Electronmicroscopical detection of iota-Carrageenan as its ruthenium red complex

Summary Specific detection of iota-Carrageenan (i-CAR) at the ultrastructural level has been obtained by coupling with ruthenium red (RR) — an electron microscopic stain. The i-CAR-RR complex showed electron density on carbon layers. Peritoneal macrophages were treated with the complex and after 3 h it caused the same morphological changes in macrophages as iota-Carrageenan alone. On the surfaces of macrophages, fine filamentous electron dense material — the i-CAR-RR complex — was detected.     

Localization,transmission, spontaneous mutations,and variation of function of the Dpp4 (Dipeptidyl-peptidase IV; CD26) gene in rats

Dipeptidyl-peptidase IV (DPPIV) is involved in endocrine and immune functions via cleavage of regulatory peptides with a N-terminal proline or alanine such as incretins, neuropeptide Y, or several chemokines. So far no systematic investigations on the localization and transmission of the Dpp4 gene or the natural variations of DPPIV-like enzymatic function in different rat strains have been conducted. Here we mapped the Dpp4 gene to rat chromosome 3 and describe a semi-dominant mode of inheritance for Dpp4 in a mutant F344/DuCrj(DPPIV-) rat substrain lacking endogenous DPPIV-like activity. This mutant F344/DuCrj(DPPIV-) rat substrain constantly exhibits a nearly complete lack of DPPIV-like enzymatic activity, while segregation of DPPIV-like enzymatic activity was observed in another DPPIV-negative F344/Crl(Ger/DPPIV-) rat substrain. Screening of 12 different inbred laboratory rat strains revealed dramatic differences in DPPIV-like activity ranging from 11 mU/microl (LEW/Ztm rats) to 40 mU/microl (BN/Ztm and DA/Ztm rats). A lack of DPPIV-like activity in F344 rats was associated with an improved glucose tolerance and blunted natural killer cell function, which indicates the pleiotropic functional role of DPPIV in vivo. Overall, the variations in DPPIV-like enzymatic activity probably represent important confounding factors in studies using rat models for research on regulatory peptides.     

The effect of the dominant grass <Emphasis Type="Italic">Festuca rupicola</Emphasis> on the establishment of rare forbs in semi-dry grasslands

Establishment is the most sensitive process in the life-cycle of plant species, and each stage – from germination to survival and growth – can be affected by environmental factors and plant traits. We hypothesized that the dominant tussock grass Festuca rupicola negatively affects forb establishment in semi-dry grasslands where it has recently expanded into. Moreover, we expected that seedling recruitment is affected by grass density and seed size, with larger seeded forbs being more successful in areas of higher grass density. In a garden experiment, we sowed seven forb individuals of differing seed size (smallest to largest: Veronica spicata < Dianthus carthusianorum < Plantago media < Biscutella laevigata < Filipendula vulgaris < Scabiosa ochroleuca < Sanguisorba minor) into pots with zero, one, two and three grass individuals, and assessed germination, survival and growth over one year. As expected, increasing grass density negatively affected germination, survival and growth of forbs; however, contrary to our expectation, seed size did not influence any of parameters measured. The response of each individual species varied from zero or weak to strong with respect to grass density. Festuca rupicola therefore acts as a strong competitor against the establishment of forbs, irrespective of their seed size, and its spread lowers species diversity in semi-dry grasslands.     

The application of GIS and spatiotemporal analyses to investigations of unusual marine mammal strandings and mortality events

In 2006–2007, an unusually high number of harbor porpoises (Phocoena phocoena) stranded along the Washington and Oregon coastlines. Spatiotemporal analyses were used to examine their ability to detect clusters of porpoise strandings during an unusual mortality event (UME) in the Pacific Northwest using stranding location data. Strandings were evaluated as two separate populations, outer coast and inland waters. The presence of global clustering was evaluated using the Knox spatiotemporal test, and the presence of local clusters was investigated using a spatiotemporal scan statistic (space–time permutation). There was evidence of global clustering, but no local clustering, supporting the hypothesis that strandings were due to more varied etiologies instead of localized causes. Further analyses at subregional levels, and concurrently assessing environmental factors, might reveal additional geographic distribution patterns. This article describes the spatial analytical tools applied in this study and how they can help elucidate the spatiotemporal epidemiology of other UMEs and assist in determining their causes. More than one spatial analytical technique should be used if the study objective is to detect and describe clustering in time and space and to generate hypotheses regarding causation of marine mammal disease and stranding events.     

The Cockroach Origin of the Termite Gut Microbiota: Patterns in Bacterial Community Structure Reflect Major Evolutionary Events

Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).     

Species-Specific Viromes in the Ancestral Holobiont Hydra

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.     

Scanning X-Ray Nanodiffraction on Dictyostelium discoideum

We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions.     

Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a M_r of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The M_r of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.     

Patent and Exclusivity Status of Essential Medicines for Non-Communicable Disease

Objective

The threat of non-communicable diseases (“NCDs”) is increasingly becoming a global health crisis and are pervasive in high, middle, and low-income populations resulting in an estimated 36 million deaths per year. There is a need to assess intellectual property rights (“IPRs”) that may impede generic production and availability and affordability to essential NCD medicines.

Methods

Using the data sources listed below, the study design systematically eliminated NCD drugs that had no patent/exclusivity provisions on API, dosage, or administration route. The first step identified essential medicines that treat certain high disease burden NCDs. A second step examined the patent and exclusivity status of active ingredient, dosage and listed route of administration using exclusion criteria outlined in this study.

Materials

We examined the patent and exclusivity status of medicines listed in the World Health Organization’s (“WHO”) Model List of Essential Drugs (Medicines) (“MLEM”) and other WHO sources for drugs treating certain NCDs. i.e., cardiovascular and respiratory disease, cancers, and diabetes. We utilized the USA Food and Drug Administration Orange Book and the USA Patent and Trademark Office databases as references given the predominant number of medicines registered in the USA.

Results

Of the 359 MLEM medicines identified, 22% (79/359) address targeted NCDs. Of these 79, only eight required in-depth patent or exclusivity assessment. Upon further review, no NCD MLEM medicines had study patent or exclusivity protection for reviewed criteria.

Conclusions

We find that ensuring availability and affordability of potential generic formulations of NCD MLEM medicines appears to be more complex than the presence of IPRs with API, dosage, or administration patent or exclusivity protection. Hence, more sophisticated analysis of NCD barriers to generic availability and affordability should be conducted in order to ensure equitable access to global populations for these essential medicines.