Diversity of Environmental Mycobacterium Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

The strength of migratory connectivity for birds en route to breeding through the Gulf of Mexico

The strength of migratory connectivity is a measure of the cohesion of populations among phases of the annual cycle, including breeding, migration, and wintering. Many Nearctic‐Neotropical species have strong migratory connectivity between breeding and wintering phases of the annual cycle. It is less clear if this strength persists during migration when multiple endogenous and exogenous factors may decrease the cohesion of populations among routes or through time along the same routes. We sampled three bird species, American redstart Setophaga ruticilla, ovenbird Seiurus aurocapilla, and wood thrush Hylocichla mustelina, during spring migration through the Gulf of Mexico region to test if breeding populations differentiate spatially among migration routes or temporally along the same migration routes and the extent to which within‐population timing is a function of sex, age, and carry‐over from winter habitat, as measured by stable carbon isotope values in claws (δ¹³C). To make quantitative comparisons of migratory connectivity possible, we developed and used new methodology to estimate the strength of migratory connectivity (MC) from probabilistic origin assignments identified using stable hydrogen isotopes in feathers (δ²H). We found support for spatial differentiation among routes by American redstarts and ovenbirds and temporal differentiation along routes by American redstarts. After controlling for breeding origin, the timing of American redstart migration differed among ages and sexes and ovenbird migration timing was influenced by carry‐over from winter habitat. The strength of migratory connectivity did not differ among the three species, with each showing weak breeding‐to‐spring migration MC relative to prior assessments of breeding‐wintering connectivity. Our work begins to fill an essential gap in methodology and understanding of the extent to which populations remain together during migration, information critical for a full annual cycle perspective on the population dynamics and conservation of migratory animals.     

Contrasting the distribution of butterflies and termites in plantations and tropical forests

In the tropics vast areas of natural forests are being converted into plantations. The magnitude of the resulting loss in arthropod biodiversity and associated ecosystem services represents a significant topic of research. In this study we contrasted the abundance, species richness and faunal turnover of butterflies, resident butterflies (i.e., whose host plants were ascertained to occur in the habitats studied) and termites between small (average 4.3 ha) 20+ year old exotic plantations (teak and Terminalia), native plantations (Cedro espino), and an old growth forest in Panama. We used Pollard walks and manual search to quantify the abundance or occurrence of butterflies and termites, respectively. In 2014 we observed 4610 butterflies representing 266 species and 108 termite encounters (out of 160 quadrats) representing 15 species. Butterflies were more abundant and diverse in plantations than in the forest, whereas this pattern was opposite for resident butterflies and termites. There was marked faunal turnover between plantations and forest. We conclude that (a) the magnitude of faunal changes between forest and plantations is less drastic for termites than for butterflies; (b) resident butterfly species are more impacted by the conversion of forest to plantations than all butterflies, including transient species; and (c) species richness does not necessarily decrease in the series forest > native > exotic plantations. Whereas there are advantages of studying more tractable taxa such as butterflies, the responses of such taxa can be highly unrepresentative of other invertebrate groups responsible for different ecological services.     

Generation and characterization of recombinant human antibodies specific for native laminin epitopes: potential application in cancer therapy

Laminins are specific cellular regulators that directly and indirectly control activities such as cell attachment and migration, differentiation and polarity, proliferation and apoptosis, and protease expression. Considering the centrality of these issues to tumor progression, the generation of human-derived antibody fragments able to modulate laminin-regulated biological functions would allow the development of new strategies to improve treatment of cancer patients. In this report, we explore the use of phage display technology to isolate human anti-laminin antibody fragments. A library of single chain antibodies was selected using intact mouse laminin, and five different clones were identified. All the antibodies were specific for their cognate antigen, as revealed by lack of cross-reactivity with other components of the basement membranes. A more extensive characterization of the panel indicated that these antibodies recognize the native protein through conformational epitopes. All of them reduced tumor cell attachment to laminin, suggesting that domains of the laminin molecule that are recognized by these antibodies likely bind to cell-surface receptors. The antibody fragments bind to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications.     

IL-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against HIV-1 Env in a dose-dependent manner.

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.     

The binding of trivalent chromium to low-molecular-weight chromium-binding substance (LMWCr) and the transfer of chromium from transferrin and chromium picolinate to LMWCr

A recent model for the role of chromium in insulin signaling requires that the oligopeptide low-molecular-weight chromium-binding substance (LMWCr) tightly bind four chromic ions before the oligopeptide obtains a conformation required for binding to the tyrosine kinase active site of the insulin receptor. To test this model, the chromium-binding constant of LMWCr was determined, and the ability of LMWCr to remove chromium from Cr2-transferrin and the nutritional supplement chromium picolinate, Cr(pic)3, was examined. These results are consistent with the model of the mode of action of LMWCr; a Hill study indicates the four chromic ions bind to apoLMWCr in a highly cooperative fashion (n =3.47) with a binding constant of 1.54x 10(21). Chromium is readily transferred from transferrin to apoLMWCr at near neutral pH. The results also suggest that reduction of the chromic center of Cr(pic)3 may be required for the supplement to release chromium; thus, release of chromium is related to a mechanism by which Cr(pic)3 may generate hydroxyl radicals in cells.     

Assay of 3-hydroxybutyrate in the picomolar range

A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.