Coprophilous fungi of the horse

A total of 1267 microfungi, including 35 Myxomycetes, were recorded from the fecal samples of the 60 horses; of these 395 were found on 20 saddle-horse feces, 363 on 20 race-horses and 509 on 20 working-horses. Eighty two species representing 53 genera were recorded; of these 7 were Zygomycetes, 18 Ascomycetes, 1 Basidiomycetes and 25 Fungi Imperfecti: 2 Myxomycetes. Common coprophilous fungi are in decreasing orderPilobolus kleinii, Saccobolus depauperatus, Mucor hiemalis, Lasiobolus ciliatus, Podospora curvula, Petriella guttulata, M. circinelloides, Coprinus radiatus, Dictyostelium mucoroides, Sordaria fimicola, C. miser, C. stercorarius, Acremonium sp., Coprotus granuliformis, Graphium putredinis, Iodophanus carneus, Chaetomium murorum, Podospora communis, P. inaequalis, P. setosa, Saccobolus versicolor andCladosporium cucumerinum. Species ofMyrothecium verrucaria, Actinomucor elegans, Kernia nitida, Spiculostilbella dendritica andMucorparvispora were found exclusively in working-horses feces.Badhamia sp., Anixiopsis stercoraria, Echinobotryum state ofD. stemonitis, Geotrichum candidum andOidiodendron sp. were found only in saddle-horses feces.Chlamidomyces palmarum andPhilocopra sp. were found exclusively in race-horses feces.Notes on infrequent or interesting fungi includeThamnostylum piriforme, Phialocephala dimorphospora, Rhopalomyces elegans andSpiculostilbella dendritica.     

In search of AKT kinase inhibitors as anticancer agents: structure-based design,docking, and molecular dynamics studies of 2,4,6-trisubstituted pyridines

The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian, and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435, and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.     

The molecular basis of venom resistance in a rattlesnake‐squirrel predator‐prey system

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long‐sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins (“venom interactive proteins” [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum‐based resistance is both population‐ and species‐specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum “resistome” involves broad‐based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co‐evolutionary interactions between species at the molecular level.     

Interactions among Triatoma sanguisuga blood feeding sources,gut microbiota and Trypanosoma cruzi diversity in southern Louisiana

Integrating how biodiversity and infectious disease dynamics are linked at multiple levels and scales is highly challenging. Chagas disease is a vector‐borne disease, with specificities of the triatomine vectors and Trypanosoma cruzi parasite life histories resulting in a complex multihost and multistrain life cycle. Here, we tested the hypothesis that T. cruzi transmission cycles are shaped by triatomine host communities and gut microbiota composition by comparing the integrated interactions of Triatoma sanguisuga in southern Louisiana with feeding hosts, T. cruzi parasite and bacterial microbiota in two habitats. Bugs were collected from resident's houses and animal shelters and analysed for genetic structure, blood feeding sources, T. cruzi parasites, and bacterial diversity by PCR amplification of specific DNA markers followed by next‐generation sequencing, in an integrative metabarcoding approach. T. sanguisuga feeding host communities appeared opportunistic and defined by host abundance in each habitat, yielding distinct parasite transmission networks among hosts. The circulation of a large diversity of T. cruzi DTUs was also detected, with TcII and TcV detected for the first time in triatomines in the US. The bacterial microbiota was highly diverse and varied significantly according to the DTU infecting the bugs, indicating specific interactions among them in the gut. Expanding such studies to multiple habitats and additional triatomine species would be key to further refine our understanding of the complex life cycles of multihost, multistrain parasites such as T. cruzi, and may lead to improved disease control strategies.     

Focus: Zoonotic Disease: Zoonotic Ocular Onchocercosis by Onchocerca lupi

The parasitic filarioid Onchocerca lupi causes ocular disease characterized by conjunctivitis and nodular lesions. This nematode was first described in 1967 in a wolf from Georgia, and since then cases of infection from dogs and cats with ocular onchocercosis and sporadically from humans also with subcutaneous and cervical lesions caused by O. lupi have been reported from the Middle East, Europe, and North America. Due to its zoonotic potential, this parasitic infection has gained attention in the past 20 years. Phylogenetic studies have highlighted the recent divergence of O. lupi from other Onchocerca spp. and the importance of domestication in the evolutionary history of this worm. Moreover, the finding of an O. lupi genotype associated with subclinical and mild infection in the Iberian Peninsula, raises important questions about the pathogenicity of this presently enigmatic parasite.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.     

Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells

We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A(2). The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A(2) activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis.     

Developmental patterns of larval growth in the edible spider crab, Maja brachydactyla (Decapoda: Majidae)

The large edible spider crab Maja brachydactyla Balss, 1922, an overexploited coastal fishery resource in Galicia (NW Spain), is considered as a potential aquaculture candidate. Patterns of its larval growth were studied under controlled laboratory conditions (constant 18 ± 1 °C; 36‰ salinity; photoperiod ca. 12:12 h; lipid-enriched Artemia metanauplii provided as food). From hatching through complete larval development and metamorphosis to the first juvenile crab instar, changes in carapace size, dry weight (DW), ash content, elemental composition (carbon, hydrogen, nitrogen; CHN), and proximate biochemical composition (total proteins, lipids, carbohydrates; Pr, L, Ch) were measured in successive stages (zoea I, II, megalopa, crab I). Body size may be described as a linear function of the number of molting cycles, whereas the amounts of DW, CHN, Pr, L, and Ch per individual increased exponentially (3 to 9 fold). The highest growth rates were observed in L, C and H, the lowest in DW, Pr and N. As a consequence of these patterns, the C:N mass ratio as well as the fractions of L, C and H (in % of DW) increased significantly, while those of Pr and N decreased from 26% to 16% of DW. Throughout development, however, Pr remained the principal biochemical component of total DW. Positive correlations between biochemical and CHN data allow for estimates of Pr from N and of L from C values per individual. The patterns of larval growth observed in M. brachydactyla are, in general, similar to those previously described for other brachyuran crabs with a planktotrophic mode of larval development.     

DNA damage response in microcephaly development of MCPH1 mouse model

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV–VI), while generate less late-born neurons that form a thinner outer layer (II–III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.