To systematically review the literature on human gene expression data of placental tissue in pre-eclampsia and to characterize a meta-signature of differentially expressed genes in order to identify novel putative diagnostic markers.Medline through 11 February 2011 using MeSH terms and keywords related to placenta, gene expression and gene expression arrays; GEO database using the term “placent*”; and reference lists of eligible primary studies, without constraints.From 1068 studies retrieved from the search, we included original publications that had performed gene expression array analyses of placental tissue in the third trimester and that reported on differentially expressed genes in pre-eclampsia versus normotensive controls. Two reviewers independently identified eligible studies, extracted descriptive and gene expression data and assessed study quality. Using a vote-counting method based on a comparative meta-profiling algorithm, we determined a meta-signature that characterizes the significant intersection of differentially expressed genes from the collection of independent gene signatures.rd trimester comprising 30 datasets on mRNA expression and 4 datasets on microRNA expression. The pre-eclamptic placental meta-signature consisted of 40 annotated gene transcripts and 17 microRNAs. At least half of the mRNA transcripts encode a protein that is secreted from the cell and could potentially serve as a biomarker.We identified 33 eligible gene expression array studies of placental tissue in the 3st trimester placenta, and three encode a secreted protein.In addition to well-known and validated genes, we identified 14 transcripts not reported previously in relation to pre-eclampsia of which the majority is also expressed in the 1 Pre-eclampsia, a pregnancy-specific disorder characterized by the development of de novo hypertension and proteinuria during the second half of gestation, is a major obstetric problem that contributes substantially to maternal and perinatal morbidity and mortality worldwide The exact mechanisms involved in the development of pre-eclampsia are not well defined, but it is generally accepted that the pathogenesis of pre-eclampsia initiates at the time of trophoblast invasion and remodeling of the spiral arteries during the first and early second trimester of pregnancy. Impaired invasion of myometrial spiral arteries relates to abnormal placentation, which in turn results in the release of various components from the intervillous space into the maternal circulation. Consequently, exaggerated maternal endothelial activation and a generalized hyper-inflammatory state precede the onset of clinical symptoms of pre-eclampsia Gene expression array analysis is an important and powerful tool to identify genes differentially expressed between tissues from normotensive and pre-eclamptic pregnancies. Some key targets involved in the pathogenesis of pre-eclampsia, such as Fms-like tyrosine kinase 1 (FLT1) and Endoglin (ENG) were discovered using this approach The aim of this study was to systematically review the literature on array-based gene-expression studies of placental tissues in pre-eclampsia in order to determine a common expression signature and identify novel putative diagnostic leads.This systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines. We performed an electronic search on 11 February, 2011 in Medline (from inception) through PubMed and GEO (Gene Expression Omnibus) without language or publication date restrictions to identify all articles reporting on differentially expressed genes in human placental samples relating to pre-eclampsia.The electronic search strategy for Medline was based on MeSH terms and keywords related to placenta, gene expression and gene expression arrays. Eligible were those original publications that had performed gene expression array analyses of human placental tissue in the third trimester and that reported on differentially expressed genes in pregnancies that had pre-eclampsia compared to normotensive controls. Only studies presenting their results as a gene signature  were included.First, two reviewers (MM and GA) independently scrutinized titles and abstracts of all retrieved references to select potentially eligible articles. Full text papers of references selected by at least one reviewer were obtained. Second, two or three reviewers  independently examined these full text papers to see whether they met the inclusion criteria. In case of multiple studies of the same set of women we included only the most recent or most complete one. Disagreements about inclusion were resolved by consensus.For each study, data on clinical characteristics of included women , diagnostic criteria for pre-eclampsia, characteristics of the gene expression array experiment , details of the statistical analysis, the gene signature and other experimental results were extracted independently by two experienced reviewers (EK and MM) using standardized data extraction forms.As there are no formal quality assessment approaches for this type of study, we assessed study quality by discussing reported details of sampling, testing and analysis processes among four of the authors . Attention was paid to the results presented and whether the reported list of differentially expressed genes had been subject to additional selection, such that not the whole list was reported.Acceptable diagnostic criteria for pre-eclampsia were persistent high systolic (≥140 mm Hg) or diastolic (≥90 mm Hg) blood pressure combined with proteinuria  of new onset after 20 weeks of gestation, according to the International Society for the Study of Hypertension in Pregnancy criteria From each included paper we extracted the published gene signature and selected the official gene symbol as gene identifier. If the gene symbol was not given, we mapped the other gene information  to the official gene symbol using the NCBI Gene database. We checked for each symbol whether this was an official symbol or an alias or both. If the symbol was not an official symbol but only an alias, we used the other gene information to obtain the official symbol.et al.On two occasions, multiple papers were published on the same set of placental samples. In four papers by Pang and Xing Step 1: For each gene mentioned, we counted how often it appeared in a signature and ordered the genes by number of appearance. This way we obtained , with Nx: the number of genes that appear x times in a signature, and S: the total number of signatures. Step 2: We created a random signature as follows. For each signature, the number of genes remained the same as in the original signature, but the gene symbols were replaced by symbols randomly drawn from a (selected) list of 7000 official gene symbols, representing the intersection of all genes targeted by the whole genome platforms included in the meta-analysis of which the NCBI GeneID is publicly available. For the microRNA data we took a set of 854 publicly available annotated microRNAs. For these random signatures we repeated Step 1, and obtained , with Ex: the number of genes that appear x times in a random signature. Step 3: Step 2 was repeated 1000 times. Step 4: For each i = 1, …, S, we computed the empirical probability distribution function for Ei+…+ES. Then we determined the significance of the observed number of genes present in at least i signatures, Ni+…+NS. The associated p-value is the probability of Ei+…+ES ≥ Ni+…+NS, reflecting the probability that the number of genes present in at least i random signatures is at least as large as the observed number. If there was an i such that the p-value was <0.05, then a significant meta-signature was found. If there were multiple i for which the p-value was <0.05, we took the smallest i. The genes forming the meta-signature were listed.Using all gene signatures presented in the included papers, we determined a significant meta-signature using a vote counting approach based on a modified version of the so-called comparative meta-profiling method We performed the analyses twice: once on those studies that presented the complete list of all genes identified as differentially expressed, and once on all studies, thereby also including studies that reported an incomplete  gene signature. All analyses were performed using R version 2.14.0 .Functional interaction networks of proteins encoded by the genes in the meta-signature were visualized using STRING 9.05 The results of the search and the selection process are shown in et al., in total 26 gene signatures were included in the analysis. A gene had to be present in 3 or more of these 26 signatures in order to be part of the significant meta-signature (p<0.001). This way, 40 of 1343 genes were incorporated in the meta-signature, in which a large majority of genes was up-regulated in pre-eclampsia . According to this criterion, 17 of the 840 genes present in at least one signature were incorporated in the meta-signature. Most genes were up-regulated in pre-eclampsia compared to normotensive placentas . The metclampsia .The 4 studies on third-trimester placental tissue microRNA expression represented 39 pre-eclampsia samples and 38 control samples. A gene had to be present in 2 or more signatures in order to be part of the significant meta-signature (p = 0.001) resulting in a meta-signature consisting of 17 out of 127 miRNAs .Using high confidence scores based on experimental data and associations in curated protein databases and text mining, 15 of the 40 signature genes could be mapped into an interaction network . SubsequThe placenta is the key organ involved in pre-eclampsia. Identification of genes differentially expressed between normotensive and pre-eclamptic placentas is essential to understand the molecular mechanisms involved in the etiology of the disease and to develop novel biomarkers or identify therapeutic targets. This systematic review provides an overview of placental tissue gene expression array studies in pre-eclampsia. It is the first study that extracts a meta-signature from these individual studies consisting of up-regulated and down-regulated genes in pre-eclampsia as identified in multiple publications.When all 30 studies generating signatures on placental tissue mRNA expression are included, the pre-eclamptic placental meta-signature consists of 40 genes. As authors reporting an incomplete list of genes may be driven by bias towards the genes they list (for instance only specific genes of the authors’ particular interest), we also generated a meta-signature from the 14 complete gene-lists by which we obtained a meta-signature of 17 genes. As the ratio of the number of times a gene is present in a signature compared to the total number of datasets is similar for both meta-signatures we conclude that the inclusion bias does not affect the overall meta-signature very strongly.Both meta-signatures have identical well-known and validated genes on top of their list. The relation of the angiogenesis-related cluster FLT1, VEGFA, and PlGF to pre-eclampsia has been extensively documented. Members of this cluster are used as biomarkers and have been shown to play a functional role in pre-eclampsia In both the meta-signature derived from complete and incomplete signatures, the most frequently occurring genes are only mentioned in approximately one third of the individual signatures. Variation in technical aspects, as reported in et al. There was good overall agreement on the direction of differential gene expression among studies. An exception seems to be the data of Mayor-Lynn Apart from transcripts previously related to pre-eclampsia, the meta-signature includes 14 genes for which no information with regard to pre-eclampsia has been reported. They encode structural proteins , plasma membrane proteins with a channel/transporter or receptor function , proteins involved in immunomodulation , a mitochondrial protein (RDH13), a nuclear protein (BHLHE40) and cytosolic proteins . With the exception of VIM, the expression of all these genes is up-regulated in pre-eclamptic placenta.st trimester placental mRNA expression Interestingly, 3 of these 14 genes encode a secreted protein that might be detectable in the maternal circulation. However, as the placenta deports large amounts of cellular material into the maternal circulation, non-secreted proteins should not be excluded as candidate biomarkers Due to the limited number of available studies, meta-analysis of the microRNA expression studies resulted in a less robust meta-signature. In particular the direction (up/down) of the microRNA expression showed a high level of ambiguity between studies. As microRNA expression is considered a regulatory layer for silencing gene expression predominantly by degradation of their target mRNAs Meta-analysis of raw whole genome expression data would partly overcome the methodological variation encountered in this paper and should be undertaken to validate and extend the novel leads with respect to diagnosis and putative therapeutic targets identified here.In addition, the combined analysis of microRNA and mRNA meta-signatures is a further necessary step to comprehend the overall deviating transcription profile of the pre-eclamptic placenta.Table S1Clinical data: characteristics of all studies and women included in this systematic review and meta-analysis, as they were reported in the original papers. Data on maternal age, parity and gestational age at delivery are reported as mean (standard deviation) or median (range), unless indicated otherwise. FGR: fetal growth restriction; IUGR: intra uterine growth restriction; PE: pre-eclampsia.(DOCX)Click here for additional data file.Table S2Experimental data: details of the sampling, testing and analysis processes of all studies, as they were reported in the original papers. ANOVA: analysis of variance; PE: pre-eclampsia. a: A complete list is defined as the complete list of genes identified as differentially expressed according to the studies’ selection criteria.(DOCX)Click here for additional data file.Table S3Meta signature of third-trimester placental tissue mRNA expression. Genes present in 3 or more complete signatures, or 3 or more signatures in total, are part of the meta-signature. Genes incorporated in the meta-signature are ordered by (1) the total number of publications that reported the gene as differentially expressed and (2) alphabetically. References printed in bold indicate publications that reported a complete signature. Enquobahrie 2008, Kang 2011, Pang (combined) and Vaiman 2005 did not report the direction of differential expression (upregulation or downregulation). For comparison, the number of times a gene is present in complete signatures below the significance threshold is shown in grey. *: Results were inconsistent among studies, the majority of studies reported the result presented here. –: No consensus among studies: equal numbers of studies reported upregulated expression and downregulated expression in pre-eclampsia. 1st trimester expression was scored using microarray data from control chorionic villous sampling (NCBI Gene Expression Omnibus GSE12767) and 1st trimester placenta samples (NCBI Gene Expression Omnibus GSE9984). Microarrays were analyzed for expression using The Gene Expression Barcode 2.0 (http://barcode.luhs.org/). ‘+’ indicates that in >90% of the samples expression of the gene was detected, ‘–’ indicates that in <10% of the samples expression of the gene was detected. The remaining samples showed a variable degree of expression (‘+/−’).(DOCX)Click here for additional data file.Document S1Electronic search strategy PubMed.(DOC)Click here for additional data file.Document S2Studies that were excluded after screening of full text and new studies that were included, as described in .(DOC)Click here for additional data file.Document S3PRISMA 2009 Checklist.(DOC)Click here for additional data file.


Following recommendations made by the Scottish Intensive Care Society , we haveWe performed a study to assess Family Satisfaction over a 10-week period in a 16-bed critical care unit (CCU). A modified version of an FS-ICU published in US and Canadian studies  was distWe received and analysed 32 completed surveys. Seventy-eight per cent of relatives reported that the treatment of the patient was excellent, including symptom control such as pain, breathlessness and agitation . Ninety-one per cent felt the care and frequency of communication provided by nurses was excellent or very good, compared with 81% by doctors. Only 9% of family members were offered spiritual support during their CCU experience. Forty-seven per cent felt they would have liked more involvement in the decision-making process.The vast majority of relatives rated their overall experience on CCU as excellent or very good. The study highlighted two main areas for improvement: provision of spiritual support as well as family and patient involvement in the decision-making process. We will arrange multi-disciplinary teaching sessions focusing on the positive impact, and therefore importance of patient satisfaction. A poster providing information about available spiritual support will be displayed around our CCU. Further studies are required to evaluate these measures.


Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi.Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in  Myosins, molecular motors that move cargo along actin filaments, occur in all eukaryotic organisms. Based on sequence homology, the myosin super family can be divided into 37 classes  appear to label the nuclear envelope in epidermal cells Figures . Even thHigh levels of expression are often achieved following agroinfiltration. When a myosin tail domain lacking the motor function is expressed, a dominant negative effect on motility can sometimes be detected. However, when we examined the vesicles labeled by the 11 YFP::At PAL sub-domains, we found that rapid motility was retained, in contrast to our prior observation of stationary vesicles following agroinfiltration of YFP::At DIL domain fusions. Motility is documented in Figures We have observed that 42-amino acid portions of 12 Arabidopsis myosin XI tail regions (the PAL sub-domain) are targeted to different subcellular organelles and membranes. Previously Li and Nebenfuhr  expresseA. thaliana class XI myosins are quite similar, there are some sequence differences in this 42 amino acid region of individual tail domains that could explain the different subcellular localizations . The primers used are listed in Table Generation of the YFP::myosin constructs was performed as described  as described . Structure homology models for the tail domain of class XI mysoins were built by SWISSMODEL (Arnold et al., GeneDoc was used for generation of alignments (Nicholas and Nicholas, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NOS are large modular enzymes present in all kingdoms which through evolution were the result of multiple gene and genome duplication events together with changes in protein architecture  circulating in the troposphere and the earth surface during the last century, correlating with the population increase. As an undesirable consequence, high levels of reactive N are polluting the environment where humans inhabit. Nitric oxide (NO) is one of the reactive N species with both positive and negative impact on life. NO synthases (NOSs) are enzymes that oxidize arginine to citrulline and generate the denitrifying intermediate NO which can be subsequently reduced to N4-Cys motif involved in Zn coordination seems to be conserved in all animal NOS described so far  were the first NOS structures to be biochemically characterized, crystallized and their complete structure deciphered by X-ray diffraction. These NOSs are functional as homodimers, each monomer consisting on an N-terminal oxygenase domain (NOSoxy) containing the binding sites for the cofactors heme, tetrahydrobiopterin (BH4) and for the substrate L-arginine, and a C-terminal reductase domain (NOSred) that binds to NADPH, FAD and FMN Figure . Mammalir Figure . The NOSr Figure . Particur Figure . The isor Figure .Ostreococcus tauri represents the first NOS characterized in photosynthetic organisms (plant kingdom)   required for NOS activity  resulted in a different and novel NOS structure. ScNOS is the only characterized bacterial NOS with a covalently attached reductase domain (NOSred). This reductase module has a 2Fe2S ferredoxin domain, a FAD- binding motif and a NAD-binding motif. Interestingly, scNOS has an inverted structure: the NOSred domain is located at the N-terminal and NOSoxy at the C-terminal , although it still remains to be confirmed the NOS activity of these proteins.Most bacterial NOSs have been described in Gram-positive bacteria and consist of the NOSoxy domain lacking of the C-terminal NOSred domain Figure . NOSoxy The lack of apparent NOS in the plants which are most commonly worked on has led to suggest that either plants have lost this gene in the course of evolution or the gene has strongly diverged to a yet unknown new type of NOS. Since several evidences support an arginine-dependent NO production in higher plants reminiscent of a NOS activity (Cueto et al., Overall, the unexpected diversity of the NOS structures that are currently reported in the literature should allow us to keep optimistic for identifying the NOS gene/s or protein/s complex responsible of NO generation from L-arginine in higher plants.


Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr).The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in  Drosophila consists of two cell types, the somatic gonadal precursor cells (SGPs) and the primordial germ cells (PGCs). The SGPs are specified by zygotic genes that pattern the mesoderm during mid-embryogenesis and sufficient to program the newly formed pole cells to assume and then maintain a distinct PGC identity The embryonic gonad of Drosophila PGCs are refractory to the effects of somatic signaling molecules during early embryogenesis has now been widely accepted, there is, in fact, evidence that these cells have the machinery needed to perceive somatic signaling molecules. One of these signaling molecules is the Bone Morphogenetic Protein (BMP) Decapentaplegic (Dpp). Studies by Dorfman and Shilo dpp. Further supporting the conclusion that newly formed PGCs express and utilize the canonical BMP signal transduction machinery, Dorfman and Shilo thickveins had to be mutant in both the mother’s germline and in the zygote in order to block pMad accumulation in PGC nuclei.Although the idea that Of course, the fact that PGCs are able to perceive BMP signals from the nearby soma does not mean that they are capable of responding to this signal or, if they do, that the response is important for PGC development. In fact, since the PGCs are transcriptionally quiescent, a plausible assumption has been that this nuclear pMad is simply inert, unable to exert any regulatory effects on its target genes. On the other hand, in the adult gonad, BMP signaling from the soma plays a critical role in the self-renewing properties of the descendents of the PGCs, the germline stem cells (GSCs). The GSCs reside at the tip of the adult male and female gonad in a special somatic microenvironment called the niche lesswrightThe fact that BMP signaling plays such a central role in maintaining GSC identity prompted us to ask whether this signaling pathway also impacts the development of the PGCs in the embryo. Here we have focused on the period after the PGCs exit the mid-gut, migrate towards the SGPs and then coalesce into the embryonic gonad. During this period, the PGCs begin to reactivate transcription and start assuming some of the morphological features characteristic of the GSCs in the adult gonad. Our studies show that BMP signaling during this period in mid-embryogenesis is critical for the maintenance of PGC identity. However, the response to external BMP signals must be delicately adjusted, as excess signaling has deleterious effects on the PGCs. We show that a component of the sumoylation pathway, Dorfman and Shilo The loss and subsequent reappearance of nuclear pMad in PGCs raises a number of questions. We address several of these below:twist-Gal4 to drive UAS-dpp expression in the mesoderm. As illustrated by the stage 12 twi-Gal4/UAS-dpp embryo in dpp induces a substantial accumulation of nuclear pMad (arrows).In the period between exiting the midgut and coalescing with the SGPs to form the embryonic gonad, PGCs have little pMad protein. The low levels of pMad in stage 10–13 PGCs could be due to the absence of a potent source of BMP ligands in the surrounding somatic cells. Alternatively, stage 10–13 PGCs might not be capable of receiving BMP signals. To distinguish between these possibilities we overexpressed one of the BMP ligand family members, Dpp, using dpp, the global inhibition of Polymerase II transcription would effectively neutralize any regulatory effects of activating the BMP signal transduction cascade. However, this does not seem to be the case as we observed three unusual PGC phenotypes. First, there is a substantial reduction in the number of PGCs in the coalesced gonad . Second, there are migration defects and a significant fraction of the PGCs scatter through the embryo instead of making contact with the SGPs. This difference is shown for stage 13 embryos in twi-Gal4/UAS-dpp embryos have a reduced level of Vasa protein (not shown: see below). We also tested two other Drosophila BMP family members, screw and glass bottom boat using the twist driver. Unlike dpp, ectopic expression of screw had no major effect on PGCs. In the case of gbb, overexpression using the twist driver resulted in a relatively minor but statistically significant reduction in total number of germ cells . Though the effects are small, this observation could be significant as gbb is expressed in the mesoderm during mid-embryogenesis around the time when PGCs migrate through the midgut epithelial wall.Since PGCs enter a “quiescent” state as soon as they are formed at the blastoderm stage, it is generally thought that they are refractory to the effects of cell- cell signaling molecules produced in the soma until the formation of the embryonic gonad. For this reason, it seemed possible that even though high levels of nuclear pMad can be generated in stage 10–13 PGCs by ectopic dpp. One is the induction of apoptosis. In wild type, expression of the eiger (egr) gene, a tumor necrosis factor ligand (TNF) homologue, is activated in stage 9 PGCs by a dpp-dependent mechanism egr then helps induce the apoptosis gene head involution defective (hid) in PGCs. We thus wondered if loss of PGCs seen upon exposure to excess Dpp is due to apoptosis. To assess this directly, we employed antibodies recognizing the cleaved Caspase 3 polypeptide. Accumulation of this activated form of Caspase 3 is a reliable marker of apoptosis. As can be seen in twi-Gal4/UAS-dpp PGCs as compared to wild type PGCs (compare panels D and E). Furthermore, more than 60% (16 out of 25) of total number of twi-Gal4/UAS-dpp PGCs were positive for cleaved Caspase 3 as opposed to 8% (2 out of 25) wild type PGCs.It seems likely that several factors contribute to the phenotypic effects of ectopic dpp demonstrates that stage 10–13 PGCs are capable of receiving and responding to BMP signals, interpretation of the phenotypic effects on PGC development is complicated by the severe somatic patterning defects induced by broad dpp expression in the mesoderm. To reduce in so far as possible the side effects of abnormal somatic patterning on PGC development we ectopically expressed dpp using a nanos- Gal4 driver . This driver is thought to be specific for the germline. In addition, its activation is delayed until stage 9–10 twist driver comes on earlier in development. As might be expected from the restricted expression of the nos driver, we see no obvious abnormalities in somatic development in nos-Gal4/UAS-dpp embryos. Moreover, as illustrated in twist driver are due to abnormalities in somatic patterning induced by the mesodermal expression of Dpp.While induction of pMad accumulation by ectopic nos driver. Like the twist driver, there is a substantial reduction in the number of germ cells in the coalesced gonad  as opposed to 12 PGCs per gonad in wild type embryos . Similarly, nearly a quarter of the germ cells in nos-Gal4/UAS-dpp embryos have noticeably reduced levels of Vasa . This reduction can be seen in the nos-Gal4/UAS-dpp embryo shown in twi-Gal4/UAS-dpp combination, the germ cell loss appears to be due to apoptosis as cleaved Caspase 3 is also elevated in nos-Gal4/UAS-dpp PGCs . Moreover, as was the case for nos-Gal4/UAS-dpp PGCs, we also observed a reduction in Vasa accumulation in Nos-Gal4/UAS-tkvA .Although expression of Dpp in PGCs using the Taken together these findings establish that stage 10–13 PGCs are competent to receive and respond to BMP signals. Moreover, it is clear that ‘too much’ BMP signaling during this stage of development has deleterious effects on PGCs, and results in a reduction in the number of germ cells that are incorporated into the embryonic gonad. Most likely this loss is due to the induction of cell death pathways by excess BMP signaling dpp.While the findings described above demonstrate that PGCs have the molecular machinery needed to sense and respond to BMP signals in mid- embryogenesis, the observed effects rely on ectopic expression and thus it is entirely possible that this signaling pathway is of no significance to the development of the PGCs at this stage. To address this issue we examined the effects of mutations in the gene encoding the BMP signaling molecule Drosophila PGCs.Properly specified PGCs have a germ cell specific organelle called the spectrosome dpp mutant embryos. Since dpp is haploinsufficient, homozygous mutant dpp embryos were generated using a dpp null allele, H46,dpp and a balancer that carries two copies of dpp. In the experiment shown in H46/dppH46dpp embryos. The newly formed spectrosomes in PGCs of stage 11–12 H46dpp embryos are similar in size and morphology to wild type of the same age (not shown). However, in older embryos differences were readily apparent and the spectrosomes in H46dpp PGCs (n = 75) fell into three classes. PGCs in the first class (39%) resembled wild type and had a single large spherical spectrosomes, which stained strongly for both Spectrin  female germline clones and mated the females to wild type males. The resulting progeny are compromised maternally for the tkv BMP receptor, but are rescued zygotically. Unlike H46dpp embryos, somatic patterning in mtkvz+− embryos appears to be entirely normal and the PGCs migrate to and coalesce properly with the SGPs to form an embryonic gonad. Although both maternal and zygotic tkv must be removed in order to completely eliminate pMad accumulation in blastoderm stage PGCs, elimination of maternal tkv reduces the level of nuclear pMad mtkvz+− PGCs would have reduced tkv activity compared to PGCs from wild type mothers. As would be expected the effects of reducing but not eliminating tkv activity are considerably less dramatic than those seen in dpp mutant embryos. We found that the spectrosomes in about half of the mtkvz+− PGCs resembled wild type (n = 50). They had the characteristic spherical shape and stained strongly for Spectrin and Hts . In other PGCs, the spectrosome is weakly labeled (arrowhead in E) fragmented (arrows in F) or missing altogether (* in F).To determine whether the requirement for BMP signaling is direct or indirect we first generated  and Hts . On the tkv germline clone embryos were similar to those seen in dpp mutants, we did not observe any obvious reductions in Vasa protein accumulation in the mtkvz+− PGCs. This could mean that BMP signaling is required for the maintenance and elaboration of the spectrosome but that it has only an indirect role, presumably via some other BMP dependent somatic signal, in ensuring Vasa protein accumulation. Alternatively since zygotic transcription is known to provide a source of tkv in mtkvz+− PGCs While the disruptions in spectrosome integrity in PGCs from nos-Gal4 to drive the expression of a UAS-tkvRNAi transgene. Since the nos driver does not come on until stage 9–10, the RNAi knockdown would be expected to first begin compromising tkv activity around or after the time when the spectrosomes are beginning to assemble. In spite of this delay, the RNAi knockdown gave abnormal spectrosome phenotypes quite similar to those seen in the tkv germline clones. We found that 59% of the nos-Gal4/UAS-tkvRNAi PGCs had spectrosomes that resembled wild type, 21% had faint and fragmented spectrosomes and 21% lacked spectrosomes altogether (n = 29). Importantly, the PGCs in nos-Gal4/UAS-tkvRNAi embryos differ from those in mtkvz+− embryos in that there were obvious reductions in Vasa protein accumulation. nos-Gal4/UAS-tkvRNAi PGCs have reduced levels of Vasa. PGCs with reduced levels of Vasa protein were also observed when BMP signal transduction was compromised in the germline using nos-Gal4 to drive the expression of RNAi specific for the pMad co-factor Medea  have fragmented, faint (16%) or missing spectrosomes (47%). Similarly, over 30% of the nos-Gal4/UAS-dSmurf PGCs had noticeably reduced levels of Vasa protein . Thus, we suspect that the reduction in Vasa levels seen in some PGCs exposed to excess dpp is not due to the loss of a PGC-like identity as in seems to be the case when BMP signaling is compromised. A more likely possibility is that it is related to the death of the PGCs The fact that we saw loss of Vasa not only when there was too little BMP signaling but also when there was too much  in the nervous system using an The experiments in the previous sections indicate that PGCs require BMP signaling to maintain spectrosome integrity and a PGC-like identity in the period after they exit the mid-gut. However, we also found that excess BMP signaling during roughly this same interval has detrimental effects on PGC development including reduced Vasa, upregulation of the apoptotic protein Egr and PGC loss. The negative effects of both too little and too much BMP signaling pose a question: how is the proper balance achieved?et al.Drosophila Ubc9 homolog Lesswright (Lwr). When Med is sumoylated in somatic cells it exits the nucleus, and if it is in a complex with pMad, it can also drag it along into the cytoplasm. This sequestration of sMed and pMad in the cytoplasm (plus the turnover of the cytoplasmic proteins) attenuates the transcriptional response of the somatic cells to BMP signals. Suggesting that such a mechanism could operate in the embryonic germline, Hashiyama et al.lwr (as well as other sumoylation pathway genes) are greatly enriched in PGCs. Although their subsequent studies uncovered a sex-specific function for lwr in PGC development A possible hint as to one of the balancing mechanisms comes from a comparison of pMad localization in the PGCs of early, blastoderm stage embryos and in the coalesced gonad. While blastoderm stage PGCs have high levels of nuclear pMad, this is not true of the PGCs in the coalesced embryonic gonad. Though pMad can be detected in the nucleus at this stage, much of it seems to be localized in the cytoplasm. Miles lwr in the germline is to attenuate BMP signaling, then reducing lwr activity should alter the pattern of accumulation of the downstream effectors. To test this prediction we used nos-Gal4 to drive the expression of a transgene, UAS-lwrDN, that encodes a dominant negative Lwr protein. The dominant negative Lwr protein should interfere with lwr activity and mimic the upregulation of the signaling pathway that is seen when dpp is ectopically expressed using a germline driver. On the other hand, since the inhibition of lwr activity is expected to require the accumulation of sufficient quantities of LwrDN, the effects on pMad accumulation are likely to smaller compared to those observed when Dpp is expressed using the same nos-Gal4 driver. A comparison of pMad accumulation in nos-Gal4/UAS-lwrDN PGCs with that in nos-Gal4/UAS-dpp and wild type PGCs is shown in nos- Gal4/UAS-lwrDN , nanos-Gal4::VP16 (stock number: BL-7303), UAS-dpp (stock number: BL-1486), UAS-screw (obtained from Kavita Arora/Rahul Warrior labs), UAS-medRNAi (stock number: BL-31928), UAS-tkvRNAi (stock number: BL-35166 and 31040), UAS-madRNAi (stock number: BL-31315), UAS-dsmurf, UAS-lwrDN (stock number: BL-9318), and UAS-lwr (stock number: BL-9324). Typically, virgin females homozygous for the Gal4 transgene were mated with males carrying the UAS transgene H46dpp was used. Germline clonal analysis was performed as described in Chou and Perrimon,1992) using 8FRT-tkv allele stock obtained from Chip Ferguson’s lab. Females carrying germline clones were mated with males of the appropriate genotype UAS-gbb and UAS-tkvA  stocks were provided by Michael O'Connor and Chip Ferguson’s labs respectively.Flies were grown at room temperature (22°C) on standard medium similar to the current “standard media” listed on the Bloomington stock center website. The following stocks were used for misexpression analysis: The stainings were performed essentially as described in Deshpande et al (1995) Figure S1Ectopic expression of the BMP ligand, DPP, induces apoptosis. Embryos of the specified genotypes were double stained with Vasa and cleaved Caspase 3 antibodies. Panels A and D: wild type, Panels B and E: twi-Gal4/UAS-dpp, Panels C and F: nos-Gal4/UAS-dpp. Panels A-C show composite images of Vasa (red) and cleaved Caspase 3 (green) whereas panels D-F show just a gray scale image of the cleaved Caspase 3.(TIF)Click here for additional data file.Figure S2Vasa levels are unaffected among the mismigrated PGCs. Wild type and elav-Gal4/UAS-hmgcr embryos were stained with Vasa antibody . Panel A: Wild-type. Panel B and C: elav-Gal4/UAS-hmgcr. As reported previously ectopic expression of hmgcr in the nervous system using the elav-Gal4 driver induces PGC migration defect. Shown in panels B and C are PGCs from the same embryo. The PGCs on one side of the embryo that are shown in panel B have coalesced into normal looking gonad. The PGCs on the other side of the embryo are scattered from the effects ectopic Hmgcr. In both cases, the levels of Vasa protein are similar in all PGCs and equivalent to that seen in wild type PGCs.(TIFF)Click here for additional data file.


TMPRSS2:ETS fusion markers with a sensitive quantitative PCR platform. The pathology reported 39 biopsy-positive cases from 92 patients (42.4%). In urine test, 10 unique combinations of fusion types were detected in 32 of 92 (34.8%) prebiopsy samples. A novel combination of fusion markers, termed Fx , was identified with a sensitivity of 51.3% and an odds ratio of 10.1 in detecting cancer on biopsy. Incorporating a categorical variable of Fx  with urine PCA3 and serum PSA, a regression model was developed to predict biopsy outcomes with an overall accuracy of 77%. Moreover, the overexpression of Fx  was shown to be an independent predictor to the high-grade cancer, with a predictive accuracy of 80% when coupled with PSA density. The individualized risk scores further stratified a high-risk group that is composed of 92% high-grade cancers and a low-risk group that harbors mainly clinically insignificant cancers. In conclusion, we have identified a novel combination of fusion types very specific to the clinically significant prostate cancer and developed effective regression models to predict biopsy outcomes and aggressive cancers at diagnosis.We explore noninvasive clinical applications of multiple disease-specific fusion markers recently discovered in prostate cancer to predict the risk of cancer occurrence and aggressiveness at diagnosis. A total of 92 men who were prostate-specific antigen (PSA) screened and scheduled for diagnostic biopsy were enrolled for this study. Prospectively collected urine was blind coded for laboratory tests. RNA from urine sediments was analyzed using a panel of 6  TMPRSS2 gene with an oncogenic ETS family transcription factor ERG gene, which is reported in 50% of surgical prostate tumors . This result was consistent with our previous observation for a strong correlation between TMPRSS2:ERG fusion and ERG expression levels in urine of prostate cancer patients  and [Bx(+)/Fx(−)] subgroups (n = 11) was also identified in this study, which had a molecular profile similar to the Bx(+)/Fx(+) subgroup. Interestingly, most cases detected with the common subtype I alone belonged to this subgroup. However, the Bx(−)/Fx(+) subgroup was shown to be associated with older age (P < 0.001) and increased incidence of high-grade prostatic intraepithelial neoplasia (PIN) lesions (P < 0.05) as compared with the double-negative subgroup  identified four distinctive molecular subgroups exhibiting different molecular  and clinpatients . In contubgroups . Thus, tsubgroup . These fPCA3 and serum PSA, a logistic regression model was developed to calculate an individualized molecular score for optimal prediction of biopsy outcomes (P < 0.001), TMPRSS2:ERG subtype I , or Fx  fusion type  in the prebiopsy cohort of 92 patients (n = 31), the intermediate-risk group , and the low-risk group  ] had an improved predictive value on cancer occurrence. By incorporating the categorical variable of fusion types Fx  with log-transformed continuous variables of outcomes . As demopatients . For cli n = 31) . As such n = 31) . On the P < 0.0001) but also associated with the high-risk-of-recurrence cases (P < 0.001) as defined by the NCCN guidelines , the number of positive cores , and the percentage of cancer involvement in a single core  when analyzed by the Spearman's ρ (P < 0.01) of the low-risk cancers based on molecular scores with the insignificant cancers based on pathology may suggest an indolent property for such cancer patients. Indeed, three Bx(+) cases (Gleason 6) from the low-risk group exhibited no progression in 12-month follow-up biopsies, while two Bx(+) cases (Gleason 6) from the intermediate-risk group failed to detected cancer cells in repeat biopsy (data not shown). However, long-term follow-up is required to determine the clinical outcomes that are beyond the scope of this study.One of the most challenging tasks of early cancer detection is to identify the aggressive forms of cancer at diagnosis. We explored risk models to predict high-grade cancers (Gleason ≥7) in the context of biopsy-positive cases. Unlike the detectability  of Fx  used in predicting biopsy outcomes, the overexpression profiles of these fusion markers were more informative to identify clinically significant cancers. Using a common calibrating sample sparked with fusion molecules, we devised a strategy that ranked the relative quantity of different informative fusion markers as a new continuous variable. Instead of using each fusion marker separately with reduced statistic power, we proposed to calculate the quantitative feature of combined fusion markers based on the sum of quantity values (or the highest relative quantity) of fusion markers in each sample. Coupling this new continuous variable of Fx  with the continuous variable of PSA density (PSAD) that we termed as Fx  + PSA density (FPd), a logistic regression model was developed to predict the risk of high-grade cancer (Gleason ≥7) with an overall accuracy of 80% . The essidelines . Indeed,rman's ρ . ConversTMPRSS2:ERG subtypes and low-prevalent TMPRSS2:ETS  fusion markers. We demonstrated for the first time the clinical utility of multiple low-prevalent fusion markers and diverse fusion types in urine-based cancer detection. Moreover, we identified multiple alternative fusion genes and subtypes very specific to clinically significant prostate cancers than the common subtype I, and developed effective regression models to predict both the risk of cancer occurrence and the risk of aggressive cancer prospectively.In this study, we report a prospective study for molecular diagnosis of PSA-screened patients who are scheduled for diagnostic biopsy (prebiopsy cohort) using a novel panel of both common TMPRSS2:ETS fusion markers, we not only detected diverse fusion types in urine of prebiopsy patients but also identified several alternative fusion markers very specific to biopsy outcomes. The informative fusion markers, Fx , are collectively more sensitive than the dominant fusion subtype I in prospective prostate cancer detection. Moreover, by incorporating the categorical variable of Fx  with urine PCA3 and serum PSA, the multivariate FPP model not only has an overall predictive accuracy of 77% to overall prostate cancer detection, but also allows individualized stratification of prebiopsy patients into distinctive risk groups. As such, the PPV on the biopsy outcome is 81% in a high-risk group, but only 16% in a low-risk group. On the other hand, the combined quantity of Fx  in urine are shown to be an independent predictor to the high-grade cancers (P < 0.022). When coupled with PSAD, the FPd model detects the aggressive prostate cancer with an overall predictive accuracy of 80% in biopsy-positive patients. The resulting FPd scores further stratify not only a high-risk group that is composed of 92% high-grade cancer, but also a low-risk group that harbors mainly clinically insignificant cancers. However, it remains to be elucidated on the basis of different clinical implications between a single versus a panel of markers as well as between detectability and overexpression among the same set of informative fusion markers. Interestingly, the less common subtypes (III and IV) are frequently accompanied by the common subtype I in Bx(+) cases, but the common subtype alone is overrepresented in the [Bx(−)/Fx(+)] subgroup. Thus, the genetic complexity of fusion types and the active expression of less common fusion markers in urine may be associated with an active state of cancer proliferation and hence serve as better predictive markers to the biopsy outcomes and the risk of aggressive cancer. This hypothesis is further supported by the identification of multiple novel fusion subtypes associated with the high-risk cancers when the same urine specimens were screened by a DNA-chip-based high-throughput method . Therefore, the panel approach is better suited to identify clinical significant cancers over a single marker-based test. It is feasible to further improve the clinical sensitivity by including additional fusion markers  into the same platform, and to simplify the multimarker analyses into a single test using a qPCR-array format. However, it is necessary to point out that the assay based the new fusion panel was a laboratory-developed test and that our prospective study is limited by a relatively small sample size and short follow-up time. Independent validation studies are necessary and current ongoing to address these limitations by the authors.Prostate cancer is characterized by its extensive clinical heterogeneity; early stratification of aggressive disease from a majority of indolent cancers at diagnosis is a critical clinical task in cancer management and treatment. Our work builds on an increasing number of disease-specific fusion genes recently discovered in prostate cancer –28. We aTMPRSS2:ETS fusion markers. The extensive diversity of fusion types identified in urine of PSA-screened men provides better genetic and molecular bases to stratify the clinical heterogeneity of prostate cancer. Clinically applicable risk models are developed to generate individualized molecular scores to identify distinctive risk groups. As such, patients identified with a high molecular score will likely lead to the detection of aggressive forms of cancer in biopsy-positive cases or “false” diagnosis in biopsy-negative cases who should be prioritized for repeat biopsies. On the other hand, patients characterized with a low-risk score may represent indolent cancer in biopsy-positive cases or truly low-risk subjects in biopsy-negative cases. The former may be benefitted by conservative management such as active surveillance, while the latter may be excluded from further screening to reduce the biopsy burden. It is logical to expect that the conceptual advances and simultaneous analysis of all existing fusion markers using high-throughput technologies will provide a new paradigm for personalized molecular diagnosis of the clinically significant prostate cancer.In conclusion, we propose a urine-based clinical test for early detection of clinically significant prostate cancer using a panel of multiple


Ciliopathies are responsible for multiple organ dysfunctions with chronic kidney disease as one of cardinal clinical features. We studied the renal phenotype in 33 patients diagnosed with Bardet-Biedl Syndrome (BBS) and found renal abnormalities to be present in 82% of patients (27/33). An impaired urinary concentrating ability was the most frequent manifestation  in non dialyzed and non transplanted patients, which could be observed even in the absence of renal failure or cystic formation identified by ultrasonographic and magnetic resonance imaging. In order to specify the pathophysiology involved in this urinary concentrating defect, we focused on the role of the primary cilia and its interaction with the anti-diuretic hormone, arginine-vasopressin (AVP) as it was previously shown that the AVP-Receptor-2 (AVPR2) was localized on the primary cilia. To do so, we studied the effects of vertebrate-specific chaperonin-like proteins  inactivation in cultured cell line HCD (Human Collecting Duct) by way of RNA interference techniques. Our results show that chaperonin-like proteins deprivation in vitro leads to primary cilia loss in HCD cells, resulting in an inability to detect luminal AVP and to activate the targeting of aquaporin-2 (AQP-2) to the apical membrane of the renal epithelial cell, thus unable to absorb water. Interestingly, water reabsorption through restored targeting of AQP-2 was achieved by forskolin treatment – a receptor-independent adenylate cyclase activator – demonstrating that intracellular machinery was present but not activated. This study highlights the major role of primary cilia in efficient water reabsorption in the collecting duct.


Cellular and molecular events can be investigated using electrophysiological techniques. In particular, the patch-clamp method provides detailed information. In addition, the patch-clamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. In this paper, recent researches on how acupuncture might modulate electrophysiological responses in the central nervous system (CNS) and affect peripheral structures are reviewed. The therapeutic mechanisms of acupuncture are based on the theory of acupuncture and meridians in traditional Chinese medicine (TCM). How to use modern medical research methods to clarify the mechanisms of acupuncture is one of the important subjects of current basic research in TCM. Currently, the microstructure of human tissue, including the complex relationship between cells and the interstitium, and the laws of information transmission in the human body are understood. However, the essence of acupuncture-effect mechanisms is still unclear.Cellular signal transduction theory is critical for understanding the mechanisms that explain the effects and actions of acupuncture. Cells constitute the basic units of living organisms, and the synergy between cells facilitates the various functions of the body. Communication between cells relies on receptors and ion channels in the cell membrane. Using patch-clamp technology, the electrical changes of cell membrane ion channels can be observed to elucidate the cellular mechanisms of acupuncture. As a well-developed cellular electrophysiological method , 2, the Acupuncture is a viable and complementary treatment for relieving pain; the treatment involves the insertion of needles to a certain depth at specific acupuncture points (acupoints) followed by manipulations or electrical stimuli –7. Despiin vitro patch-clamp technique more precisely demonstrates the interactions between multiple receptors [Researchers use different pain models to investigate the role of neuronal membrane receptors in the sensation, transmission, and modulation of pain , 11. Unleceptors .μg/15 mL) enhanced the EA effect [μ, δ, and κ) in the spinal cord of rats play important roles in mediating the analgesia induced by EA [μ- or κ-opioid-receptor-mediated depressant effect on the voltage-gated calcium current. The CCK receptors were presynaptically coexpressed with the opioid receptors in the same neuron [The mechanisms of acupuncture-induced analgesia have been investigated since the 1950s as a key project of the Natural Science Development Plan in China . The resA effect –19. All ed by EA . Second,e neuron , 22. Thie neuron . Becausee neuron .α,β-methylene-ATP  evoked similar fast desensitizing inward currents in the DRG neurons. The amplitudes of the currents in the CCI group were larger than in the sham group and were significantly attenuated by the EA treatment . EA may induce an apparent analgesic effect by decreasing the expression of and inhibiting P2X3 receptors in the DRG neurons of rats with CCI [In neuropathic pain, adenosine 5′-triphosphate disodium- (ATP-) gated P2X receptors, especially the P2X3 subtype, play an important role in the transmission of pain signals , 26. Forwith CCI .Visceral pain is difficult to be localized and easily transferred to cutaneous structures. Some natural stimuli are clearly associated with multisourced pain from the viscera, such as hollow organ distention, muscle spasm, ischemia, and inflammation . EA is cin vitro application of the μ-opioid receptor agonist (D-Ala (2), N-MePhe (4), and Gly (5)-Ol) enkephalin (DAMGO) mimicked the EA-mediated suppression. These findings revealed the endogenous opioid-related analgesic effects at the cellular level during EA treatment of visceral pain in functional gastrointestinal disorders.To investigate the effect of EA on colon-specific DRG neurons in rats with CVH and to clarify the interactions between somatic and visceral nociceptive inputs in the spinal dorsal horn, electrophysiological measurements were made . Before in vitro, the effects of acupuncture on target cells can be directly observed because the blood-brain barrier is bypassed. With the proper conditions  content), the metabolic and biological properties of the brain slices can be maintained in vitro for a couple of hours [Patch-clamp recordings from brain slices have been utilized to analyze CNS function since the 1980s –34. By aof hours , which fμm) were sectioned from the acupuncture and sham groups and superfused with the aCSF oxygenated at 31°C. In vitro ischemia was generated by reducing the perfusion rate from 2 mL/min to 0.2 mL/min for 6 min. Ischemia generally induces increases in the threshold potential and absolute refractory periods of firing spikes in neurons that are measured in the whole-cell recording mode with sequential spikes induced by depolarizing current pulses; acupuncture prevented these increases. Meanwhile, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in the GFP-labeled GABAergic neurons. The impairment of active transmissions between neurons as a result of ischemia was significantly prevented with acupuncture. Acupuncture improves ischemic stroke via preventing this dysfunction by targeting voltage-gated sodium channels [δ-opioid receptors in the periaqueductal gray results in inhibition of GAT1-mediated current as measured in whole-cell recordings.An imbalance between the excitation and inhibition of gamma-aminobutyric acidergic (GABAergic) neurons caused by ischemia-induced neuron dysfunction has been suggested as an alternative mechanism for neuronal excitotoxicity . Stimulachannels . Modulatchannels . As discchannels ; it was in vivo patch-clamp technique is mainly used to study the characteristics and mechanisms of the sensory system in response to environmental stimuli [in vivo for whole-cell recording. Later, the success rate was significantly improved by two-photon targeted patching (TPTP), in which the patch clamp is performed under direct visual control by imaging the fluorescence with two-photon microscopy (2PM) [in vivo patch clamp has been broadened. Compared with in vitro isolated neurons, in vivo measurements on brain or spinal cord slices have a greater advantage: one can obtain not only the final outputs but also the subthreshold responses. With this information, analyzing the interaction between excitatory and inhibitory synaptic inputs activated by cutaneous stimulation becomes possible [The  stimuli . The propy (2PM) , 43. Thepossible , 45. TheIn vivo whole-cell voltage-clamp recordings were applied during acupuncture to the SG neurons, which were morphologically identified by injection of neurobiotin [VH = 0 mV) and sEPSCs (VH = − 70 mV) were recorded from the same neurons by altering the holding potentials. In the CFA model group, sEPSCs with higher magnitudes were observed in the majority of SG neurons. The application of EA to the contralateral ST-36 point slightly decreased the amplitude and frequency of the sEPSCs. In contrast, a larger amplitude and higher frequency of sIPSCs were elicited with acupuncture frequencies from 2 to 10 Hz. This in vivo patch-clamp recording technique permits functional analyses of modality-dependent synaptic responses evoked by acupuncture. Therefore, the behavioral changes at the single-cell level can be explained with more certainty [The substantia gelatinosa  may be a major site for the modification and integration of noxious sensation. A chronic pain model was established by complete Freund's adjuvant- (CFA-) induced inflammation in the right hind paw of rats. A lumbar laminectomy was performed on an anaesthetized rat at the L4 or L5 level. The surface of the spinal cord was exposed by removal of the pia-arachnoid membrane and was irrigated with oxygenized Krebs solutions at 38°C. robiotin . Spontanertainty .As the basic elements in meridian structure, acupoints are suggested to serve as the initiating points for acupuncture treatments . Despite+, Na+, and Ca2+, TREK and TRAAK [− channels are the primary mechanosensitive anion channels that play an important role in volume regulation [Sensitivity to mechanical stimulation is an inherent property of many tissues and cells. The patch-clamp technique can directly study the responses of mechanosensitive (MS) ion channels , which and TRAAK , TRPV2 [nd TRAAK , and TRPnd TRAAK  are typigulation .2O applied via a patch pipette). This stress induced an inward-directed current along with cell degranulation [− concentration, indicating that this current component may be mediated by Cl− channels, possibly from the CLC family [in situ in connective tissue slices obtained from the ST-36 acupoint in rats. The resulting current to pressure gradient exhibited a similar outward rectification as for the activation of HMC-1 [− channels might be input receptors that contribute to the transduction process of the mechanical signal from acupuncture stimulations.Research on MS ion channels and the proteins that might play roles in the acupuncture effect was conducted at the cellular level using a human leukemia mast cell line (HMC-1) , 67. In nulation . The actnulation . The TRPof HMC-1 , 67. Givμm in thickness) isolated from the surface of abdominal organs in rats. The small round PN cells had a low resting membrane potential (−39 mV) and spontaneous activities [+ contributed to the resting membrane potential of these cells.As widely questioned in histological studies, the primo vascular system was first introduced as the anatomical structure of acupuncture meridians by Kim  and recetivities , 74. Of tivities . The TEAThe patch-clamp technique allows for the study of the properties of single channels expressed in specific cell membranes; progress has also occurred in research on the mechanisms of acupuncture. However, the depth (and breadth) is insufficient for a clear understanding of the processes that occur in acupoints and along meridians. By applying specific agonists, antagonists, and allosteric modulators to different parts of the membrane (inside or outside) at different times with fast perfusion, differentiated ionic currents through single channels can be recorded. These currents, together with the dynamic properties of the receptors, may help to demonstrate the existence and function of the detected receptor. in vivo or in vitro tissues [Based on the technical potential of the patch-clamp method, the use of serum from acupunctured animals or humans  as a per tissues –79. It win vivo patch-clamp technology, the functions and relationships of intracerebral nuclei or spinal cords can also be observed during acupuncture; thus, the acupuncture pathway and the signal transduction process can be explored. Compared with investigations using patch clamp on isolated cells, there are only a few reports of in vivo studies because there are still difficulties in controlling factors, such as the mechanical fixation, anesthesia, and physiological interference of animals.Additionally, by using The application of electrophysiological techniques to study the mechanisms of acupuncture at the molecular level first requires the establishment of an animal model corresponding to the disease at the organ level. Then, single cells must be obtained from crucial organs or acupoints using cell separation and cultivation. Finally, cellular models must be built from different organs under pathological circumstances. By combining that information with specific agonists and antagonists, it is possible to determine the pathway of electrophysiological signal changes caused by acupuncture and to preliminarily explore the mechanisms of disease at the molecular level.Using modern measurement techniques to study the mechanisms of acupuncture is one of the most important demonstrations of the modernization of Chinese medicine. The patch clamp can be combined with additional recordings to increase the analysis capabilities: with fluorescence measurement , the tec


Ricinus communis). The phylogenetic and domain analysis indicated that these genes were divided into two groups (group I and II). The group II was a big branch and was further classified into three subgroups (subgroup II-1 to 3) based on the phylogeny, gene structures and conserved motifs. It was observed that the genes of subgroup II-1 had distinct evolutionary features from those of the other two subgroups, however, were more similar to those of group I. Therefore, we inferred that group I and subgroup II-1 might retain ancient signals, whereas the subgroup II-2 and 3 exhibited the divergence during evolutionary process. Estimation of evolutionary parameters (dN and dN/dS) further supported our hypothesis. At first, the group I was more constrained by strong purifying selection and evolved slowly with a lower substitution rate than group II. As regards the three subgroups, subgroup II-1 had the lowest rate of substitution and was under strong purifying selection. By contrast, subgroups II-2 and 3 evolved more rapidly and experienced less purifying selection. These results indicated that the different evolutionary rates and selection strength caused the different evolutionary patterns of the members of SBP-box genes in castor bean. Taken together, these results provide better insights into understanding evolutionary divergence of the members of SBP-box gene family in castor bean and provide a guide for future functional diverse analyses of this gene family.Genes in the SQUAMOSA promoter-binding-protein (SBP-box) gene family encode transcriptional regulators and perform a variety of regulatory functions that involved in the developmental and physiological processes of plants. In this study, a comprehensive computational analysis identified 15 candidates of the SBP-box gene family in the castor bean ( Arabidopsis thalianaPrecise and coordinate gene expression is essential for organism growth and development. Regulation of gene transcription is the primary one among the many mechanisms that operate to control gene expression. Transcriptional control relies on transcription factors (TFs), which are usually defined as proteins that show sequence-specific DNA binding and are capable of activating and/or repressing transcription. Most known transcription factors can be grouped into families according to their DNA binding domain SQUAMOSA (SQUA) promoter-binding-like (SPL) genes represent one such family of plant-specific transcription factors SQUAMOSA promoter-binding-protein (SBP) domain) with two separate zinc-binding sites  Cucumis sativus) Glycine max) Citrullus lanatus) Ricinus communis) an important oil-producing member of the Spurge family (Euphorbiaceae) that is the only species its genus and has no immediate relatives The draft genome sequence has been published for the castor bean . Twenty-seven genes were initially identified as candidates that possess possibly the SBP domain. Following the removal of redundant sequences and SMART Oryza sativa) Vitis vinifera) Arabidopsis homolog. A similar method has been used to name the SPL genes in the tomato plant (Solanum lycopersicum) RcSBP1 to RcSBP15) in this study.To identify the SBP-box genes present in the castor bean, we used multiple BLAST algorithms to compare against the genome and protein data sets, using the representative SBP domains of RcSBP12). In our previous study RcSBP12 was classified as group I and the remaining 14 proteins were assigned to group II  . This DBp II see .Arabidopsis genes, we generated a phylogenetic tree based on the highly conserved SBP domains. Castor bean SPL genes were not clustered together in a species-specific manner , suggesting that the castor bean SPL genes might have originated up to 400 million years ago. In addition, we observed that each phylogenetic branch contained at least one Arabidopsis and castor bean SBP-box protein. This suggests that SPL genes from the two species have similar functions. To better understand their evolutionary relationships and functions, we analyzed a synteny map to compare the two genomes. The resulting, putative ortholog pairs were: RcSBP12-AtSPL7; RcSBP10/11-AtSPL1; RcSBP13-AtSPL11; RcSBP14-AtSPL13; RcSBP5-AtSPL4; RcSBP7/8-AtSPL6; RcSBP15-AtSPL17; RcSBP3-AtSPL14  was classified as part of group I along with AtSPL7, while the remainder constituted group II  and nonsynonymous (dN) substitution rates for the SPL genes of two groups and three subgroups, separately. Synonymous substitutions usually accumulate more rapidly than nonsynonymous ones, which leads to a differential saturation of dS and dN, thus potentially biasing estimates of their values. On the other hand, the saturation of synonymous substitutions could mask the information provided by nonsynonymous ones. To avoid that problem with synonymous substitution rates, we used only nonsynonymous substitution rates. The mean dN value for group I was two times lower than that for group II (N/dS using the tree topology suggested by neighbor-joining (NJ) analysis of the domain sequences. We found that dN/dS values were much lower than 1.0 for the two groups, suggesting that purifying selection was operating: the ratio was estimated as 0.08 for group I and 0.31 for group II. We analyzed the three subgroups in the same way. First, we found that the substitution rate was significantly slower in the genes of subgroup II-1 than in those of subgroups II-2 and 3 , which was consistent with results of the dN analysis. This suggests that the genes of subgroup II-1 were subjected to stronger selection pressures and have evolved more slowly than those of subgroup II-2 and 3.We hypothesized that the genes most likely to retain ancient evolutionary signals should be in the most slowly evolving positions. To test this, we estimated synonymous . Therefore, we inferred that the SPL ancestor gene might have a complex gene structure with many exons and introns. The genes of subgroup II-2 and 3 might have suffered exon and intron loss during the evolutionary process.When we analyzed the SPL genes of the castor bean alone, a similar trend was observed. Previous studies have examined that many plausible, functional constraints have been related to the different evolutionary patterns (in particular the evolutionary rate) These results provide strong supporting evidence for the evolutionary diversity of SPL genes in the castor bean. Our novel study provides a foundation for analyzing the functional diversification of the members in the SBP-box gene family in castor bean.Arabidopsis SBP-box gene family were downloaded from the PlnTFDB database Arabidopsis SPL gene for each group/subgroup defined by us http://castorbean.jcvi.org/downloads.php)  . As a fiArabidopsis were first aligned using ClustalX http://evolution.genetics.washington.edu/phylip.html). The amino acid substitution matrix used was the Jones-Taylor-Thornton (JTT) model, and local support values were assessed using 1000 replicate bootstrap tests.The SBP-box domain sequences from castor bean and http://castorbean.jcvi.org/downloads.php). A diagram of exon/intron structures was created using the online Gene Structure DiSPLay Server . Motifs were detected using MEME (version 4.9.0) Exon/intron site and length data were extracted based on the genome annotation GFF files (S) and nonsynonymous (dN) substitution rates per protein were estimated using the program codeml in PAML version 4.4 S estimates will be systematically lower than the real synonymous substitution levels. To avoid the problem of saturation of synonymous substitutions, we applied a threshold that is usually set around dS>1.5 in many studies.Synonymous Click here for additional data file.Figure S2Conserved synteny of castor bean and Arabidopsis SPL genes. The scaffolds of castor bean and the chromosomes of Arabidopsis are depicted as horizontal gray and blue bars, respectively. Castor bean and Arabidopsis SPL genes are indicated by vertical black lines. The marked regions in the scaffolds of castor bean and the chromosomes of Arabidopsis denote syntenic regions. Note: The length of the bars is scaled based on the length of scaffolds of castor bean. However, the length of chromosomes of Arabidopsis is too long and denoted by “//”.(TIF)Click here for additional data file.Figure S3Schematic diagram of motif architectures of every group or subgroup. The length of the motif can be estimated using the scale at bottom.(TIF)Click here for additional data file.Table S1Summary of information on ArabidopsisSPL genes used in this study.(DOC)Click here for additional data file.


Ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as a ubiquitous post-translational modification (PTM) whose function extends far beyond its original role as a tag for protein degradation identified three decades ago. Although sharing parallel properties with phosphorylation, ubiquitination distinguishes itself in important ways. Nevertheless, the interplay and crosstalk between ubiquitination and phosphorylation events have become a recurrent theme in cell signalling regulation. Understanding how these two major PTMs intersect to regulate signal transduction is an important research question. In this review, we first discuss the involvement of ubiquitination in the regulation of the EGF-mediated ERK signalling pathway via the EGF receptor, highlighting the interplay between ubiquitination and phosphorylation in this cancer-implicated system and addressing open questions. The roles of ubiquitination in pathways crosstalking to EGFR/MAPK signalling will then be discussed. In the final part of the review, we demonstrate the rich and versatile dynamics of crosstalk between ubiquitination and phosphorylation by using quantitative modelling and analysis of network motifs commonly observed in cellular processes. We argue that given the overwhelming complexity arising from inter-connected PTMs, a quantitative framework based on systems biology and mathematical modelling is needed to efficiently understand their roles in cell signalling. Cell signalling crucially depends on a repertoire of posttranslational modification (PTM) mechanisms for its regulation. Protein ubiquitination, the covalent attachment of the short protein modifier ubiquitin to target proteins, has emerged as a prevalent modification utilised by signalling processes to regulate a range of functional behaviours. First recognised as a targeting signal to send proteins to the proteosomal degradation pathway, ubiquitUnlike phosphorylation where the addition of the phosphate group to the modified targets is a rather straightforward single step, ubiquitination occurs in a three-step reaction requiring three different enzymes: an ubiquitin-activating enzyme E1), an ubiquitin conjugating enzyme (E2), and an ubiquitin ligase enzyme (E3). Ubiquitin is first activated by E1, followed by conjugation to an E2 before finally ligated to the lysine residues of target proteins by the E3 ligase  endocytosis was one of the early findings of the non-proteolytic roles of this PTM in cell signalling,22. UbiqUbiquitination-related mechanisms regulating the adaptor proteins also play crucial roles in the functioning of the endocytotic pathway, including cargo recognition and delivering. These adaptors include proteins at the plasma membrane including the clathrin coat, the EGFR substrate 15 (EPS15), a member of the EPS15-interacting protein family (EPSIN1–EPSIN3), and hepatocyte growth factor-regulated Tyr kinase substrate (HRS) at the endosomes. Adaptor proteins, which contain ubiquitin binding domains (UBD) such as the ubiquitin-interacting motif (UIM) can recognise the ubiquitin molecules on the ubiquitinated EGFR. This leads to the assembly of active receptors in clathrin-coated pits of the plasma membrane, endosomes and the multivesicular bodies (MVBs). AdaptorUbiquitin-mediated EGFR endocytosis affects the signalling dynamics of the downstream pathways, thereby modulating the cellular decisions. Cells have evolved ways to reverse ubiquitination events through de-ubiquitinating enzymes. The STARas is a small GTPase that connects RTK activation to the triggering of many downstream effector pathways including MAP kinase cascades. Ras exists in three isoforms: H-Ras, N-Ras and K-Ras which, despite sharing some regulators and effectors due to similar interaction domains, exhibit divergent functional properties and involvement in carcinogenesis. In certain cell types, K-Ras is the most potent activator of Raf-1,41, wherin vivo and in vitro ubiquitination assays along with RNAi technology, the authors showed that Rabex-5 is necessary and sufficient to catalyse H/N-Ras ubiquitination, promoting their endosomal localisation and resulting in suppressed ERK activation , known previously as a GEF for Rab5,47, is a Figure 48]. Ov. Ovin vi Figure [48]. Bein vivo[Although the studies by the Bar-Sagi group,57,58 diin vivo. Combinein vivo. In concThe transduction of a cellular signal as it propagates through the MAPK cascades, exemplified by the Raf/MEK/ERK module, is predominantly controlled by phosphorylation events where typically, each kinase in the cascade is activated by an upstream kinase and inactivated by relevant phosphatases. However, accumulating evidence has revealed that components of this cascade also can undergo ubiquitination, which not only leads to the degradation of the substrate proteins but also appears to regulate their activity and/or localisation.Raf proteins are the main effectors of Ras,64 and dAlthough evidence pointing to an ubiquitination-related mechanism involving MEK in mammalian cells is sparse, the yeast MEK protein Ste7 has been shown by multiple studies to undergo ubiquitination and regulate MAPK specificity-73. The ITCH is the HECT E3 ubiquitin ligase belonging to the NEDD4 protein family. It is characterised by the N-terminal C2 domain responsible for membrane localisation, 2 to 4 WW domains involved in substrate recognition, and the C-terminal catalytic HECT ligase domain. AlthougITCH can catalyse its own ubiquitination. However, the self-ubiquitinated conjugates of ITCH do not have K48-linked polyubiquitin chains, which would target the protein for degradation like most other E3 ligases. Instead they have K63 linkages, which serve to promote ITCH ligase activity, establiITCH was demonstrated to interact with the truncated form of the proapototic protein Bid (tBid), ubiquitinate tBid and induce its proteosomal degradation Figure . tBid isAnother route through which ITCH links EGFR/Raf/ERK signalling to apoptosis is via the MST2/LATS1 pathway Figure . Our groJNK is not the only kinase identified so far to target ITCH. Previous studies has indicated that Src-family tyrosine kinases are targeted for degradation by HECT-domain E3 ligase. Yang et al. therefore set out to investigate whether the Src-family kinase Fyn is ubiquitinated by ITCH in T cells, but instead discovered that ITCH is a substrate for Fyn. Fyn phoThe last decade has witnessed an unprecedented explosion of biological knowledge and large data sets acquired for many signalling processes at the cellular level, largely due to the development of sophisticated and high-throughput biochemical techniques in proteomics and other omics. As part of this trend, the studies reviewed in the previous section, although still limited, have revealed a rather complex picture of how ubiquitination and phosphorylation interplay to regulate signal transduction pathways such as the EGFR. The huge complexity hampers our ability to interpret and predict the regulation of the network as a whole, which is essential to better understand EGFR signalling and its role in diseases. To unravel this complexity and obtain a systems-level understanding of network signalling, systems biology approaches employing quantitative frameworks in forms of mathematical and computational models are emerging as promising solutions. These mathematical models provide a platform for the description, prediction and understanding of the various regulatory mechanisms in a quantitative and integrative way-98. In tA recurring theme in the interplay between phosphorylation and ubiquitination is that phosphorylation often influences the ubiquitination and thus degradation of the modified protein, such as in the case of c-Myc,100, andDespite the simplicity of these motifs, intuitive predictions regarding dynamical behaviour of the network components at various abundances of the regulatory proteins  would be a nontrivial task without the employment of mathematical models. We thus constructed models based on ordinary differential equations (ODEs) and the law of enzyme kinetics for these motifs, whose details are given in the Additional fileFeedback loops controlling signalling pathways are commonly seen in ubiquitination-phosphorylation networks. Here, we assume that the output target in motif 1 is an E3 ligase, which can ubiquitinate S* and pS* (displayed in Figure Self-ubiquitination is often observed among the E3 ligases. While often it is a mechanism to self-control the ligase abundance, it can also serve non-proteolytic functions and can dramatically influence the ligase activity, as in the case of ITCH discussed earlier. Degradation of ITCH is independent of its self-ubiquitination, which occurs through K63 linkages and results in stronger catalytic activity; whereas canonical K48-linked chains generated by other ligases target ITCH for degradation. LikewisSelf-modification reactions can occur in either an intra-molecular or inter-molecular fashion, as depicted in Figure As discussed earlier, ubiquitination is a multi-step process which depends not only on the abundance and properties of the E3 ligase involved but also on other factors involving the preceding steps, including loading of ubiquitin onto conjugating enzymes E2s and ubiquitin transferring to the substrate. Consideration of these factors may be necessary for a detailed model of the control of the EGFR pathway by ubiquitination. Such work however would require comprehensive experimental effort to provide the missing kinetic data and other quantitative information to calibrate and validate the model.In vitro experimental design based on the model analysis results could be the first step in confirming the predictions about the dynamics of the components of interest. An advantage of an in vitro system with purified forms of relevant E3 ligase, kinases and phosphatases is that it can be used to explore wide ranges of precisely set enzyme concentrations. To mimic the in vivo situation, some of these proteins may be embedded into a phospholipid membrane bilayer or liposomes if required, which can also facilitate the formation of protein complexes and increase reaction rates[in vitro is challenging due to a multitude of factors at play, including the adequate level of ubiquitin and the essential participation of the relevant E1/E2 enzymes. Therefore, direct in vivo approaches like imaging techniques using microscopy-based binding assay can be exploited for high temporal resolution measurements of components kinetics and may be a more favourable option[Our findings of potentially bistable and oscillatory behaviour of the ubiquitination-phosphorylation motifs await experimental testing. on rates. For inse option. On the In summary, we have constructed mathematical models and carried out analysis for a number of commonly seen motifs of ubiquitination-phosphorylation crosstalk. The motifs, although simplified, show diverse dynamics including sustained oscillations and bistability. More importantly, the models have facilitated the identification of the conditions under which these dynamics may realise, which would have been infeasible if such models are not used. Modelling therefore provides a useful and necessary tool for efficient analysis of ubiquitination-phosphorylation crosstalk, thereby potentially improving our systems-level understanding of the integrated EGFR signalling.Since the first discovery of protein ubiquitination more than three decades ago, extensive work has revolutionized our perception of its role in signalling networks. Not only protein ubiquitination serves as a main mechanism for protein degradation, emerging evidence has revealed that different types of ubiquitin chains can induce a variety of non-proteolytic functions and can dramatically alter the biological activities of a target protein. On top of that, ubiquitination is frequently observed to interplay with other PTMs such as phosphorylation or sumoylation to coordinate regulation of signalling processes in intricate manners. Such complexity arising from interconnected PTM networks poses enormous challenges for the systems level analysis of signalling processes. Mathematical modelling is emerging as a valuable tool to provide insight into their dynamic behaviour that would otherwise not be possible. Mathematical models help combine the mechanistic, molecular knowledge with rigorous analysis of the complex output dynamics of the PTM networks.The expanding roles of ubiquitylation and phosphorylation in cell signalling, to large extent, have been uncovered thanks to recent advances in proteomics technologies which have enabled new ways for in-depth, unbiased and quantitative analysis of different PTMs on a global scale-110. TecDUB: De-ubiquitinating enzyme; EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; ERK: Extracellular signal-regulated kinase; Cbl: Casitas b-lineage lymphoma; RTK: Receptor tyrosine kinase; EPS15: Epidermal growth factor receptor substrate 15; MVB: Multivesicular body; UIM: Ubiquitin-interacting motif; HRS: Hepatocyte growth factor-regulated tyrosine kinase substrate; HECT: Homologous to the E6-AP carboxyl terminus; STAMP: Signal transducing adaptor molecule; STAMBP: STAM binding protein; USP8: Ubiquitin specific peptidase 8; Rab5: Ras-related protein Rab5; GEF: Guanine nucleotide exchange factor; GA: Benzoquinone ansamycin Geldanamycin; JNK: c-Jun N-terminal kinase; MEKK1: MEK kinase 1; ITCH Itchy: E3 ubiquitin protein ligase; NEDD4: Neural precursor cell expressed developmentally down-regulated protein 4; Rpn4: Regulatory particle non-ATPase; RING1B: Really interesting new gene 1 protein.The authors declare that they have no competing interests.LKN and BK designed the project. LKN carried out model simulations and analysis. LKN, WK and BK wrote the paper. All authors read and approved the final manuscript.Mathematical models for the investigated motifs.Click here for file