Functional and comparative genome analysis of novel virulent actinophages belonging to Streptomyces flavovirens.

BACKGROUND: Next Generation Sequencing (NGS) technologies provide exciting possibilities for whole genome sequencing of a plethora of organisms including bacterial strains and phages, with many possible applications in research and diagnostics. No Streptomyces flavovirens phages have been sequenced to date; there is therefore a lack in available information about S. flavovirens phage genomics. We report biological and physiochemical features and use NGS to provide the complete annotated genomes for two new strains (Sf1 and Sf3) of the virulent phage Streptomyces flavovirens, isolated from Egyptian soil samples. RESULTS: The S. flavovirens phages (Sf1 and Sf3) examined in this study show higher adsorption rates (82 and 85%, respectively) than other actinophages, indicating a strong specificity to their host, and latent periods (15 and 30 min.), followed by rise periods of 45 and 30 min. As expected for actinophages, their burst sizes were 1.95 and 2.49 virions per mL. Both phages were stable and, as reported in previous experiments, showed a significant increase in their activity after sodium chloride (NaCl) and magnesium chloride (MgCl2.6H2O) treatments, whereas after zinc chloride (ZnCl2) application both phages showed a significant decrease in infection. The sequenced phage genomes are parts of a singleton cluster with sizes of 43,150 bp and 60,934 bp, respectively. Bioinformatics analyses and functional characterizations enabled the assignment of possible functions to 19 and 28 putative identified ORFs, which included phage structural proteins, lysis components and metabolic proteins. Thirty phams were identified in both phages, 10 (33.3%) of them with known function, which can be used in cluster prediction. Comparative genomic analysis revealed significant homology between the two phages, showing the highest hits among Sf1, Sf3 and the closest Streptomyces phage (VWB phages) in a specific 13Kb region. However, the phylogenetic analysis using the Major Capsid Protein (MCP) sequences highlighted that the isolated phages belong to the BG Streptomyces phage group but are clearly separated, representing a novel sub-cluster. CONCLUSION: The results of this study provide the first physiological and genomic information for S. flavovirens phages and will be useful for pharmaceutical industries based on S. flavovirens and future phage evolution studies.

Gut complex carbohydrates and intestinal microflora in broiler chickens fed with oregano (Origanum vulgare L.) aqueous extract and vitamin E.

One hundred and seventy one-day-old female broiler chicks were randomly divided into three groups fed with different dietary treatments: basal control diet (C); C supplemented (2 g/kg) with an oregano aqueous extract (O); C supplemented (150 mg/kg) with vitamin E (E). Growth performance was evaluated at 21 (T1) and 42 days (T2). On the same days, morphological, histochemical and microbiological analyses were performed. The O group showed the highest (p < 0.01) body weight at T1, while no differences were observed at T2. Light microscopic observation and conventional histochemistry showed no differences with regard to the two sampling times, whereas significant differences emerged among the treatments. The O treatment generally enhanced goblet cell reactivity more than both the C and E treatments. Coliform count was lower in the ileum tract of the O group at both T1 and T2 (p < 0.05) and increased with age in all groups. Escherichia coli showed the lowest values in the caecum of the O group (p < 0.001) at both sampling times. Enterococci, lactobacilli and staphylococci populations showed no differences among the different experimental groups in the caecum. In the ileum, the O group did not exhibit the sharp decline (p < 0.001) in the lactic acid bacteria population observed in the other two experimental groups. In conclusion, oregano aqueous extract supplementation seemed to elicit the best response among treatments, enabling better growth performance, enhancing both the quantity and quality of glycoconjugates involved in indirect defence actions and significantly reducing both the coliform and E. coli counts.

Genetic diversity and population structure of an Italian landrace of runner bean (Phaseolus coccineus L.): inferences for its safeguard and on-farm conservation.

The landraces are considered important sources of valuable germplasm for breeding activities to face climatic changes as well as to satisfy the requirement of new varieties for marginal areas. Runner bean (Phaseolus coccineus L.) is one of the most cultivated Phaseolus species worldwide, but few studies have been addressed to assess the genetic diversity and structure within and among landrace populations. In the present study, 20 different populations of a runner bean landrace from Central Italy named "Fagiolone," together with 41 accessions from Italy and Mesoamerica, were evaluated by using 14 nuclear SSRs to establish its genetic structure and distinctiveness. Results indicated that "Fagiolone" landrace can be considered as a dynamic evolving open-pollinated population that shows a significant level of genetic variation, mostly detected within populations, and the presence of two main genetic groups, of which one distinguished from other Italian runner bean landraces. Results highlighted also a relevant importance of farmers' management practices able to influence the genetic structure of this landrace, in particular the seed exchanges and selection, and the past introduction in cultivation of landraces/cultivars similar to seed morphology, but genetically rather far from "Fagiolone." The most suitable on-farm strategies for seed collection, conservation and multiplication will be defined based on our results, as a model for threatened populations of other allogamous crop species. STRUCTURE and phylogenetic analyses indicated that Mesoamerican accessions and Italian landraces belong to two distinct gene pools confirming the hypothesis that Europe could be considered a secondary diversification center for P. coccineus.

Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Immunohistochemical distribution of leptin receptor in the major salivary glands of horses.

The presence of the leptin receptor (ObR) has already been highlighted in the human major salivary glands and it has been hypothesized that leptin may act by regulating the gland's growth. No data are reported on domestic animals so, considering the important role that these glands play, not only related to food ingestion and digestion, and the important functional role hypothesized to explain the presence of ObR in humans salivary glands, the aim of the present work was to investigate the presence and the distribution of the leptin receptor in horse parotid and mandibular glands, by immunohistochemical techniques. The presence of ObR was evidenced in parotid and mandibular glands, exclusively localized in duct epithelial cells; their positivity was localized in the cytoplasm and was most evident near its apical portion. Immuno-positivity not only affects the intralobular ducts (intercalated and striated) but also the interlobular ones. Our results indicate that horse major salivary glands, like those of humans, are likely targets of leptin actions, suggesting a functional role of leptin on these glands.

Immunohistochemical detection of the orexin system in the placenta of cats.

The aim of the present study was to investigate the presence and distribution of cells containing orexin A (OXA), and orexin type 1 and 2 receptors (OX1R and OX2R, respectively) in the feline placenta by means of immunohistochemical technique. OXA was identified in several decidual and syncytiotrophoblastic cells present in the lamellar portion of the placenta. In the same placental structures, few decidual and syncytiotrophoblastic cells showed the presence of OX1R-like immunoreactivity. Characteristically, immunopositivity for OX2R, but not for OX1R, was evidenced in the cells of the glandular layer. The orexinic system was not expressed in the uterine structures that were not engaged by the chorion. Our results provide the first evidence of the presence of a placental orexinic system in a mammalian species. Orexin A and both OX1R and OX2R are unequally distributed within the cat placenta. Local OXA production and the presence of specific receptors, differentially expressed in the placental structures of the cat, suggest that the orexinic system may participate in placental growth and development as well as in the regulation of its steroidogenic capacity via endocrine, paracrine and/or autocrine mechanisms.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.

Immunohistochemical localization of CB1 receptor in canine salivary glands.

CB1 is a member of the G-protein-linked receptor superfamily that is present in the central nervous system as well as in certain peripheral neuronal and non-neuronal tissues. Recently, the presence of CB1 was found in the ductal system of the major salivary glands of laboratory animals, but no data are available for domestic mammals. Thus, in the present study, we examined the presence and distribution of CB1 in the major salivary glands of dogs using immunohistochemical techniques. CB1 was found in the parotid and mandibular glands of adult dogs; positive immunoreaction was localized to the cells of the striated ducts, with a peculiar localization on or near the apical membrane. This particular localization may be explained by the characteristics of this receptor as membrane-associated. The acinar structures were completely negative for CB1. We conclude that CB1 is involved in the control of dog salivary secretion via endogenous substances, likely endocannabinoids. The localization of CB1 highlights that endocannabinoids promote qualitative and/or quantitative changes of the primary saliva in the ductal system.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs.

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.

Intraluteal regulation of prostaglandin F2 alpha-induced prostaglandin biosynthesis in pseudopregnant rabbits.

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

[Genetic polymorphism of biotransforming enzymes and genotoxic effects of styrenes]. / Polimorfismi genetici degli enzimi della biotrasformazione ed effetti genotossici dello stirene.

A cross-sectional study was carried out on laminators producing glass-fibre reinforced plastics, to evaluate the role of genetic polymorphism of xenobiotic metabolising enzymes on the genotoxicity of styrene. Clastogenic effects, evaluated by the micronucleus test, are related with end-of-shift urinary concentration of 4-vinylphenol and seem to be modulated by NQO1 polymorphism; aneuploidogenic effects, evaluated by the identification of centromers in micronuclei using the fluorescence in situ hybridisation technique with a pancentromeric probe, are related with before-shift urinary levels of mandelic and phenylglyoxylic acids and seem to be modulated by the GSTM1 polymorphism.