Molecular analysis of fungal diversity associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica

The fungal communities associated with three bryophytes species (the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata) in the Fildes Region, King George Island, maritime Antarctica, were studied using clone library analysis. Fungal communities showed low diversity; the 680 clones belonged to 93 OTUs. Of these, 78 belonged to the phylum Ascomycota, 13 to the phylum Basidiomycota, 1 to the phylum Zygomycota, and 1 to an unknown phylum. Among the OTUs, the most common orders in the Ascomycota were Helotiales (42 OTUs) and Chaetothyriales (14 OTUs) and the most common orders in the Basidiomycota were Sebacinales (3 OTUs) and Platygloeales (3 OTUs). Most OTUs clustered within clades that contained phylotypes identified from samples in Antarctic or Arctic ecosystems or from bryophytes in other ecosystems. In addition, we found that host-related factor may shape the fungal communities associated with bryophytes in this region. This is the first systematic study of the fungal community in Antarctic bryophytes to be performed using culture-independent method and the results may improve understanding of the endophytic fungal evolution and ecology in the Antarctic ecosystem.     

Nitrate signals determine the sensing of nitrogen through differential expression of genes involved in nitrogen uptake and assimilation in finger millet

In order to understand the molecular basis of high nitrogen use efficiency of finger millet, five genes (EcHNRT2, EcLNRT1, EcNADH-NR, EcGS, and EcFd-GOGAT) involved in nitrate uptake and assimilation were isolated using conserved primer approaches. Expression profiles of these five genes along with the previously isolated EcDof1 was studied under increased KNO₃ concentrations (0.15 to 1,500 μM) for 2 h as well as at 1.5 μM for 24 h in the roots and shoots of 25 days old nitrogen deprived two contrasting finger millet genotypes (GE-3885 and GE-1437) differing in grain protein content (13.76 and 6.15 %, respectively). Time kinetics experiment revealed that, all the five genes except EcHNRT2 in the leaves of GE-3885 were induced within 30 min of nitrate exposure indicating that there might be a greater nitrogen deficit in leaves and therefore quick transportation of nitrate signals to the leaves. Exposing the plants to increasing nitrate concentrations for 2 h showed that in roots of GE-3885, NR was strongly induced while GS was repressed; however, the pattern was found to be reversed in leaves of GE-1437 indicating that in GE-3885, most of the nitrate might be reduced in the roots but assimilated in leaves and vice-versa. Furthermore, compared with the low-protein genotype, expression of HNRT2 was strongly induced in both roots and shoots of high-protein genotype at the least nitrate concentration supplied. This further indicates that GE-3885 is a quick sensor of nitrogen compared with the low-protein genotype. Furthermore, expression of EcDof1 was also found to overlap the expression of NR, GS, and GOGAT indicating that Dof1 probably regulates the expression of these genes under different conditions by sensing the nitrogen fluctuations around the root zone.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.     

Ve1‐mediated resistance against Verticillium does not involve a hypersensitive response in Arabidopsis

The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co‐expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co‐expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1‐induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance.     

Mesenchymal stem cell sheet transplantation combined with locally released simvastatin enhances bone formation in a rat tibia osteotomy model

Nonunion of fractured bones is a common clinical problem for orthopedic surgeons. This study aimed to investigate the effects of simvastatin locally applied from calcium sulfate (CS) combined with a mesenchymal stem cell (MSC) sheet on fracture healing. In vitro, the proliferation and differentiation of rat bone marrow–derived MSCs stimulated by simvastatin were investigated. In vivo, an osteotomy model was made in rat tibia, and fractured tibias were treated with CS, CS/simvastatin, CS/MSC sheet or simvastatin-loaded CS with MSC or untreated (control). Tibias were harvested at 2 or 8 weeks and underwent real-time quantitative polymerase chain reaction, x-ray, micro-CT and histological analysis. The expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor of simvastatin-induced MSCs increased with the concentrations of the simvastatin, significantly higher than those in the MSCs group. At 2 weeks, the CS/simvastatin/MSC sheet group showed significantly higher expressions of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor, with more callus formation around the fracture site compared with the other four groups. At 8 weeks, complete bone union was obtained in the CS/simvastatin/MSC sheet group. By contrast, newly regenerated bone tissue partially bridged the gap in the CS/simvastatin group and the CS/MSC sheet group; the control and CS group showed nonunion of the tibia. These results show that both simvastatin and the MSC sheet contributed to the formation of new bone and that the tibia fracture was completely healed by transplantation of the MSC sheet with locally applied simvastatin. Such MSC sheet with locally applied simvastatin might contribute to the treatment of fractures, bone delayed unions or nonunions in clinical practice.     

DDIG-in: discriminating between disease-associated and neutral non-frameshifting micro-indels

Micro-indels (insertions or deletions shorter than 21 bps) constitute the second most frequent class of human gene mutation after single nucleotide variants. Despite the relative abundance of non-frameshifting indels, their damaging effect on protein structure and function has gone largely unstudied. We have developed a support vector machine-based method named DDIG-in (Detecting disease-causing genetic variations due to indels) to prioritize non-frameshifting indels by comparing disease-associated mutations with putatively neutral mutations from the 1,000 Genomes Project. The final model gives good discrimination for indels and is robust against annotation errors. A webserver implementing DDIG-in is available at http://sparks-lab.org/ddig.     

Expression and role of Toll-like receptors on human umbilical cord mesenchymal stromal cells

Background aimsToll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs).MethodsIn the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined.ResultsAt the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon β, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs.ConclusionsTaken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs.