706 research outputs found
Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs
Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen
Discordant Population Structure Among Rhizobium Divided Genomes and Their Legume Hosts
Symbiosis often occurs between partners with distinct life history characteristics and dispersal mechanisms. Many bacterial symbionts have genomes comprising multiple replicons with distinct rates of evolution and horizontal transmission. Such differences might drive differences in population structure between hosts and symbionts and among the elements of the divided genomes of bacterial symbionts. These differences might, in turn, shape the evolution of symbiotic interactions and bacterial evolution. Here we use whole genome resequencing of a hierarchically structured sample of 191 strains of Sinorhizobium meliloti collected from 21 locations in southern Europe to characterize population structures of this bacterial symbiont, which forms a root nodule symbiosis with the host plant Medicago truncatula. S. meliloti genomes showed high local (within-site) variation and little isolation by distance. This was particularly true for the two symbiosis elements, pSymA and pSymB, which have population structures that are similar to each other, but distinct from both the bacterial chromosome and the host plant. Given limited recombination on the chromosome, compared to the symbiosis elements, distinct population structures may result from differences in effective gene flow. Alternatively, positive or purifying selection, with little recombination, may explain distinct geographical patterns at the chromosome. Discordant population structure between hosts and symbionts indicates that geographically and genetically distinct host populations in different parts of the range might interact with genetically similar symbionts, potentially minimizing local specialization
N-Palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids
Article discussing how the N-palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids
Combining GWAS and Population Genomic Analyses to Characterize Coevolution in a Legume-rhizobia Symbiosis
The mutualism between legumes and rhizobia is clearly the product of past coevolution. However, the nature of ongoing evolution between these partners is less clear. To characterize the nature of recent coevolution between legumes and rhizobia, we used population genomic analysis to characterize selection on functionally annotated symbiosis genes as well as on symbiosis gene candidates identified through a two-species association analysis. For the association analysis, we inoculated each of 202 accessions of the legume host Medicago truncatula with a community of 88 Sinorhizobia (Ensifer) meliloti strains. Multistrain inoculation, which better reflects the ecological reality of rhizobial selection in nature than single-strain inoculation, allows strains to compete for nodulation opportunities and host resources and for hosts to preferentially form nodules and provide resources to some strains. We found extensive host by symbiont, that is, genotype-by-genotype, effects on rhizobial fitness and some annotated rhizobial genes bear signatures of recent positive selection. However, neither genes responsible for this variation nor annotated host symbiosis genes are enriched for signatures of either positive or balancing selection. This result suggests that stabilizing selection dominates selection acting on symbiotic traits and that variation in these traits is under mutation-selection balance. Consistent with the lack of positive selection acting on host genes, we found that among-host variation in growth was similar whether plants were grown with rhizobia or N-fertilizer, suggesting that the symbiosis may not be a major driver of variation in plant growth in multistrain contexts
Annotation of two large contiguous regions from the Haemonchus contortus genome using RNA-seq and comparative analysis with Caenorhabditis elegans
The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes
Search for Nucleon Decays induced by GUT Magnetic Monopoles with the MACRO Experiment
The interaction of a Grand Unification Magnetic Monopole with a nucleon can
lead to a barion-number violating process in which the nucleon decays into a
lepton and one or more mesons (catalysis of nucleon decay). In this paper we
report an experimental study of the effects of a catalysis process in the MACRO
detector. Using a dedicated analysis we obtain new magnetic monopole (MM) flux
upper limits at the level of for
, based on the search for
catalysis events in the MACRO data. We also analyze the dependence of the MM
flux limit on the catalysis cross section.Comment: 12 pages, Latex, 10 figures and 2 Table
Final results of magnetic monopole searches with the MACRO experiment
We present the final results obtained by the MACRO experiment in the search
for GUT magnetic monopoles in the penetrating cosmic radiation, for the range
. Several searches with all the MACRO sub-detectors
(i.e. scintillation counters, limited streamer tubes and nuclear track
detectors) were performed, both in stand alone and combined ways. No candidates
were detected and a 90% Confidence Level (C.L.) upper limit to the local
magnetic monopole flux was set at the level of cm
s sr. This result is the first experimental limit obtained in
direct searches which is well below the Parker bound in the whole range
in which GUT magnetic monopoles are expected.Comment: 12 pages, Latex, 9 figures and 2 Table
A combined analysis technique for the search for fast magnetic monopoles with the MACRO detector
We describe a search method for fast moving ()
magnetic monopoles using simultaneously the scintillator, streamer tube and
track-etch subdetectors of the MACRO apparatus. The first two subdetectors are
used primarily for the identification of candidates while the track-etch one is
used as the final tool for their rejection or confirmation. Using this
technique, a first sample of more than two years of data has been analyzed
without any evidence of a magnetic monopole. We set a 90% CL upper limit to the
local monopole flux of in the
velocity range and for nucleon decay
catalysis cross section smaller than .Comment: 29 pages (12 figures). Accepted by Astroparticle Physic
Muon Energy Estimate Through Multiple Scattering with the Macro Detector
Muon energy measurement represents an important issue for any experiment
addressing neutrino induced upgoing muon studies. Since the neutrino
oscillation probability depends on the neutrino energy, a measurement of the
muon energy adds an important piece of information concerning the neutrino
system. We show in this paper how the MACRO limited streamer tube system can be
operated in drift mode by using the TDC's included in the QTPs, an electronics
designed for magnetic monopole search. An improvement of the space resolution
is obtained, through an analysis of the multiple scattering of muon tracks as
they pass through our detector. This information can be used further to obtain
an estimate of the energy of muons crossing the detector. Here we present the
results of two dedicated tests, performed at CERN PS-T9 and SPS-X7 beam lines,
to provide a full check of the electronics and to exploit the feasibility of
such a multiple scattering analysis. We show that by using a neural network
approach, we are able to reconstruct the muon energy for 40 GeV. The
test beam data provide an absolute energy calibration, which allows us to apply
this method to MACRO data.Comment: 25 pages, 11 figures, Submitted to Nucl. Instr. & Meth.
The Observation of Up-going Charged Particles Produced by High Energy Muons in Underground Detectors
An experimental study of the production of up-going charged particles in
inelastic interactions of down-going underground muons is reported, using data
obtained from the MACRO detector at the Gran Sasso Laboratory. In a sample of
12.2 10^6 single muons, corresponding to a detector livetime of 1.55 y, 243
events are observed having an up-going particle associated with a down-going
muon. These events are analysed to determine the range and emission angle
distributions of the up-going particle, corrected for detection and
reconstruction efficiency. Measurements of the muon neutrino flux by
underground detectors are often based on the observation of through-going and
stopping muons produced in interactions in the rock below the
detector. Up-going particles produced by an undetected down-going muon are a
potential background source in these measurements. The implications of this
background for neutrino studies using MACRO are discussed.Comment: 18 pages, 9 figures. Accepted by Astrop. Physic
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