Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.     

A species-level total evidence phylogeny of the microteiid lizard family Alopoglossidae (Squamata: Gymnophthalmoidea)

Alopoglossidae is a family of Neotropical lizards composed of 23 species allocated in two genera (Alopoglossus and Ptychoglossus). There is a lack of knowledge about the phylogenetic relationships and systematics of this family. Published phylogenies that include alopoglossid species have very low taxon coverage within the family, and are usually based on limited character sampling. Considering these shortcomings, we infer the phylogenetic relationships of Alopoglossidae—including all but one species in the family—based on the combined analyses of DNA sequences and morphological characters. We use four loci (the mitochondrial 12S, 16S and ND4; the nuclear C-mos) and a matrix of 143 phenotypic characters from scutellation, tongue morphology, hemipenis morphology, and osteology. The dataset is analyzed with Maximum Parsimony, with four alternative weighting schemes: three under Extended Implied Weighting, and one with equal weighting. The respective resulting topologies are compared in a sensitivity analysis framework. Our analyses support the paraphyly of Ptychoglossus, with Alopoglossus nested within it. We provide an updated classification for the family, where Ptychoglossus Boulenger, 1890 is considered a junior synonym of Alopoglossus Boulenger, 1885.     

Assessing avian diversity and community composition along a successional gradient in traditional Lacandon Maya agroforests

Evidence regarding the ability of agroforests to conserve biological diversity has been mixed; they tend to maintain avian communities with species richness similar to that of undisturbed forest ecosystems but generally do not completely preserve community composition. Using a combination of occupancy modeling and non-metric multidimensional scaling on point-count data, we assessed changes in avian community diversity and composition along a successional gradient in traditional Lacandon Maya agroforests and compared them to protected areas in the region. Bird species richness and diversity in Lacandon agroforests peaked in early secondary forest stages. These agroforests' mean Shannon–Weiner diversity was 5% higher than that of nearby protected areas, but their species richness was similar. Community composition in Lacandon agroforests changed throughout succession, with earlier stages supporting communities distinctly characterized by generalist species, while subsequent, less-intensively managed stages tended to support more forest-dwellers. The bird community observed in even the most mature secondary forest stages in Lacandon agroforests differed from that of undisturbed rain forest ecosystems. These results demonstrate the potential of traditional Lacandon agroforestry management to conserve avian biodiversity while ensuring food sovereignty for farmers. However, because the community composition of early-successional stages was different than later stages, shortening fallow cycles and reducing forest cover to increase agricultural production will limit the species this system can support. This study illustrates the value of incorporating traditional agroecosystems into conservation planning as well as maintaining protected areas, because the latter serve as refugia for species that require undisturbed forest habitat in an agroecological matrix.     

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1? null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.     

Movement patterns of Antillean manatees in Chetumal Bay (Mexico) and coastal Belize: A challenge for regional conservation

Information from 15 satellite‐tracked Antillean manatees (Trichechus manatus manatus) was analyzed in order to assess individual movements, home ranges, and high‐use areas for conservation decisions. Manatees were captured in Chetumal Bay, Mexico, and tagged with Argos‐monitored satellite transmitters. Location of the manatees and physical characteristics were assessed to describe habitat properties. Most manatees traveled to freshwater sources. The Maximum Area Size (MAS) for each manatee was determined using the observation‐area method. Additional kernel densities of 95% home range and 50% Center of Activity (COA) were also calculated, with manatees having 1–3 COAs. Manatees exhibited two different movement patterns: remaining in Chetumal Bay, and long‐distance (up to 240 km in 89 d). The residence time in Chetumal Bay was higher for females (89.6% of time) than for males (72.0%), but the daily travel rate (0.4–0.5 km/d) was similar for both sexes. Most of the COAs fell within Natural Protected Areas (NPA). However, manatees also travel for long distances into unprotected areas, where they face uncontrolled boat traffic, fishing activities, and habitat loss. Conservation of movement corridors may promote long‐distance movements and facilitate genetic exchange.     

The Multiple-Minima Problem in Small Peptides Revisited. The Threshold Accepting Approach

Abstract

A recently reported optimization method, known as Threshold Accepting, was tested for the purpose of locating the structure of several peptide molecules with the lowest conformational energy. A comparison with previous results obtained with the Simulated Annealing technique was made. Our study indicate Threshold Accepting as a better technique in locating such structures.     

Contribution of Double-Stranded RNA and CPSF30 Binding Domains of Influenza Virus NS1 to the Inhibition of Type I Interferon Production and Activation of Human Dendritic Cells

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.     

A New Green Species of Frog,Genus Eleutherodactylus,from Bolivia and Perú (Amphibia,Anura, Leptodactylidae)

Eleutherodactylus olivaceus sp. n., a member of the E. unistrigatus group is described. The new species is known from montane rainforests of the Departamento Cochabamba, Bolivia, as well as from lowland rainforest of the Departamento Madre de Dios, Perú. E. olivaceus is mainly characterized by its predominantly olive green dorsal color and a papilla on the tip of the snout. Advertisement call and habitat of E. olivaceus are described. The occurrence of Eleutherodactylus mendax in Bolivia is briefly discussed.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.