Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.     

Micropipette Aspiration‐Based Assessment of Single Channel Water Permeability

Measurements of the unitary hydraulic conductivity of membrane channels, p_f, may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for p_f measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving p_f values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. p_f amounted to 2.4 ± 0.1 × 10^?13 cm³ s^?1 for aquaporin‐1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on p_f on a single sample.     

Association of IL4R single-nucleotide polymorphisms with rheumatoid nodules in African Americans with rheumatoid arthritis

Introduction

To determine whether IL4R single-nucleotide polymorphisms (SNPs) rs1805010 (I50V) and rs1801275 (Q551R), which have been associated with disease severity in rheumatoid arthritis (RA) patients of European ancestry, relate to the presence of rheumatoid nodules and radiographic erosions in African Americans.

Methods

Two IL4R SNPs, rs1805010 and rs1801275, were genotyped in 749 patients from the Consortium for Longitudinal Evaluation of African-Americans with Early Rheumatoid Arthritis (CLEAR) registries. End points were rheumatoid nodules defined as present either by physical examination or by chest radiography and radiographic erosions (radiographs of hands/wrists and feet were scored using the modified Sharp/van der Heijde system). Statistical analyses were performed by using logistic regression modeling adjusted for confounding factors.

Results

Of the 749 patients with RA, 156 (20.8%) had rheumatoid nodules, with a mean age of 47.0 years, 84.6% female gender, and median disease duration of 1.9 years. Of the 461 patients with available radiographic data, 185 (40.1%) had erosions (score >0); their mean age was 46.7 years; 83.3% were women; and median disease duration was 1.5 years. Patients positive for HLA-DRB1 shared epitope (SE) and autoantibodies (rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP)) had a higher risk of developing rheumatoid nodules in the presence of the AA and AG alleles of rs1801275 (odds ratio (OR)_adj = 8.08 (95% confidence interval (CI): 1.60-40.89), P = 0.01 and OR_adj = 2.97 (95% CI, 1.08 to 8.17), P = 0.04, respectively). Likewise, patients positive for the HLA-DRB1 SE and RF alone had a higher risk of developing rheumatoid nodules in presence of the AA and AG alleles of rs1801275 (OR_adj = 8.45 (95% CI, 1.57 to 45.44), P = 0.01, and OR_adj = 3.57 (95% CI, 1.18 to 10.76), P = 0.02, respectively) and in the presence of AA allele of rs1805010 (OR_adj = 4.52 (95% CI, 1.20 to 17.03), P = 0.03). No significant association was found between IL4R and radiographic erosions or disease susceptibility, although our statistical power was limited by relatively small numbers of cases and controls.

Conclusions

We found that IL4R SNPs, rs1801275 and rs1805010, are associated with rheumatoid nodules in autoantibody-positive African-American RA patients with at least one HLA-DRB1 allele encoding the SE. These findings highlight the need for analysis of genetic factors associated with clinical RA phenotypes in different racial/ethnic populations.     

Exploring the catalytic mechanism of the first dimeric Bcp: Functional,structural and docking analyses of Bcp4 from Sulfolobus solfataricus

The detoxification from peroxides in Sulfolobus solfataricus is performed by the Bacterioferritin comigratory proteins (Bcps), Bcp1 (Sso2071), Bcp2 (Sso2121), Bcp3 (Sso2255) and Bcp4 (Sso2613), antioxidant enzymes belonging to one of the subfamilies of the Peroxiredoxins. In this paper we report on the functional, structural and docking analyses of Bcp4, characterized by the CXXXXC motif in the active site. Bcp4 represents the first dimeric Bcp so far investigated. Biochemical studies showed that the protein has a non-covalent dimeric structure and adopts an atypical 2-Cys catalytic mechanism. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, described the protein dimeric arrangement. Finally, concurrent availability of the crystallographic structure of the monomeric Bcp1 allowed comparative analysis of the interaction with Protein Disulfide Oxidoreductase SsPDO (Sso0192), involved in the reduction of both Bcp1 and Bcp4, through a protein–protein docking approach.     

Regulation of redox forms of plasma thiols by albumin in multiple sclerosis after fasting and methionine loading test

Increases in plasma concentrations of total homocysteine (tHcy) have recently been reported in multiple sclerosis (MS) as the alteration of the methionine cycle for the onset of autoimmune diseases. Homocysteine (Hcy) and cysteine (Cys) are generated by the methionine cycle and transsulfuration reactions. Their plasma levels are subjected to complex redox changes by oxidation and thiol/disulfide (SH/SS) exchange reactions regulated by albumin. The methionine loading test (MLT) is a useful in vivo test to assay the functionality of the methionine cycle and transsulfuration reactions. Time courses of redox species of Cys, cysteinylglycine (CGly), Hcy, and glutathione have been investigated in plasma of MS patients versus healthy subjects after an overnight fasting, and 2, 4, and 6 h after an oral MLT (100 mg/kg body weight), to detect possible dysfunctions of the methionine cycle, transsulfuration reactions and alterations in plasma distribution of redox species. After fasting, the MS group showed a significant increase in cysteine-protein mixed disulfides (bCys) and total Cys (tCys). While plasma bCys and tCys in MS group remained elevated after methionine administration when compared to control, cystine (oxCys) increased significantly with respect to control. Although increased plasma concentrations of bCys and tCys at fasting might reflect an enhance of transsulfuration reactions in MS patients, this was not confirmed by the analysis of redox changes of thiols and total thiols after MLT. This study has also demonstrated that albumin-dependent SH/SS exchange reactions are a potent regulation system of thiol redox species in plasma.     

Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O₂ atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling.Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well the level of AMPK phosphorylation may be considered as predictors of the tumor sensitivity to anti-angiogenic drugs.     

N-(2-alkylaminoethyl)-4-(1,2,4-oxadiazol-5-yl)piperazine-1-carboxamides as highly potent smoothened antagonists

Smoothened (Smo) antagonists are emerging as new therapies for the treatment of neoplasias with aberrantly reactivated hedgehog (Hh) signaling pathway. A novel series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as smoothened antagonists was recently described, herein the series has been further optimized through the incorporation of a basic amine into the urea. This development resulted in identification of some exceptionally potent smoothened antagonists with low serum shifts, however, reductive ring opening on the 1,2,4-oxadiazole in rats limits the applicability of these compounds in in vivo studies.     

Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay

Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates.     

Identification and SAR of novel pyrrolo[1,2-a]pyrazin-1(2H)-one derivatives as inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1)

Herein we describe the discovery of a novel series of pyrrolo[1,2-a]pyrazin-1(2H)-one PARP inhibitors. Optimization led to compounds that display excellent PARP-1 enzyme potency and inhibit the proliferation of BRCA deficient cells in the low double-digit nanomolar range showing excellent selectivity over BRCA proficient cancer cells.