Effect of the concentration of protein and nanoparticles on the structure of biohybrid nanocomposites

We present colloidal nanocomposites formed by incorporating magnetite Fe₃O₄ nanoparticles (MNPs) with lysozyme amyloid fibrils (LAFs). Preparation of two types of solutions, with and without addition of salt, was carried out to elucidate the structure of MNPs-incorporated fibrillary nanocomposites and to study the effect of the presence of salt on the stability of the nanocomposites. The structural morphology of the LAFs and their interaction with MNPs were analyzed by atomic force microscopy and small-angle x-ray scattering measurements. The results indicate that conformational properties of the fibrils are dependent on the concentration of protein, and the precise ratio of the concentration of the protein and MNPs is crucially important for the stability of the fibrillary nanocomposites. Our results confirm that despite the change in fibrillary morphology induced by the varying concentration of the protein, the adsorption of MNPs on the surface of LAF is morphologically independent. Moreover, most importantly, the samples containing salt have excellent stability for up to 1 year of shelf-life.     

Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698^T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.     

Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

Mitochondrial DNA and Y-chromosomal diversity in ancient populations of domestic sheep (Ovis aries) in Finland: comparison with contemporary sheep breeds

Background

Several molecular and population genetic studies have focused on the native sheep breeds of Finland. In this work, we investigated their ancestral sheep populations from Iron Age, Medieval and Post-Medieval periods by sequencing a partial mitochondrial DNA D-loop and the 5’-promoter region of the SRY gene. We compared the maternal (mitochondrial DNA haplotypes) and paternal (SNP oY1) genetic diversity of ancient sheep in Finland with modern domestic sheep populations in Europe and Asia to study temporal changes in genetic variation and affinities between ancient and modern populations.

Results

A 523-bp mitochondrial DNA sequence was successfully amplified for 26 of 36 sheep ancient samples i.e. five, seven and 14 samples representative of Iron Age, Medieval and Post-Medieval sheep, respectively. Genetic diversity was analyzed within the cohorts. This ancient dataset was compared with present-day data consisting of 94 animals from 10 contemporary European breeds and with GenBank DNA sequence data to carry out a haplotype sharing analysis. Among the 18 ancient mitochondrial DNA haplotypes identified, 14 were present in the modern breeds. Ancient haplotypes were assigned to the highly divergent ovine haplogroups A and B, haplogroup B being the major lineage within the cohorts. Only two haplotypes were detected in the Iron Age samples, while the genetic diversity of the Medieval and Post-Medieval cohorts was higher. For three of the ancient DNA samples, Y-chromosome SRY gene sequences were amplified indicating that they originated from rams. The SRY gene of these three ancient ram samples contained SNP G-oY1, which is frequent in modern north-European sheep breeds.

Conclusions

Our study did not reveal any sign of major population replacement of native sheep in Finland since the Iron Age. Variations in the availability of archaeological remains may explain differences in genetic diversity estimates and patterns within the cohorts rather than demographic events that occurred in the past. Our ancient DNA results fit well with the genetic context of domestic sheep as determined by analyses of modern north-European sheep breeds.     

Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis

Background

Fungal rhinosinusitis has become an increasingly recognized disease, being Aspergillus species responsible for most of the cases. Its diagnosis is quite difficult because of the non-specific symptoms and low sensitivity of the current diagnostic methods.

Comparison of sensory structures on the antenna of different species of Philopotamidae (Insecta: Trichoptera)

Structure and distribution of sensilla were studied in sixteen species of the caddisfly family Philopotamidae. Their antennae bear numerous curved trichoid and pseudoplacoid sensilla and fewer coronal, styloconic and chaetoid sensilla on the flagellar segments. The most numerous pseudoplacoid sensilla have non-specific localization. The curved trichoid sensilla form clusters ventrally on each antennal segment. Sensilla belonging to coronal, styloconic and chaetoid types have specific positions. Long grooved trichoid sensilla are located nonspecifically in all the studied species. The average number of sensilla per segment decreases from the proximal to distal part of the flagellum. Scapus and pedicellum are devoid of most types of sensilla, however, they bear the Böhm bristles and long trichoid sensilla. A positive correlation between antenna dimensions and its cuticular structures is found.     

Effect of Geographic Origin and Ex Situ Growing Site on Phenology,Morphology, and Seed Yield of Yarrow (Achillea millefolium L.) Germplasm from the Rhaetian Alps,Italy

Species adapted to prevailing soil and climatic conditions and native to the same geographic context are increasingly recommended for ecological restoration at high altitude. Better knowledge is required on the level of variation within these species for morpho‐physiological traits and seed yield. Adequate and affordable seed production is a prerequisite for native species to be widely adopted for restoration interventions. This study evaluated the variation of yarrow (Achillea millefolium L.) germplasm collected in the Rhaetian Alps, Italy. The worth of yarrow for restoration at high altitude has been repeatedly noted. The main goal of the study was to identify promising materials for selection purposes. The explored mountain section harbored valuable variation for traits of interest. Altitude, climate, and soil should frame an ecological correspondence between the collection site and the target restoration site, to secure the value of the native germplasm outside the boundaries of its collection area. The work also assessed any interaction between the germplasm and the ex situ growing environments, represented by a mountain and a lowland site, and verified the feasibility of the latter for seed multiplication. The growing site remarkably affected several morpho‐physiological characters. The lack of difference in mean seed yield between the two altitude‐contrasting sites suggested that seed multiplication of yarrow could also be conveniently carried out in lowland environments, with advantages in terms of production costs. Nonetheless, different yield responses in the two sites were observed, emphasizing the need of choosing the material to multiply based on the environment adopted for seed production.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.