Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors

We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent.     

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V_max of 3.9 ± 0.2 μmol/min/mg and K _m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.     

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomic tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.     

On the evolution of protamines in bony Fish: Alternatives to the “Retroviral horizontal transmission” hypothesis

Fish protamines are highly specialized molecules which are responsible for chromatin condensation during the last stages of spermatogenesis (spermiogenesis). However, not all fish contain protamines in their sperm nuclei; rather, there seems to be a random distribution of protamines within this group. The origin of this sporadic presence of protamines in the sperm and its significance have not yet been precisely determined. In this paper we have conducted an exhaustive survey of the literature available on the different types of nuclear protein composition of the sperm of teleost fish in order to try to correlate these data with what is presently known about the taxonomy of this group. The results of this analysis have allowed us to make the following observations. The divergence between protamines and histones has occurred several times during the evolution of the bony fish. However, the relative frequency of this divergence is almost negligible during the differentiation of genera and species (intrafamily variation) and is very small during the differentiation of families (interfamily variation). Nevertheless, the divergence is very noticeable among the different orders. It is therefore possible to conclude from all this that the sporadic distribution of protamines in bony fish is not a random event as initially believed. Furthermore, such a heterogeneous distribution of protamines cannot be easily accounted for by a mechanism of horizontal retroviral transmission through repeated and independent acquisition of a prot amine gene as has been recently proposed (Jankowski, Stater, Dixon (1986) J Mol Evol 23:1–10). Rather, it could possibly be explained by a repeated and independent loss of the expression of the protamine gene (or loss of the gene itself) which mainly occurred during the diversification of the orders of this group.Correspondence to: J. Ausio     

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5–L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

ROI-Based Compression Strategy of 3D MRI Brain Datasets for Wireless Communications

ObjectivesBecause of the large amount of medical imaging data, the transmission process becomes complicated in telemedicine applications. Thus, in order to adapt the data bit streams to the constraints related to the limitation of the bandwidths a reduction of the size of the data by compression of the images is essential. Despite the improvements in the field of compression, the transmission itself can also introduce errors. For this reason, it is important to develop an adequate strategy which will help reduce this volume of data without having to introduce some distortion and resist the errors introduced by the channel noise during transmission. Thus, in this paper, we propose a ROI-based coding strategy and unequal bit stream protection to meet this dual constraint.Material and methodsThe proposed ROI-based compression strategy with unequal bit stream protection is composed of three parts: the first one allows the extraction of the ROI region, the second one consists of a ROI-based coding and the third one allows an unequal protection of the ROI bit stream.First, the Regions Of Interest (ROI) are extracted by hierarchical segmentation of these regions according to a segmentation method based on the technique of Marker-based-watershed combined with the technique of active contours by level set. The resulting regions are selectively encoded by a 3D coder based on a shape adaptive discrete wavelet transform 3D-BISK, where the compression ratio of each region depends on its relevance in diagnosis. These obtained regions of interest are protected with an error-correcting code of Reed-Solomon type with a code rate that varies according to the relevance of the region by an unequal protection strategy (UEP).ResultsThe performance of the proposed compression scheme is evaluated in several ways. First, tests are performed to study the impact of errors on the different bit streams. In the first place, these tests are carried out in order to study the effect of the variation of the compression rates on the different bit streams. Secondly, different Reed Solomon error-correcting codes of different code rates are tested at different compression rates on a BSC channel. Finally, the performances of this coding strategy are compared with those of SPIHT 3D in the case of transmission on a BSC channel.ConclusionThe obtained results show that the proposed method is quite efficient in transmission time reduction. Therefore, our proposed scheme will reduce the volume of data without having to introduce some distortion and resist the errors introduced by the channel noise in the case of telemedicine.     

尿蛋白定性与定量检测结果的差异性和相关性分析

目的：探讨尿液干化学法及免疫透射比浊法检测尿白蛋白结果的差异性及相关性。方法：对514例住院患者随机尿标本进行尿液干化学法及免疫透射比浊法尿蛋白的检测。结果：尿液干化学法阳性率为82．1％,免疫透射比浊法阳性率为72．8％。两种方法检测结果均为阴性标本的符合率为98．9％,为（±）的标本二者符合率为69．7％,为㈩的标本二者符合率为75．6％,为（＋＋）的标本二者符合率为67．2％,为（卅）标本中二者符合率为42．5％,为（＋＋H）标本二者的符合率为37．5％。两种方法的检测结果有显著性差异（P〈O．05）;UmAlb／Ucr、NAG、和NAG／Ucr与UmAlb具有显著相关（P〈0．05）,且UmAlb／Ucr与UmAlb的相关性最高。两种方法所得等级结果比较,＋＋～卅之间差异有统计学意义（P〈0．05）,-～±、±～＋、＋～＋＋、＋＋＋～＋＋＋＋之间差异均无统计学意义（P〉0．05）。结论：尿蛋白定性与定量检测结果存在显著性差异,而UmAlb／Ucr与UmAlb相关性较高。在泌尿系统疾病的诊断中,检测尿中UmAlb比尿常规更有意义。