Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Video activity extraction and reporting with incremental unsupervised learning

The present work presents a new method for activity extraction and reporting from video based on the aggregation of fuzzy relations. Trajectory clustering is first employed mainly to discover the points of entry and exit of mobiles appearing in the scene. In a second step, proximity relations between resulting clusters of detected mobiles and contextual elements from the scene are modeled employing fuzzy relations. These can then be aggregated employing typical soft-computing algebra. A clustering algorithm based on the transitive closure calculation of the fuzzy relations allows building the structure of the scene and characterises the ongoing different activities of the scene. Discovered activity zones can be reported as activity maps with different granularities thanks to the analysis of the transitive closure matrix. Taking advantage of the soft relation properties, activity zones and related activities can be labeled in a more human-like language. We present results obtained on real videos corresponding to apron monitoring in the Toulouse airport in France.

Puroindoline-a, a lipid binding protein from common wheat, spontaneously forms prolate protein micelles in solution

The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 +/- 4.5 A ˚ and minor axial radius of 40.4 +/- 0.18 A ˚ . These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5–11) and at elevated temperatures (20–65 degC). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-b-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a.

Simultaneous observations of reconnection pulses at Cluster and their effects on the cusp aurora observed at the Chinese Yellow River station

While the Cluster spacecraft were located near the high-latitude magnetopause, between 1010 and 1040 UT on 16 January 2004, three typical flux transfer event (FTE) signatures were observed. During this interval, simultaneous and conjugated all‐sky camera measurements, recorded at Yellow River Station, Svalbard, are available at 630.0 and 557.7 nm that show poleward‐moving auroral forms (PMAFs), consistent with magnetic reconnection at the dayside magnetopause. Simultaneous FTEs seen at the magnetopause mainly move northward, but having duskward (eastward) and tailward velocity components, roughly consistent with the observed direction of motion of the PMAFs in all‐sky images. Between the PMAFs meridional keograms, extracted from the all‐sky images, show intervals of lower intensity aurora which migrate equatorward just before the PMAFs intensify. This is strong evidence for an equatorward eroding and poleward moving open‐closed boundary associated with a variable magnetopause reconnection rate under variable IMF conditions. From the durations of the PMAFs, we infer that the evolution time of FTEs is 5–11 minutes from its origin on the magnetopause to its addition to the polar cap.

Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNAtargeted fluorescence in situ hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridised population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted (FACS) -recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz® method for the extraction of bacterial cells from soil.

Sudden onset stuttering in an adult: Neurogenic and psychogenic perspectives

A healthy 33 year old man with no previous history of speech language problems was referred to speech language therapy services following an episode which left him with a pronounced stutter, and which worsened over the next ten days. A range of neurological and psychological assessments failed to find any abnormality, as did MRI testing, and a diagnosis of psychogenic stuttering was made. This client was seen for three sessions of fluency therapy without significant improvement, after which he ceased attending. This paper considers the relationship between psychogenic and neurogenic stuttering generally, then more specifically in regard to this client, and the treatment he received. The paper concludes by considering problems in differentially diagnosing neurogenic from psychogenic stuttering.

Equidistributing Meshes with Constraints

Adaptive methods which “equidistribute” a given positive weight function are now used fairly widely for selecting discrete meshes. The disadvantage of such schemes is that the resulting mesh may not be smoothly varying. In this paper a technique is developed for equidistributing a function subject to constraints on the ratios of adjacent steps in the mesh. Given a weight function $f \geqq 0$ on an interval $[a,b]$ and constants $c$ and $K$, the method produces a mesh with points $x_0 = a,x_{j + 1} = x_j + h_j ,j = 0,1, \cdots ,n - 1$ and $x_n = b$ such that\[ \int_{xj}^{x_{j + 1} } {f \leqq c\quad {\text{and}}\quad \frac{1} {K}} \leqq \frac{{h_{j + 1} }} {{h_j }} \leqq K\quad {\text{for}}\, j = 0,1, \cdots ,n - 1 . \] A theoretical analysis of the procedure is presented, and numerical algorithms for implementing the method are given. Examples show that the procedure is effective in practice. Other types of constraints on equidistributing meshes are also discussed. The principal application of the procedure is to the solution of boundary value problems, where the weight function is generally some error indicator, and accuracy and convergence properties may depend on the smoothness of the mesh. Other practical applications include the regrading of statistical data.

The upper end of climate model temperature projections is inconsistent with past warming

Climate models predict a large range of possible future temperatures for a particular scenario of future emissions of greenhouse gases and other anthropogenic forcings of climate. Given that further warming in coming decades could threaten increasing risks of climatic disruption, it is important to determine whether model projections are consistent with temperature changes already observed. This can be achieved by quantifying the extent to which increases in well mixed greenhouse gases and changes in other anthropogenic and natural forcings have already altered temperature patterns around the globe. Here, for the first time, we combine multiple climate models into a single synthesized estimate of future warming rates consistent with past temperature changes. We show that the observed evolution of near-surface temperatures appears to indicate lower ranges (5–95%) for warming (0.35–0.82 K and 0.45–0.93 K by the 2020s (2020–9) relative to 1986–2005 under the RCP4.5 and 8.5 scenarios respectively) than the equivalent ranges projected by the CMIP5 climate models (0.48–1.00 K and 0.51–1.16 K respectively). Our results indicate that for each RCP the upper end of the range of CMIP5 climate model projections is inconsistent with past warming.