Mitochondrial phylogeny of Locustella and related genera

We used maximum likelihood analysis of complete mitochondrial ND2 sequences (1041 bp) to clarify the taxonomy and relationships of various species and genera of grass and bush warblers. The tree revealed two clades of grass and bush warblers. One clade was comprised of all four western Palearctic Locustella and two species of Asian Bradypterus . The other clade included five eastern Palearctic Locustella (including the distinctive Sakhalin warbler Locustella amnicola ) and the marsh grassbird Megalurus pryeri . African Bradypterus and Australian little grassbird Megalurus gramineus were distantly related to their Asian congeners. Therefore, current taxonomy of these genera does not reflect their evolutionary history and needs revision. It is proposed that a phylogenetic analysis of morphology and ecological preferences would show that the current taxonomy of grass and bush warblers reflects species' habitat preferences and morphology related to locomotion and foraging in their habitats, rather than their shared ancestry. Distinct clades were found in grasshopper warbler Locustella naevia and Pallas's grasshopper warbler L. certhiola . Detailed phylogeographic studies are needed to elucidate the species status of the clades within these two species.     

Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.     

Gene Expression Profiling Reveals Epithelial Mesenchymal Transition (EMT) Genes Can Selectively Differentiate Eribulin Sensitive Breast Cancer Cells

Objectives

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a mechanistically unique inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with eribulin activity compared to paclitaxel, which stabilizes microtubules, based on gene expression profiling of cell line panels of breast, endometrial, and ovarian cancer in vitro.

Results

We determined the sets of genes that were differentially altered between eribulin and paclitaxel treatment in breast, endometrial, and ovarian cancer cell line panels. Our unsupervised clustering analyses revealed that expression profiles of gene sets altered with treatments were correlated with the in vitro antiproliferative activities of the drugs. Several tubulin isotypes had significantly lower expression in cell lines treated with eribulin compared to paclitaxel. Pathway enrichment analyses of gene sets revealed that the common pathways altered between treatments in the 3 cancer panels were related to cytoskeleton remodeling and cell cycle regulation. The epithelial-mesenchymal transition (EMT) pathway was enriched in genes with significantly altered expression between the two drugs for breast and endometrial cancers, but not for ovarian cancer. Expression of genes from the EMT pathway correlated with eribulin sensitivity in breast cancer and with paclitaxel sensitivity in endometrial cancer. Alteration of expression profiles of EMT genes between sensitive and resistant cell lines allowed us to predict drug sensitivity for breast and endometrial cancers.

Conclusion

Gene expression analysis showed that gene sets that were altered between eribulin and paclitaxel correlated with drug in vitro antiproliferative activities in breast and endometrial cancer cell line panels. Among the panels, breast cancer provided the strongest differentiation between eribulin and paclitaxel sensitivities based on gene expression. In addition, EMT genes were predictive of eribulin sensitivity in the breast and endometrial cancer panels.     

Structure Driven Design of Novel Human Ether-A-Go-Go-Related-Gene Channel (hERG1) Activators

One of the main culprits in modern drug discovery is apparent cardiotoxicity of many lead-candidates via inadvertent pharmacologic blockade of K⁺, Ca²⁺ and Na⁺ currents. Many drugs inadvertently block hERG1 leading to an acquired form of the Long QT syndrome and potentially lethal polymorphic ventricular tachycardia. An emerging strategy is to rely on interventions with a drug that may proactively activate hERG1 channels reducing cardiovascular risks. Small molecules-activators have a great potential for co-therapies where the risk of hERG-related QT prolongation is significant and rehabilitation of the drug is impractical. Although a number of hERG1 activators have been identified in the last decade, their binding sites, functional moieties responsible for channel activation and thus mechanism of action, have yet to be established. Here, we present a proof-of-principle study that combines de-novo drug design, molecular modeling, chemical synthesis with whole cell electrophysiology and Action Potential (AP) recordings in fetal mouse ventricular myocytes to establish basic chemical principles required for efficient activator of hERG1 channel. In order to minimize the likelihood that these molecules would also block the hERG1 channel they were computationally engineered to minimize interactions with known intra-cavitary drug binding sites. The combination of experimental and theoretical studies led to identification of functional elements (functional groups, flexibility) underlying efficiency of hERG1 activators targeting binding pocket located in the S4–S5 linker, as well as identified potential side-effects in this promising line of drugs, which was associated with multi-channel targeting of the developed drugs.     

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.     

Effect of p-nitrophenol metabolites on microbial cell electro-optical characteristics

The major cell wall-associated protein of the equine pathogen Streptococcus equi subsp. equi is a fibrinogen-binding protein (FgBP) which binds horse fibrinogen and equine IgG-Fc avidly through residues located in the N-terminal half and central regions of the molecule, respectively. The molecule is a major virulence factor for the organism and displays protective potential. In the present study, we use circular dichroism spectroscopy to investigate the secondary structure of the protein and show through the analysis of a panel of recombinant FgBP truncates that the C-terminal portion of FgBP contains an extensive alpha-helical coiled-coil structure that contributes to the thermal stability of the molecule.     

Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

Global transcriptome analysis of human bone marrow stromal cells (BMSC) reveals proliferative, mobile and interactive cells that produce abundant extracellular matrix proteins, some of which may affect BMSC potency

Background aimsBone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality.MethodsEarly passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34⁺ cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes.ResultsBMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/β catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (α5 and β5 integrin chains, fibronectin and collagen type IIIα1 and Vα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)].ConclusionsGlobal analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.