The Pathway of Assembly of Intermediate Filaments from Recombinant α-Internexin

The pathway of filament assembly from the neuronal intermediate filament α-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37°C. Short filaments formed upon dialysis at 24°C, which elongated further when incubated at 37°C. Soluble forms of α-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, α-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first “unit-length half-filaments” (Herrmann, H., and Aebi, U. (1998)Curr. Opin. Struct. Biol.8, 177–185) and then “unit-length filaments,” which subsequently elongate by annealing.     

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-¹³C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained ¹³C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (¹³C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Assessment of Environmental Hazards to Public Health in Temperate Urban Argentina

EcoHealth - Human health risk in urban areas depends on multiple environmental features. We performed a year-round survey in a highly urbanized district located in temperate Argentina (General San...     

Simplifier: a web tool to eliminate redundant NGS contigs

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms.

Availability

Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.     

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun‐protected and sun‐exposed body sites

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun‐protected) and photoaged (sun‐exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun‐protected and sun‐exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun‐exposed‐derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.     

Autosomal and mtDNA Markers Affirm the Distinctiveness of Lions in West and Central Africa

The evolutionary history of a species is key for understanding the taxonomy and for the design of effective management strategies for species conservation. The knowledge about the phylogenetic position of the lion (Panthera leo) in West/Central Africa is largely based on mitochondrial markers. Previous studies using mtDNA only have shown this region to hold a distinct evolutionary lineage. In addition, anthropogenic factors have led to a strong decline in West/Central African lion numbers, thus, the conservation value of these populations is particularly high. Here, we investigate whether autosomal markers are concordant with previously described phylogeographic patterns, and confirm the unique position of the West/Central African lion. Analysis of 20 microsatellites and 1,454 bp of the mitochondrial DNA in 16 lion populations representing the entire geographic range of the species found congruence in both types of markers, identifying four clusters: 1) West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India. This is not in line with the current taxonomy, as defined by the IUCN, which only recognizes an African and an Asiatic subspecies. There are no indications that genetic diversity in West/Central Africa lions is lower than in either East or Southern Africa, however, given this genetic distinction and the recent declines of lion numbers in this region, we strongly recommend prioritization of conservation projects in West/Central Africa. As the current taxonomic nomenclature does not reflect the evolutionary history of the lion, we suggest that a taxonomic revision of the lion is warranted.     

Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.