Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.     

Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus

Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.     

Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.     

Nucleobase-containing peptides: an overview of their characteristic features and applications

Reports on nucleobase-containing chiral peptides (both natural and artificial) and achiral pseudopeptides are reviewed. Their synthesis, structural features, DNA and RNA-binding ability, as well as some other interesting applications which make them promising diagnostic/therapeutic agents of great importance in many areas of biology and therapy are taken into critical consideration.     

A novel functional role of iduronate-2-sulfatase in zebrafish early development

Sulfated glycosaminoglycan chains of extracellular matrix and cell membrane-tethered proteoglycans exert specific cellular functions by interacting with a broad spectrum of morphogens and growth factors.In humans, a congenital impaired catabolism of sulfated glycosaminoglycans is associated with severe metabolic disorders. Here, we report on the identification and characterization of a zebrafish iduronate sulfatase orthologue. By knocking down its function with antisense morpholino oligos, we demonstrate that iduronate sulfatase plays a critical role during early vertebrate development and its downregulation may be responsible for severe developmental defects, including a misshapen trunk and abnormal craniofacial cartilages. We show that the altered cartilage patterning is mediated by depauperation of sox10-expressing neural crest cell precursors. Through the application of a transactivation reporter assay, we also provide a molecular proof that increased TGFβ (Transforming Growth Factor β) signalling is tightly associated with downregulation of iduronate sulfatase function. Our results provide an insight into the early biological impairments underlying the Hunter syndrome and suggest the use of zebrafish as a novel tool to better understand lysosomal storage disorder pathogenesis.     

Addressing challenges in non invasive capture-recapture based estimates of small populations: a pilot study on the Apennine brown bear

It is often difficult to determine optimal sampling design for non-invasive genetic sampling, especially when dealing with rare or elusive species depleted of genetic diversity. To address this problem, we ran a hair-snag pilot study on the remnant Apennine brown bear population. We used occupancy models to estimate the performance of an improved field protocol, a meta-analysis approach to indirectly model capture probability, and simulations to evaluate the effect of genotyping errors on the accuracy of capture-recapture population estimates. In spring 2007 we collected 70 bear hair samples in 15 5 × 5 km cells, using 5 10-day trapping sessions. Bear detectability was higher in 2007 than in a previous attempt on the same population in 2004, reflecting improved field protocols and sampling design. However, individual capture probability was 0.136 (95% CI = 0.120–0.152), still below the minimum requirements of capture-mark-recapture closed population models. We genotyped hair samples (n = 63) at 9 microsatellite loci, obtaining 94% Polymerase Chain Reaction success, and 13 bear genotypes. Estimated P_IDsib was 0.00594, and per-genotype error rate was 0.13, corresponding to a 99% probability of correct individual identification. Simulation studies showed that the effect of non-corrected or filtered genetic errors on the accuracy of population estimates was negligible only when individual capture probability was >0.2. Our results underline how the interaction among field protocols, sampling strategies and genotyping errors may affect the accuracy of DNA-based estimates of small and genetically depleted populations, and warned us about the feasibility of a survey using only traditional hair-snag sampling. In this and similar cases, indications from pilot studies can provide cost-effective means to evaluate the efficiency of designed sampling and modelling procedures.     

Construction and validation of a yeast model system for studying in vivo the susceptibility to nucleoside analogues of DNA polymerase gamma allelic variants

Mitochondrial dysfunctions have been observed in subjects treated with antiretroviral nucleoside analogues, such as stavudine, as they can interfere with the activity of DNA polymerase gamma. Recently, stavudine-induced mitochondrial toxicity was associated to POLG mutations R964C and E1143G. A yeast model system useful to evaluate the association between D4T toxicity and mutations in MIP1, the yeast ortholog of POLG, was constructed and validated as a tool for pharmacogenetics research. We showed that mutant Mip1p^R964C and possibly Mip1p^E1143G are more sensitive to stavudine, and that stavudine has the potential to cause mitochondrial toxicity in heterozygous subjects harboring recessive mutations.     

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.     

Muscle mechanism: The acceleration of the load

The load (force/cross-section) determines the response of muscle power output, force and speed of contraction). The force is the product of the mass by the acceleration, thus the same force is generated by an infinite number of mass and acceleration couples and each one of these couples displays different physical and biological effects. Therefore, the load must be defined both by the mass and by the acceleration. Early muscle investigators were well aware of this situation as it is indicated by the work of Hill on the flexion of the arm against the “heavy fly-wheel”. By making use of a model of sarcomere contraction we show here that the acceleration of the load is the first determinant of the time course of the process of generation of the isometric tension. We also propose that, in order to reproduce the rapid release, it is not necessary to invoke the presence of a distinct elastic element in the contractile machinery. It is sufficient to assume that the stiffness of the same machinery increases with the contractile force.