Chromatin-modifying agents in anti-cancer therapy

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Molecular systematics in Aratinga parakeets: species limits and historical biogeography in the 'solstitialis' group, and the systematic position of Nandayus nenday

The parrot genus Aratinga comprises 20 species that can be separated, based on morphological characters, in at least three distinct groups. We performed a phylogenetic analysis based on mtDNA sequences of individuals belonging to the solstitialis group with the objectives of: (1) assessing the genetic differences among individuals in order to clarify their specific status; (2) testing the monophyly of the group and establishing its phylogenetic position relative to other Aratinga species, (3) making inferences about temporal and geographical patterns of diversification in the Neotropics. As a result of the analysis, the three taxa belonging to the Aratinga solstitialis complex were found to be diagnosable phylogenetic species, the monotypic genus Nandayus was found to be included in the solstitialis group and the non-monophyly of the genus Aratinga was confirmed. Most of the speciation events occurred during the Pliocene and Pleistocene and may be related to habitat shifts associated to climate oscillation during these periods.     

Alona iheringula Sinev & Kotov, 2004 (Crustacea,Anomopoda, Chydoridae,Aloninae): Life Cycle and DNA Barcode with Implications for the Taxonomy of the Aloninae Subfamily

Knowledge of reproductive rates and life cycle of the Cladocera species is essential for population dynamic studies, secondary production and food webs, as well as the management and preservation of aquatic ecosystems. The present study aimed to understand the life cycle and growth of Alona iheringula Kotov & Sinev, 2004 (Crustacea, Anomopoda, Chydoridae), a Neotropical species, as well as its DNA barcoding, providing new information on the Aloninae taxonomy. The specimens were collected in the dammed portion of the Cabo Verde River (21°26′05″ S and 46°10′57″ W), in the Furnas Reservoir, Minas Gerais State, Brazil. Forty neonates were observed individually two or three times a day under controlled temperature (25±1°C), photoperiod (12 h light/12 h dark) and feeding (Pseudokirchneriella subcapitata at a concentration of 10⁵ cells.mL⁻¹ and a mixed suspension of yeast and fish feed in equal proportion). Individual body growth was measured daily under optical microscope using a micrometric grid and 40× magnification. The species had a mean size of 413(±29) µm, a maximum size of 510 µm and reached maturity at 3.24(±0.69) days of age. Mean fecundity was 2 eggs per female per brood and the mean number of eggs produced per female during the entire life cycle was 47.6(±6.3) eggs per female. The embryonic development time was 1.79(±0.23) days and the maximum longevity was 54 days. The species had eight instars throughout its life cycle and four instars between neonate and primipara stage. The present study using molecular data (a 461 bp smaller COI fragment) demonstrated a deep divergence in the Aloninae subfamily.     

Mitochondria as sensors and regulators of calcium signalling

During the past two decades calcium (Ca(2+)) accumulation in energized mitochondria has emerged as a biological process of utmost physiological relevance. Mitochondrial Ca(2+) uptake was shown to control intracellular Ca(2+) signalling, cell metabolism, cell survival and other cell-type specific functions by buffering cytosolic Ca(2+) levels and regulating mitochondrial effectors. Recently, the identity of mitochondrial Ca(2+) transporters has been revealed, opening new perspectives for investigation and molecular intervention.     

Phylobetadiversity among Forest Types in the Brazilian Atlantic Forest Complex

Phylobetadiversity is defined as the phylogenetic resemblance between communities or biomes. Analyzing phylobetadiversity patterns among different vegetation physiognomies within a single biome is crucial to understand the historical affinities between them. Based on the widely accepted idea that different forest physiognomies within the Southern Brazilian Atlantic Forest constitute different facies of a single biome, we hypothesize that more recent phylogenetic nodes should drive phylobetadiversity gradients between the different forest types within the Atlantic Forest, as the phylogenetic divergence among those forest types is biogeographically recent. We compiled information from 206 checklists describing the occurrence of shrub/tree species across three different forest physiognomies within the Southern Brazilian Atlantic Forest (Dense, Mixed and Seasonal forests). We analyzed intra-site phylogenetic structure (phylogenetic diversity, net relatedness index and nearest taxon index) and phylobetadiversity between plots located at different forest types, using five different methods differing in sensitivity to either basal or terminal nodes (phylogenetic fuzzy weighting, COMDIST, COMDISTNT, UniFrac and Rao’s H). Mixed forests showed higher phylogenetic diversity and overdispersion than the other forest types. Furthermore, all forest types differed from each other in relation phylobetadiversity patterns, particularly when phylobetadiversity methods more sensitive to terminal nodes were employed. Mixed forests tended to show higher phylogenetic differentiation to Dense and Seasonal forests than these latter from each other. The higher phylogenetic diversity and phylobetadiversity levels found in Mixed forests when compared to the others likely result from the biogeographical origin of several taxa occurring in these forests. On one hand, Mixed forests shelter several temperate taxa, like the conifers Araucaria and Podocarpus. On the other hand, tropical groups, like Myrtaceae, are also very representative of this forest type. We point out to the need of more attention to Mixed forests as a conservation target within the Brazilian Atlantic Forest given their high phylogenetic uniqueness.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G₁, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent K_m for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower K_m constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

Exploring Leptin Antagonism in Ophthalmic Cell Models

Background

Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.

Methods and Results

The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.

Conclusions

Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.