295 research outputs found
Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis
<p>Abstract</p> <p>Background</p> <p>Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium <it>Bacillus subtilis </it>has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.</p> <p>Findings</p> <p>While observing the recruitment of the transcription machinery into the forespore of sporulating <it>Bacillus subtilis</it>, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.</p> <p>Conclusions</p> <p>Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.</p
D* Production in Deep Inelastic Scattering at HERA
This paper presents measurements of D^{*\pm} production in deep inelastic
scattering from collisions between 27.5 GeV positrons and 820 GeV protons. The
data have been taken with the ZEUS detector at HERA. The decay channel
(+ c.c.) has been used in the study. The
cross section for inclusive D^{*\pm} production with
and is 5.3 \pms 1.0 \pms 0.8 nb in the kinematic region
{ GeV and }. Differential cross
sections as functions of p_T(D^{*\pm}), and are
compared with next-to-leading order QCD calculations based on the photon-gluon
fusion production mechanism. After an extrapolation of the cross section to the
full kinematic region in p_T(D^{*\pm}) and (D^{*\pm}), the charm
contribution to the proton structure function is
determined for Bjorken between 2 10 and 5 10.Comment: 17 pages including 4 figure
Plastisol Foaming Process. Decomposition of the Foaming Agent, Polymer Behavior in the Corresponding Temperature Range and Resulting Foam Properties
The decomposition of azodicarbonamide, used as foaming agent in PVC - plasticizer (1/1) plastisols was studied by DSC. Nineteen different plasticizers, all belonging to the ester family, two being polymeric (polyadipates), were compared. The temperature of maximum decomposition rate (in anisothermal regime at 5 K min-1 scanning rate), ranges between 434 and 452 K. The heat of decomposition ranges between 8.7 and 12.5 J g -1. Some trends of variation of these parameters appear significant and are discussed in terms of solvent (matrix) and viscosity effects on the decomposition reactions. The shear modulus at 1 Hz frequency was determined at the temperature of maximum rate of foaming agent decomposition, and differs significantly from a sample to another. The foam density was determined at ambient temperature and the volume fraction of bubbles was used as criterion to judge the efficiency of the foaming process. The results reveal the existence of an optimal shear modulus of the order of 2 kPa that corresponds roughly to plasticizer molar masses of the order of 450 ± 50 g mol-1. Heavier plasticizers, especially polymeric ones are too difficult to deform. Lighter plasticizers such as diethyl phthalate (DEP) deform too easily and presumably facilitate bubble collapse
Observation of Scaling Violations in Scaled Momentum Distributions at HERA
Charged particle production has been measured in deep inelastic scattering
(DIS) events over a large range of and using the ZEUS detector. The
evolution of the scaled momentum, , with in the range 10 to 1280
, has been investigated in the current fragmentation region of the Breit
frame. The results show clear evidence, in a single experiment, for scaling
violations in scaled momenta as a function of .Comment: 21 pages including 4 figures, to be published in Physics Letters B.
Two references adde
Observation of hard scattering in photoproduction events with a large rapidity gap at HERA
Events with a large rapidity gap and total transverse energy greater than 5
GeV have been observed in quasi-real photoproduction at HERA with the ZEUS
detector. The distribution of these events as a function of the
centre of mass energy is consistent with diffractive scattering. For total
transverse energies above 12 GeV, the hadronic final states show predominantly
a two-jet structure with each jet having a transverse energy greater than 4
GeV. For the two-jet events, little energy flow is found outside the jets. This
observation is consistent with the hard scattering of a quasi-real photon with
a colourless object in the proton.Comment: 19 pages, latex, 4 figures appended as uuencoded fil
Measurement of Jet Shapes in Photoproduction at HERA
The shape of jets produced in quasi-real photon-proton collisions at
centre-of-mass energies in the range GeV has been measured using the
hadronic energy flow. The measurement was done with the ZEUS detector at HERA.
Jets are identified using a cone algorithm in the plane with a
cone radius of one unit. Measured jet shapes both in inclusive jet and dijet
production with transverse energies GeV are presented. The jet
shape broadens as the jet pseudorapidity () increases and narrows
as increases. In dijet photoproduction, the jet shapes have been
measured separately for samples dominated by resolved and by direct processes.
Leading-logarithm parton-shower Monte Carlo calculations of resolved and direct
processes describe well the measured jet shapes except for the inclusive
production of jets with high and low . The observed
broadening of the jet shape as increases is consistent with the
predicted increase in the fraction of final state gluon jets.Comment: 29 pages including 9 figure
Measurement of the diffractive structure function in deep inelastic scattering at HERA
This paper presents an analysis of the inclusive properties of diffractive
deep inelastic scattering events produced in interactions at HERA. The
events are characterised by a rapidity gap between the outgoing proton system
and the remaining hadronic system. Inclusive distributions are presented and
compared with Monte Carlo models for diffractive processes. The data are
consistent with models where the pomeron structure function has a hard and a
soft contribution. The diffractive structure function is measured as a function
of \xpom, the momentum fraction lost by the proton, of , the momentum
fraction of the struck quark with respect to \xpom, and of . The \xpom
dependence is consistent with the form \xpoma where
in all bins of and
. In the measured range, the diffractive structure function
approximately scales with at fixed . In an Ingelman-Schlein type
model, where commonly used pomeron flux factor normalisations are assumed, it
is found that the quarks within the pomeron do not saturate the momentum sum
rule.Comment: 36 pages, latex, 11 figures appended as uuencoded fil
A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores
Background
Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile.
Methods
Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity.
Results
Deletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy.
Conclusions
We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen
Long-Baseline Neutrino Facility (LBNF) and Deep Underground Neutrino Experiment (DUNE) Conceptual Design Report Volume 2: The Physics Program for DUNE at LBNF
The Physics Program for the Deep Underground Neutrino Experiment (DUNE) at
the Fermilab Long-Baseline Neutrino Facility (LBNF) is described
Reduced Expression of Brain-Enriched microRNAs in Glioblastomas Permits Targeted Regulation of a Cell Death Gene
Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs) made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK) gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers
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