Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.     

Spherical vs. granular immobilization support selection and performance on an optical flow cell immunosensor based on the fluorescence of Cyanine-5

A spherical porous glass support Trisoperl (TRISO) with four pore diameters (? 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (-NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(-NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(-NCS) and TRISO(-NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti-Goat IgG conjugated with Cyanine-5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(-NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(-NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Can amphibians take the heat? Vulnerability to climate warming in subtropical and temperate larval amphibian communities

Predicting the biodiversity impacts of global warming implies that we know where and with what magnitude these impacts will be encountered. Amphibians are currently the most threatened vertebrates, mainly due to habitat loss and to emerging infectious diseases. Global warming may further exacerbate their decline in the near future, although the impact might vary geographically. We predicted that subtropical amphibians should be relatively susceptible to warming‐induced extinctions because their upper critical thermal limits (CT_max) might be only slightly higher than maximum pond temperatures (T_max). We tested this prediction by measuring CT_max and T_max for 47 larval amphibian species from two thermally distinct subtropical communities (the warm community of the Gran Chaco and the cool community of Atlantic Forest, northern Argentina), as well as from one European temperate community. Upper thermal tolerances of tadpoles were positively correlated (controlling for phylogeny) with maximum pond temperatures, although the slope was steeper in subtropical than in temperate species. CT_max values were lowest in temperate species and highest in the subtropical warm community, which paradoxically, had very low warming tolerance (CT_max–T_max) and therefore may be prone to future local extinction from acute thermal stress if rising pond T_max soon exceeds their CT_max. Canopy‐protected subtropical cool species have larger warming tolerance and thus should be less impacted by peak temperatures. Temperate species are relatively secure to warming impacts, except for late breeders with low thermal tolerance, which may be exposed to physiological thermal stress in the coming years.     

CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment

Loss of spindle-pole integrity during mitosis leads to multipolarity independent of centrosome amplification. Multipolar-spindle conformation favours incorrect kinetochore-microtubule attachments, compromising faithful chromosome segregation and daughter-cell viability. Spindle-pole organization influences and is influenced by kinetochore activity, but the molecular nature behind this critical force balance is unknown. CLASPs are microtubule-, kinetochore- and centrosome-associated proteins whose functional perturbation leads to three main spindle abnormalities: monopolarity, short spindles and multipolarity. The first two reflect a role at the kinetochore-microtubule interface through interaction with specific kinetochore partners, but how CLASPs prevent spindle multipolarity remains unclear. Here we found that human CLASPs ensure spindle-pole integrity after bipolarization in response to CENP-E- and Kid-mediated forces from misaligned chromosomes. This function is independent of end-on kinetochore-microtubule attachments and involves the recruitment of ninein to residual pericentriolar satellites. Distinctively, multipolarity arising through this mechanism often persists through anaphase. We propose that CLASPs and ninein confer spindle-pole resistance to traction forces exerted during chromosome congression, thereby preventing irreversible spindle multipolarity and aneuploidy.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Differential interactions of the antimicrobial peptide,RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes

BackgroundAntimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus.MethodsA physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations.ResultsRQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces.ConclusionsRQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application.General significanceThese results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.     

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.     

The role of landscape change and paleoclimatic events in shaping the evolutionary history of the Polioptila gnatcatchers (Passeriformes,Polioptilidae) with emphasis on species associated with open habitats

We conducted a large‐scale phylogenetic and biogeographical inference of the Poliptila gnatcatchers and investigated the evolutionary history of two closely related neotropical bird species linked to open habitats, Polioptila dumicola and Polioptila plumbea. A Bayesian inference was employed based on the NADH subunit 2 gene to reconstruct the phylogenetic relationship of the gnatcatchers, and ancestral area reconstructions were estimated using BioGeoBEARS. For the phylogeographic analysis, we analyzed two mitochondrial genes, cytochrome b and ND2, of 102 individuals from P. dumicola and P. plumbea distributed throughout the complete range of both species. To reconstruct the dates related to the splitting events, we included a subset of sequences from the nuclear gene beta‐fibrinogen intron‐7. A striking result was the recovery of the sister relationship between the lineages of P. dumicola /plumbea and the paraphyly among the subspecies of P. plumbea: the first group was formed by P. dumicola, P. p. plumbea, P. p. parvirostris, P. p. atricapilla and P. lactea, occurring mainly on the Brazilian shield; while the second group consisted of lineages from north of the Amazon, west of the Andes, and Central America, and included P. maior, P. p. cinericia, P. p. bilineata and P. p. innotata. Significant phylogeographic structure was evident within lineages attributed to P. plumbea, with high levels of differentiation in the well‐defined clades according to all phylogenetic analyses. Our biogeographic analyses support distinct evolutionary histories related to founder events and vicariance, occurring during the late Pliocene and early Pleistocene. Several dispersal episodes between North/Central America and South America led to the establishment of populations which became differentiated due to landscape changes, such as the establishment of riverine barriers, the uplift of the Andes and the formation of the Panama Isthmus.     

Chromosome Segregation Is Biased by Kinetochore Size

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