fibronectin and type IV collagen) were coated on the scaffold and their effects on the number of MSC that adhered were recorded. Results. When used alone CXCL12 and CXCL13 enhanced MSC adhesion, as did VCAM-1, P-selectin, fibronectin and collagen, but not heparin. The effects of VCAM-1, P-selectin and heparin were enhanced by the addition of CXCL12. Incubation of MSC with antibodies to integrins α4 and α5β1 inhibited their adhesion to VCAM-1 and fibronectin-treated PLLA respectively, suggesting that these integrins were involved in the MSC interactions. Conclusions. The use of certain chemokines and adhesion and extracellular matrix molecules, alone or in combination, is beneficial for the attachment of MSC to PLLA, and may be helpful as natural molecules in scaffolds for regenerative medicine.     

Electrochemically mediated CO<Subscript>2</Subscript> reduction for bio-methane production: a review

A number of methods for carbon capture, more specifically, CO₂ capture have been researched in the past few years. One such method is electrochemical CO₂ reduction to biomethane which also serves the purpose of biogas upgradation using microbial electrosynthesis systems. This technology is also known as Power to Gas technology and the review starts with the importance and requirement of PtG in the modern world by studying energy production and consumption patterns in Europe, with a focus on Norway. The paper summarises the recent works and concepts in the field of bioelectrochemical systems with a focus on electron transfer mechanisms, biocatalysts and reactor designs. Works and gaps in the studies of direct interspecies electron transfer and biocathode developments are discussed in detail. This is followed by a discussion explaining various reactor designs, the advantages of single chambered microbial reactors and the importance of reactors that combine anaerobic digestion with microbial electrolysis cells.     

The release of microparticles by Jurkat leukemia T cells treated with staurosporine and related kinase inhibitors to induce apoptosis

Microparticles (MPs) are small membrane-bound vesicles released from cells undergoing activation or cell death. These particles display potent biological activities that can impact on physiologic and pathologic processes. Previous studies with the Jurkat T leukemia cell line demonstrated that staurosporine (STS) induces the release of MPs as cells undergo apoptosis. To investigate further this process, we tested the effects of STS, its analogue, 7-hydroxystaurosporine (UCN-01), and other protein kinase C (PKC) and cyclin-dependent kinase (CDK) inhibitors. FACS analysis was used to assess MP release. Results of these studies indicate that STS and UCN-01 induce MP release by Jurkat cells; in contrast, other PKC and CDK inhibitors failed to induce comparable release, suggesting that release does not result from simple inhibition of either kinase alone. Time course experiments indicated that STS-induced particle release occurred as early as 2 h after treatment, with the early release MPs displaying low levels of binding of annexin V and propidium iodide (PI). Early-release MPs, however, matured in culture to an annexin V- and PI-positive phenotype. Together, these results indicate that STS and UCN-01 induce MPs that are phenotypically distinct and reflect specific patterns of kinase inhibition during apoptosis.     

Interneurons of the neocortical inhibitory system

Mammals adapt to a rapidly changing world because of the sophisticated cognitive functions that are supported by the neocortex. The neocortex, which forms almost 80% of the human brain, seems to have arisen from repeated duplication of a stereotypical microcircuit template with subtle specializations for different brain regions and species. The quest to unravel the blueprint of this template started more than a century ago and has revealed an immensely intricate design. The largest obstacle is the daunting variety of inhibitory interneurons that are found in the circuit. This review focuses on the organizing principles that govern the diversity of inhibitory interneurons and their circuits.     

Antiosteopenic Effect of Buffalo Milk Casein-Derived Peptide (NAVPITPTL) in Ovariectomized Rats

International Journal of Peptide Research and Therapeutics - In the last decade, several studies have reported health beneficial effects of milk derived bioactive peptides in several degenerative...     

Dissection of chilling requirement and bloom date QTLs in peach using a whole genome sequencing of sibling trees from an F2 mapping population

Chilling requirement (CR) for floral bud dormancy release is one of the major limiting factors for geographical adaptation of fruiting trees. Using a whole genome sequencing approach (Illumina platform), we explored polymorphism underlying phenotypic differences among individuals in a peach F₂ cross segregating for chilling requirement and bloom date. Allelic configuration of individuals, which represented phenotypic extremes in the cross (300 vs. 1,100 chill hours) allowed reconstruction of low- and high-chill haplotypes within three most significant quantitative trait locus (QTL) intervals on the Prunus G1, G4, and G7. We detected single nucleotide polymorphic sites (SNPs), small deletions and insertions (DIPs), and large structural variants (SVs) associated with low-chill haplotypes and created a prioritized list of candidate genes based on functionally characterized homologs from Arabidopsis thaliana. Two dormancy associated genes PpeDAM5 and PpeDAM6 are the strongest candidate genes for the major QTL signal at the lower end of G1. Also, key functional genes involved in the Polycomb repressive mechanism, cell cycle progression, and hormone regulation were evident as strong candidate genes underlying QTL intervals in this peach cross.