A receptors since effects were blocked by iontophoretically application of APV and bicuculline, respectively. In contrast, stimulation of secondary somatosensory cortex did not affect most of the Pr5 neurons; however both cortices inhibited the nociceptive responses in the Sp5C nucleus through activation of glycinergic or GABAA receptors because effects were blocked by iontophoretically application of strychnine and bicuculline, respectively. These and anatomical results demonstrated that the somatosensory cortices projects to Pr5 nucleus to modulate tactile responses by excitatory and inhibitory actions, while projections to the Sp5C nucleus control nociceptive sensory transmission by only inhibitory effects. Thus, somatosensory cortices may modulate innocuous and noxious inputs simultaneously, contributing to the perception of specifically tactile or painful sensations.The sensory information flow at subcortical relay stations is controlled by the action of topographic connections from the neocortex. To determinate the functional properties of the somatosensory corticofugal projections to the principal (Pr5) and caudal spinal (Sp5C) trigeminal nuclei, we performed unitary recordings in anesthetized rats. To examine the effect of these cortical projections we used tactile stimulation of the whisker and electrical stimulation of somatosensory cortices. Corticofugal anatomical projections to Pr5 and Sp5C nuclei were detected by using retrograde fluorescent tracers. Neurons projecting exclusively to Pr5 were located in the cingulate cortex while neurons projecting to both Sp5C and Pr5 nuclei were located in the somatosensory and insular cortices (>75% of neurons). Physiological results indicated that primary somatosensory cortex produced a short-lasting facilitating or inhibiting effects (<5 min) of tactile responses in Pr5 nucleus through activation of NMDA glutamatergic or GABA In fact, hyperalgesia evoked by infraorbital nerve constriction induce an increase of excitability of Sp5C neurons  than S1 neurons . We have studied whether or not a single somatosensory cortical neuron projects to both the Pr5 and Sp5C nuclei and the electrophysiological effect this may have in these pre-thalamic nuclei.ad libitum. All animal procedures were approved by the Ethical Committee of the Autonomous University of Madrid, in accordance with European Community Council Directive 2010/63/UE. Efforts were made to minimize animal suffering as well as to reduce the number of animals used.Electrophysiological experiments were performed on 49 adult Sprague-Dawley rats of either sexes weighing 250–300 g . Anatomical experiments were performed in 11 adult Sprague-Dawley rats (RE-1 to R-11) weighing 230–290 g. Animals were housed under standard colony conditions and food and water were supplied The anatomical pathways linking cortical areas with the Pr5 and Sp5C trigeminal nuclei were studied by injecting or depositing the neuroanatomical fluorescent retrograde tracers Fluoro-Gold  and Fast Blue  respectively in the Pr5 and Sp5C nuclei in rats.n = 11) were anesthetized with an intraperitoneal injection of a mixture of ketamine (70 mg/Kg) and xylazine (5 mg/Kg) with supplementary doses when necessary (35 mg/Kg and 2.5 mg/Kg respectively) and placed in the stereotaxic frame. After appropriate craniotomy, 60 nl of a 4% saline dilution of FlGo was injected in the Pr5 nucleus by means of a 1 µl Hamilton syringe at stereotaxic coordinates: antero-posterior −9.5 from Bregma, lateral 2.8 and vertical 8.8 according to Paxinos Atlas . After a survival period of 1 week animals were anesthetized with an overdose of the same anesthesia and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.3 followed by increasing concentrations of sucrose solutions  in the same buffer. Brains were stored in 30% sucrose for at least 5 days for tissue cryopreservation and frozen sectioned on the coronal plane at 40 µm; sections were collected in three consecutive ordered series devoted to Nissl staining, cytochrome-oxidase staining, and fluorescent visualization. Sections containing the cerebral cortex of the fluorescent visualization series were studied under a Nikon Axioskop fluorescent microscope. Sections for cytochrome oxidase staining were incubated for 3 h in the complex solution , Cytochrome C (Sigma C-7752) and sucrose (Microbiologie) before being studied under an optical/fluorescent microscope, so both cytochrome and fluorochrome labeled cells could be observed. Series processed for Nissl staining were used for delimiting structures.Animals  using the Tile Scan tool of LAS AF software; samples were analyzed under both lin 405 mm UV and lin Ar 488 mm using a 10x objective. Images are a stack of sections in maximal projection, but neurons were counted in each individual layer.2 and heart rate were monitorized.Data were obtained from urethane-anesthetized (1.6 g/Kg i.p.) Sprague-Dawley rats. Supplemental doses of urethane (0.5 g/Kg i.p.) were given to maintain areflexia. Animals were placed in a Kopf stereotaxic device in which surgical procedures and recordings were performed. The body temperature was maintained at 37°C; the end-tidal COSingle unit recordings were performed by using tungsten microelectrodes  in the Pr5 nucleus  a macroelectrode (120 µm diameter bluntly cut insulated nichrome wire) was lowered 0.5 mm from the cortical surface into the somatosensory cortex. The EEG was filtered between 0.3 and 30 Hz and sampled at 100 Hz. EEG was used to test the level of anesthesia and to correlate the trigeminal neuronal firing with the EEG activity.2, 20 ms duration; Picospritzer) delivered at 0.5 Hz through a 1 mm inner diameter polyethylene tube. To avoid complex responses due to deflections of multiple vibrissae, these were trimmed to 5 mm long, allowing reproducible responses to be evoked. RFs were defined by the limits at which stimuli elicited spike responses.Whisker deflection was performed with a brief, electronically gated, air puff  was aimed at the cortex  to locate a site with a vigorous multiunit response to tactile stimulation of the whisker. After detecting the RF in the S1 or S2 cortex, a bipolar stimulating electrode (120 mm diameter blunt cut stainless steel wire) was aimed at the same site as the recording electrode . Single pulses  or barrages of pulses (3 trains of stimuli at 50 Hz during 500 ms repeated each 2 s) were applied to the S1 or S2 cortex through bipolar electrodes  by a Grass S88 stimulator coupled to a photoelectric stimulus isolation unit. The experimental protocol consisted in a period of control tactile stimulation  followed by 5 min of continuous tactile stimulation after the cortical stimulation barrages.A bipolar stimulating electrode was positioned at stereotaxic coordinates in the ventroposterior medial nucleus of the thalamus  in order to identify thalamic projecting neurons. Pulses of electrical current  were applied through the stimulating electrode. Antidromically-activated neurons were identified by a constant response latency to VPM stimulation and the ability to follow high frequency stimulus train (>100 Hz).In some cases small electrolytic lesions  were made at the end of the recording session to identify the stimulating and recording electrodes in Nissl stained brain sections.The activity of Pr5 and Sp5C neurons was evaluated in control conditions and after unilateral application of capsaicin cream (1.6%) on the vibrissa pad, affecting one or two whiskers. The cream did not impede the movement of the whiskers. Topical application of capsaicin cream induces excitation of nociceptors and, consequently, pain lasting several hours  or the glycinergic receptor antagonist strychnine hydrochloride  were applied iontophoretically with a multibarrel micropipette (20 µm tip diameter). The NMDA receptor antagonist D-2-amino-5-phosponovaleric acid  was also applied iontophoretically. A barrel was filled with 1 M NaCl for extracellular recording and a second micropipette was filled with APV, strychnine or bicuculline. The remaining micropipette, filled with 1 M NaCl solution, balanced the currents. Each barrel of the three-barreled pipette was connected via a silver wire to a channel of a microiontophoresis current generator (WPI current generator). The current generator controlled retention and ejection currents for the drug-filled micropipette. Drugs were ejected with negative (for APV) or positive (for bicuculline and strychnine) current, using a single 10–30 s pulse of up to 200 nA. Retaining currents of 10–20 nA were used to delay drug leakage from the barrel. Since the current pulse used to inject drugs had a small intensity, they only affected the close area around the multibarrel micropipette.The selective GABAt-test was used for comparisons. All data are shown as mean ± standard error.Recordings were accepted for statistical analysis when the spike amplitude fluctuations were lower than 10% throughout the experiment. Peristimulus-time histograms  were calculated from 30 stimuli using the Spike 2 software. The mean tactile response was measured from the PSTH as the number of spikes evoked at 0–50 ms after stimulus onset divided by the number of stimuli. We considered that neurons responded to tactile stimulation when the cell discharged at least one spike every two stimuli. The autocorrelogram time histogram (ACH) was also calculated to reveal oscillatory activity. A paired In order to establish if there are individual or shared corticofugal projections from S1 and S2 to Pr5 and Sp5C trigeminal nuclei, we have injected two different fluorescent retrograde tracers in both trigeminal nuclei. This anatomical study was performed on 11 rats. The locations of the FlGo injection site in Pr5, as well as the FB deposit in Sp5C were confirmed on the sections reserved for fluorescence study of those trigeminal nuclei. Both injections and deposits were confined to the desired site without signals of diffusion in any case  or both (double labeled neurons) tracers used in the experiment have been studied in the cerebral cortex of the animals. In every case the retrogradely labeled neurons were located in layer V, mainly in the cingulate, S1, S2 and insular (Ins) cortices Figure . Single For the quantitative analysis, single FlGo and FB labeled neurons were counted separately; first double-labeled neurons were counted in all sections and then result were contrasted to the superposition of single labeled neurons in all the layers of the confocal stack. The mean percentages of labeled neurons have been reported as the total FlGo, FB and double-labeled neurons in each cortical area Figure . The meaSingle FlGo labeled neurons were present in all the rostro-caudal levels of the cingulate cortex Figure ; howeverThe percentage of single FB labeled neurons (projecting to Sp5C nucleus) was similar in the S1, S2 and Ins cortices (<2%). This means that most of the cortical neurons that project to the Sp5C nucleus are double-labeled cells that also project to the Pr5 nucleus.n = 104), sampled throughout the entire dorsoventral extension of the nucleus, or from lamina III–V of the Sp5C nucleus (at 800–1200 mm deep from the surface n = 70). The Pr5 and Sp5C neurons were silent under spontaneous conditions or displayed a low firing rate (<1 spikes/s) that followed slow EEG activity. Note that spikes tended to occur in the positive wave of the EEG . Eleven out of 14 Pr5 neurons (78%) and 11 of 18 Sp5C neurons (61%) could be activated antidromically. Antidromic spikes were identified by the fixed latency  and the ability to follow stimuli above 100 Hz  or 5.7 ± 0.4 ms in Sp5C neurons  and 5.8 ± 0.6 ms (n = 19) in Pr5 and in Sp5C nuclei, respectively.Electrical stimulation of contralateral S1 cortex with a single stimulus  induced orthodromic discharges of 1–2 spikes at latencies of 4.9 ± 0.5 ms in Pr5 neurons  induced long-lasting changes in tactile responses according to the stimulated cortical RF. When cortical and Pr5 RFs were different (unmatched condition) most of the Pr5 neurons  showed a statistically significant decrease in tactile responses after S1 stimulation trains  at 1 min, returning to control levels 5 min after cortical stimulation (1.6 ± 0.3 spikes/stimulus).Multiunit recordings in S2 cortex revealed larger RFs in the whisker pad than in S1 cortex, so the RFs received inputs from several whiskers. Electrical stimulation of contralateral S2 cortex (3 trains of stimuli at 50 Hz during 500 ms repeated each 2 s) in the unmatched condition induced long-lasting inhibition of tactile responses in 69% of Pr5 neurons  1 min after the cortical stimulating train, and control levels were recovered 5 min later  most Pr5 neurons increased their tactile response . Bicuculline strongly increased the spontaneous firing rate of Pr5 neurons  and tactile responses  and tactile responses in 7 Pr5 neurons  also increased the spontaneous firing rate  in the Pr5 nucleus reduced tactile responses in Pr5 neurons  and Sp5C neurons  5 min after capsaicin application  or 2.2 ± 0.19 spikes/stimulus in Sp5C (n = 22) neurons in control conditions. Tactile responses decreased 5 min after capsaicin application to 1.2 ± 0.15 spikes/stimulus (p = 0.03) or 1.3 ± 0.18 spikes/stimulus (p = 0.007), respectively  or S2 (n = 4) cortical stimulation . In contrast, tactile responses were inhibited when the cortical and trigeminal RFs were non-overlapping. Similar effects have been described in the dorsal column nuclei cells  (Rauschecker, An interesting point is that nociceptive modulation could be performed at different levels of the somatosensory pathway. Recent results suggest that nociceptive responses in the Sp5C may be modulated by supraspinal mechanisms such as serotoninergic projections to Sp5C (Okubo et al., Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nuñez conception and design of research; Eduardo Malmierca and Irene Chaves-Coira performed experiments and analyzed data; Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nuñez interpreted results of experiments and drafted manuscript; Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nuñez approved final version of manuscript.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.


Nonalcoholic fatty liver disease (NAFLD) is a risk factor for Hepatocellular carcinoma (HCC), but he transition from NAFLD to HCC is poorly understood. Feature selection algorithms in human and genetically modified mice NAFLD and HCC microarray data were applied to generate signatures of NAFLD progression and HCC differential survival. These signatures were used to study the pathogenesis of NAFLD derived HCC and explore which subtypes of cancers that can be investigated using mouse models. Our findings show that: (I) HNF4 is a common potential transcription factor mediating the transcription of NAFLD progression genes (II) mice HCC derived from NAFLD co-cluster with a less aggressive human HCC subtype of differential prognosis and mixed etiology (III) the HCC survival signature is able to correctly classify 95% of the samples and gives Fgf20 and Tgfb1i1 as the most robust genes for prediction (IV) the expression values of genes composing the signature in an independent human HCC dataset revealed different HCC subtypes showing differences in survival time by a Logrank test. In summary, we present marker signatures for NAFLD derived HCC molecular pathogenesis both at the gene and pathway level. Nonalcoholic fatty liver disease (NAFLD) is a condition where fat deposits in the liver. NAFLD refers to a wide spectrum of liver diseases such as fatty liver (steatosis) and inflammation derived nonalcoholic steatohepatitis (NASH). This condition can advance to fibrosis and cirrhosis producing a progressive, irreversible liver scarring that in the 15% of the cases progress into a liver hepatocellular carcinoma (HCC). Th. Th30]. Five-fold crossvalidation scheme was used because it preserves a reduced bias in comparison with resubstitution, it estimates the error with lower variance and uses less computational time compared to the leave-one-out crossvalidation . The feaTo assess the stability of a feature selection technique, variation in the distribution of features present in the subsets selected under different partitioning of the training/input data was calculated. The measure used to assess the stability of the selected subsets was the Normalized Average Hamming distance (NAHD) , 31 betwThis analysis design where there are five runs of each of the different methods allowed to further explore the produced signatures in each of the algorithms in terms of their gene composition frequency and frequency of the enriched deregulated KEGG pathways. By selecting the minimum amount of genes and overrepresented KEGG pathway which expression patterns maximized the classification performance of the phenotypes in their corresponding classes, each of the feature selection runs in the external five-fold crossvalidation procedure produced a genomic signature of genes and another one of pathways. These expression signatures showed phenotype and sample discrimination capabilities. To provide more robust feature subsets it was made a solution to the instability of the feature selection method based on the frequency aggregation of the five subsets resulting from the five runs of the crossvalidation which is essentially an ensemble solution that can be called rank summation . FinallyBootstrap resampling techniques were used to assess the uncertainty in hierarchical cluster analysis by calculating probability values  for each cluster in the dendrogram that represents the possibility that the cluster is the true cluster. Two types of p-values were available: bootstrap probability (BP) value and approximately unbiased (AU) p-value. In both cases thousands of bootstrap samples were generated by randomly sampling with replacement elements of the data and bootstrap replicates of the dendrogram were obtained by repeatedly applying cluster analysis to them. BP is biased as discussed in –39 and mClusters with AU larger than 95% were highlighted by rectangles, which are strongly supported by data as in a cluster with AU p-value > 95%, the hypothesis that "the cluster does not exist" is rejected with significance level 0.05 Figs .For the signatures of NAFLD progression microarray samples from different stages of the disease from human and mouse were collected to perform a time course analysis. Using a battery of 14 newly adapted feature selection approaches  robust sInitially raw expression values were used for the 14 supervised clustering feature selection algorithms . Then, tFor the genes composing these signatures enrichment of transcription factor binding sites were explored by the OPPOSUM program using a Fisher exact test (p<0.05)  S1 Tabl. The posBy selecting the orthologous genes using homologene database  the mousVenn diagrams were defined to capture genes that were differentially expressed in human steatosis and NASH when comparing cases and controls . Then we identified the common genes in the prognostic signature present between the differentially expressed genes in human steatosis and NASH.The signature holding the genes determining statistical differences in survival length was further validated by a Logrank test with an independent human HCC dataset having a HBV etiology and for which survival data was available . HierarcIn this study a series of newly adapted feature selection approaches was used to define different robust signatures holding the pathways and genes involved in NAFLD progression as well as a signature of differential survival in HCC common for human and mouse.The NAFLD progression signatures were used to study the pathogenesis of NAFLD derived HCC. Gene expression and pathway deregulation signatures with the genes and pathways that can distinguish different disease stages in human and mice were found using as input 471 genes having twofold regulation or more in 20% of the samples. The signatures produced by the 14 supervised clustering feature selection algorithms described in material and methods were aggregated to generate more robust solutions . WeighteUsing this preprocessed data the 14 supervised clustering and external validation feature selection algorithms were run in order to find signatures which produce the optimal cluster . Both thThe signatures produced by the 14 supervised feature selection methods for raw and smoothed data were aggregated separately and then the resulting pathway signatures were compared with the two feature selection methods which produced the optimal clustering result. It was possible to investigate the functional convergence in these signatures in terms of the overrepresented deregulated pathways whose expression levels can distinguish different disease stages and it was found that the different methods converge in similar functional solutions . The actHepatocyte accumulation of triglyceride is known to be a key component in the development of steatosis and NASH. Hepatic steatosis results from abnormal hepatocyte lipid metabolism that was found to be altered along the disease progression . TriglycThe arachidonic acid metabolism was upregulated in all mouse KO HCC samples and in all human samples  were upregulated in all HCC cases  or posterior refinement selection around the selection solution (y = number of refinement iterations) should be specified. It uses the class vector as input. Stage2: Evaluate the selected gene subset. Stage3a. If the process does not take into account the redundancy of the features (RFE): calculates the sample by sample MI excluding each gene. For each excluded gene defines a coefficient I as the difference of the sum of the sample by sample MI between classes and the sum of the sample by sample MI within groups. Stage 3b1: If the process takes into account the redundancy of the features (RFE_MR): for each gene calculates the average gene pairwise mutual information. Stage3b2: For each gene calculates the Coefficient II value by adding the average gene pairwise MI to the coefficient II. Stage4: Remove the m worst coefficient values and their corresponding genes and expression values. Stage5: Find the minimum error rate along the iterations and get the selected genes. S2 Fig. Flow diagram of the steps performed by the MRMR method. Stage1: As it is a forward search procedure, it starts from an empty set of selected genes. The process is iterative where the number of iterations should be specified and uses the class vector as input. Stage2: Calculate the normalized mutual information of the class vector with the vector containing each gene expression values along the samples. Stage3: a. For each gene calculate the average gene pairwise mutual information. b. For each gene in the subset of selected genes calculate the average gene pairwise mutual information. Stage4: For each gene define a coefficient value by dividing the value of the normalized mutual information with the average gene pairwise mutual information. Stage5: Store the gene having the maximum coefficient value and remove from the matrix the corresponding gene. Stage6: Evaluate. Stage7: Find the minimum error rate along the iterations and get selected genes. S3 Fig. Flow diagram of the GA procedure. Stage 1: The procedure initially creates a number of random variable sets (chromosomes). These variable sets form a population of chromosomes. Each random set is created with an initialization that randomly selects 70 genes from the total 504. Stage 2: Each chromosome in the population is evaluated for its ability to predict the group membership of each sample in the dataset (fitness function). Stage 3: Elitism: select the fittest individual intact for the next generation. Stage 4: The population of chromosomes is replicated. The roulette wheel selection ensures that chromosomes with a higher fitness score will generate a more numerous offspring. Stage 5: The genetic information contained in the replicated parent chromosomes is combined through genetic crossover with a crossover probability (For the parameters see supplementary Parameters in the Genetic Algorithm” supplementary section). The chromosomes are ranked according to their fitness value. Above the crossover probability the best chromosomes are maintained intact for the next generation. Below the crossover probability two randomly selected parent chromosomes are used to create two new chromosomes. This crossover mechanism allows a better exploration of possible solutions recombining good chromosomes. Stage 6: Mutations are then introduced in the new chromosomes generated by crossover randomly with a mutation probability. These mutations produce that new genes are used in chromosomes. Stage 7: The process is repeated from stage 2 until the number of generations exceeds certain threshold (100) and the regression between the population of chromosome’s minimum error rate and the generation is less than 0.05. The cycle of replication (stage 3), genetic crossover (stage 4) and mutations (stage 5) is called generation. S4 Fig. Tree structure where each of the stages of the disease has been clustered in a single cluster. Tree structure where each of the stages of the disease has been clustered in a single cluster using the GS1_clust_FOM algorithm to select the variables used as input in pvclust used to perform hierarchical clustering. S5 Fig. A: Ovalbumin serpin expression along the NAFLD progression.B: Positional gene enrichment. A: Ovalbumin serpin expression along the NAFLD progression. MAT1A_15 and GNMT_ko8 are HCC mice samples where the serpins are overexpressed.B: Positional gene enrichment analysis using PGE program [S6 Fig. 91 human HCC data clustering. Using complete hierarchical clustering using the Pearson correlation as a similarity measure it is possible to distinguish two stable clusters, cluster A and B that show statistical significant differences of survival length using by Kaplan-Meier plots and log-rank statistics analysis. S7 Fig. HNF4 alpha expression (log2 mouse KO vs wild type) in 3 and 8 month GNMT and MAT1A; and 15 month MAT1A . S8 Fig. Expression trend (log2 mouse KO vs wild type) of NAFLD progression genes regulated by HNF4a in 3 and 8 month GNMT and MAT1A; and 15 month MAT1A . S9 Fig. HNF4 alpha expression (log2 disease vs control) in human steatosis and NASH. S10 Fig. Expression trend (log2 mouse KO vs wild type) of NAFLD progression genes regulated by HNF4a in human steatosis and NASH. S11 Fig. Expression trend (log2 mouse KO vs wild type) of biosynthesis of unsaturated fatty acids in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S12 Fig. Expression (log2 mouse KO vs wild type) of stearoyl-CoA desaturase in human steatosis and NASH in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S13 Fig. Expression trend (log2 mouse KO vs wild type) of phenylalanine, tyrosine and tryptophan biosynthesis in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S14 Fig. Expression trend (log2 mouse KO vs wild type) of androgen and estrogen metabolism in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S15 Fig. Expression trend (log2 mouse KO vs wild type) of arachidonic acid metabolism in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S16 Fig. Expression (log2 mouse KO vs wild type) of cyclooxygenase in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S17 Fig. Expression trend (log2 mouse KO vs wild type) of PPAR signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S18 Fig. Expression trend (log2 mouse KO vs wild type) of drug metabolism cytochrome P450 in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S19 Fig. Expression trend (log2 mouse KO vs wild type) of metabolism of xenobiotics by cytochrome P450 in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S20 Fig. Expression trend (log2 mouse KO vs wild type) of toll-like receptor signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S21 Fig. Expression trend (log2 mouse KO vs wild type) of p53 signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S22 Fig. Expression trend (log2 mouse KO vs wild type) of MAPK signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S23 Fig. Expression trend (log2 mouse KO vs wild type) of bile acid biosynthesis in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S1 Table. Summary of the most established biomarkers in NAFLD. S2 Table. Dunn and FOM indexes of the Signatures of NAFLD progression. Dunn and FOM indexes of the Signatures of NAFLD progression resulting from the 14 different supervised clustering based feature selection methods on smoothed data; Ensemble error rate and stability in terms of Hamming distance of the Signatures of NAFLD progression resulting from the 7 different supervised clustering based feature selection methods that minimise the FOM index on smoothed data. S3 Table. Enriched Transcription Factor binding sites. Enriched Transcription Factor binding sites by means of Fisher exact test (p<0.05) in the signatures of NAFLD progression resulting from the two supervised clustering based feature selection methods which produced the optimal clustering result and the two ensemble signatures from raw and smoothed data. S4 Table. Ensemble error rate and the number of the different feature selection methods used to build survival signature. Ensemble error rate and the number of selected genes resulting from the different feature selection methods used to build the survival signatures common for human and mouse. program  shows th(DOCX)Click here for additional data file.


Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.Kisspeptin1 (product of the  KISS1 mutation Kiss1 and Kiss1r genetic knockdown models also show significant deficits in pubertal development and reproductive functions Kiss1r is expressed widely in the brain, including on GnRH1 neurons that form the final common pathway regulating the pituitary-gonadal axis KISS1 and its receptor play major roles controlling the reproductive system in vertebrates kiss gene  and the kiss receptor gene  have been identified in the nervous system of teleost fishes, including the species used in the present study – zebrafish (Danio rerio) Two forms of the Vertebrates express 2–3 forms of GnRH, with distinct genes, locations and functions kiss1 and kiss2 mRNAs are expressed during embryonic and larval development Given that zebrafish Brass GnRH3:EMD transgenic zebrafish were maintained in a zebrafish aquarium system on a 14L:10D photoperiod at 28°C. They were fed twice daily with flake food and live brine shrimp. Sexually mature males and females were maintained in separate tanks until the day before breeding. To control the breeding time, each male-female pair was set in one tank with a divider to separate them. The divider was removed shortly after lights on to allow timed breeding, and fertilized eggs were collected. Embryos were maintained in a 28°C incubator. All embryos for a single set of experiments were from the same parents.All procedures were carried out in accordance with and approved by the Animal Care and Use Committee of UCLA .YNLNSFGLRY-NH2) and Kiss2 (FNYNPFGLRF-NH2) were synthesized by Bachem Inc. , based on the sequence described by Kitahashi and co-workers Biologically-active, 10-amino acid fragments of zebrafish Kiss1  until they were sacrificed, and then fixed with 4% paraformaldehyde (PFA) at various developmental stages for morphological analysis by confocal microscopy. A comparison between the presence and absence of the chorion made no difference to the outcome of kisspeptin treatments on GnRH3 neuron development (data not shown). This indicates that kisspeptin diffuses through the chorion efficiently. In the study on adult zebrafish, embryos were treated with kisspeptins starting at 5 hpf until 3 dpf, and then raised to adulthood in the zebrafish aquarium facility starting at 5 dpf. Adult brain imaging studies were performed at about 8 months of age. For electrophysiology experiments, following the protocol described by Zhao and Wayne (2012) kiss1 and kiss2, embryos were collected daily from 1–7 days post fertilization (dpf) in four replicate experiments. Using RNeasy Plus Mini Kit , total RNA was extracted from homogenates of 20–50 zebrafish embryos or larvae collected at different developmental stages. Reverse transcriptase reaction was performed from 0.5 µg of total RNA using the MultiScribe Reverse Transcriptase . The following is the Reverse Transcriptase (RT) reaction mixture and procedure: 0.5 µg RNA, 10 µl of 2× RT buffer, 1 µl of 20× RT enzyme mixture and RNAase-free water to a final volume of 20 µl. The mixture was incubated at 37°C for 60 minutes and then heat inactivated at 95°C for 5 minutes.To test the early expression of kiss1 and kiss2 primers were previously described by Kitahashi, and co-workers (2009) Elongation factor 1a (ef1a) was used as an internal reference gene for each tested sample. Each sample was processed for quantitative PCR in triplicate. A cycle threshold (Ct)-based relative quantification with efficiency correction normalizing to ef1a was calculated by the 2−ΔΔCt method. The relative quantity of kiss1 or kiss2 gene expression was presented as the fold difference to that of 1 dpf embryos. The sequences of the primers used for quantitative RT-PCR are shown in ef1a, kiss1 and kiss2 primer sets were 88%, 100%, and 108%, respectively, as determined by serial dilution experiments.Quantitative real-time PCR was conducted using the SYBR Green PCR Master Mix  following the manufacturer's instructions in an Mx 3000P System . The reaction was composed of the following: 1.5 µl of the RT reaction, 10 µl of 2× qPCR mixture with the appropriate forward and reverse primers to a final volume of 20 µl. The final concentrations of the primers in the qPCR reaction were 1.25 µM. The nucleotide sequences of the qPCR Synaptic vesicle protein 2 (SV2) is a marker of synaptic transmission, including in zebrafish Images were acquired and analyzed with Fluoview software using an upright Olympus microscope . Three sets of experiments were performed: 1) analyze neuron number from the different GnRH3 neuronal populations to assess the effects of kisspeptins on early embryonic development of the GnRH3 neural circuit (18–25 hpf); 2) analyze the number of synaptic boutons on the cell bodies and along the neuronal processes of the bilateral clusters of TN-GnRH3 neurons to evaluate the effects of kisspeptins on potential synapse formation during embryogenesis (50 hpf) by means of whole mount immunohistochemical staining with SV2 antibody; 3) analyze the number of hypothalamic GnRH3 neurons from adult zebrafish that were treated with kisspeptins as embryos to determine if early changes continued into adulthood. Embryos were mounted in 0.8% agarose in two positions, lateral-side up  or ventral-side up , then viewed and imaged using water immersion 5×, 20× or 40× objectives . Z-stacks of images were taken at the interval of 0.5 µm.To count the number of boutons, we overlaid the projection images of both green-488 (EMD) and red-594 (SV2-ir) channels of bilateral TN-GnRH3 neuron clusters and their processes taken with 40× water objective with 3× digital zoom, counted the yellow  spots (>0.1 µm) on the cell bodies and along the neuronal processes projecting from the bilateral clusters, section by section through the projection stacks To assess whether or not embryonic treatment with kisspeptins had long lasting effects into adulthood, embryos were treated 100 nM Kiss1, 100 nM Kiss2, or control solution (5 hpf- 3 dpf) and maintained in a zebrafish aquarium system for eight months until sacrifice. Adult intact brains were removed and fixed in 4% PFA/10% sucrose in PBS at 4°C overnight. After fixation they were washed three times with 20% sucrose in PBS and were incubated at 4°C overnight. The brains then were left in the mixture of 20% sucrose and OCT compound (Tissue-Tek) (1∶2) again at 4°C overnight. The next day, the brains were sliced horizontally (50 µm thickness) using a cryostat (Leica CM1850) at −20°C. Fluoview software was used to image and to count the number of GnRH3-EMD neurons in hypothalamus using confocal imaging . Analysis of numbers of cell bodies was performed by one of the authors (MAL) and another researcher (MF) who were blind to the treatments.Whole-cell patch electrophysiology was conducted as previously described p<0.05.Values are shown as mean ± SEM. Data were analyzed using Prism 6 . Statistical significances among experimental groups in the imaging studies were determined by one-way ANOVA, followed by the Tukey's multiple-comparison test. Paired t-test was used for analysis of electrophysiological data. Differences were considered significant if kiss1 and kiss2 gene expression from 1–7 dpf  kiss2 mRNA increased more dramatically than kiss1 mRNA between 1 and 7 dpf .Using quantitative RT-PCR, we tested the dynamic changes of both  1–7 dpf . Both kiOur previous work Our previous study showed that multiple populations of GnRH3 neurons emerged in this transgenic zebrafish model by 30 hpf Our earlier work showed that TN-GnRH3 neurons express synaptic vesicle protein (SV2) on their neural processes during development, suggesting that these neurons are capable of synaptic communication during embryogenesis The functions of GnRH3 neurons are dependent upon their locations in zebrafish kiss1 and kiss2 genes are expressed by 1 dpf, and their mRNA levels increase progressively during embryonic and larval development in zebrafish. These findings corroborate earlier work in zebrafish showing increased kiss1 and kiss2 mRNA expression between 1–7 dpf, with peak levels achieved just prior to or during the time of puberty kiss1/2 during zebrafish embryogenesis is similar to what was observed in cultured embryonic brain explants from mice in which Kiss1 was also expressed early in development kiss1 and kiss2 mRNA expression peak at somite stage 19 or 1 dpf before declining to low levels by stage 26 or 2 dpf. The expression of both kiss mRNAs stayed low through 8 dpf We show that Both Kiss1 and Kiss2 stimulated the number of GnRH3 neurons in the developing trigeminal neuron population. This was the only effect of Kiss2 observed on any aspect of GnRH3 neuron biology during early development that we investigated. The trigeminal population of GnRH3 neurons is unusual because it is located in the peripheral nervous system. Trigeminal neurons are part of the sensory system and, in part, mediate the effects of mechanosensory stimuli on escape behavior in zebrafish Recent work from our laboratory showed the expression of SV2 on GnRH3 neurons during early embryogenesis in zebrafish, indicating the potential for synaptic transmission while the GnRH3 neural network was still developing Recent work showed that Kiss1, but not Kiss2, could stimulate spike frequency of TN-GnRH3 neurons in the zebrafish embryo – but only in those neurons that showed a mature tonic pattern of action potential firing. TN-GnRH3 neurons that showed the more immature bursting pattern of action potential firing were unresponsive to the stimulatory actions of Kiss1 kiss1, kiss2, and their receptors in adult zebrafish. The anatomical data suggests that Kiss2, and not Kiss1, is the primary regulator of the brain-pituitary-gonadal axis in zebrafish The physiology data in the present study is at odds with anatomical data showing localization of mRNAs for kiss1/kiss1r and kiss2/kiss2r in medaka indicated that kiss1 and both kiss1r and kiss2r play critical roles in neurulation and embryonic development, but not kiss2gnrh1 and gpr54-2 expression. However, during gonadal recrudescence, Kiss1 was more potent than Kiss2 in elevating plasma luteinizing hormone levels; while Kiss2 down-regulated gnrh1 and gpr-54-2 expression in uteroOverall, our findings that Kiss1 is the dominant stimulator of GnRH3-neuron morphological (present study) and electrophysiological development In summary, the present morphological findings in embryos show that kisspeptins modulate the development of multiple populations of GnRH3 neurons, suggesting it may play a role as a neurotrophic factor and possibly coordinate maturation of this neural network. The electrophysiology data in adult zebrafish suggests that both Kiss1 and Kiss2 regulate the activities of hypophysiotropic GnRH3 neurons. Kiss1 is an activator, while Kiss2 is an inhibitory RFamide. The functional balance of these two neuropeptides may be crucial in central control of reproduction in zebrafish.


Atoh1 (Math1) was the first gene discovered in ear development that showed no hair cell (HC) differentiation when absent and could induce HC differentiation when misexpressed. These data implied that Atoh1 was both necessary and sufficient for hair cell development. However, other gene mutations also result in loss of initially forming HCs, notably null mutants for Pou4f3, Barhl1, and Gfi1. HC development and maintenance also depend on the expression of other genes  and several genes have been identified that can induce HCs when misexpressed (Jag1) or knocked out (Lmo4). In the ear Atoh1 is not only expressed in HCs but also in some supporting cells and neurons that do not differentiate into HCs. Simple removal of one gene, Neurod1, can de-repress Atoh1 and turns those neurons into HCs suggesting that Neurod1 blocks Atoh1 function in neurons. Atoh1 expression in inner pillar cells may also be blocked by too many Hes/Hey factors but conversion into HCs has only partially been achieved through Hes/Hey removal. Detailed analysis of cell cycle exit confirmed an apex to base cell cycle exit progression of HCs of the organ of Corti. In contrast, Atoh1 expression progresses from the base toward the apex with a variable delay relative to the cell cycle exit. Most HCs exit the cell cycle and are thus defined as precursors before Atoh1 is expressed. Atoh1 is a potent differentiation factor but can differentiate and maintain HCs only in the ear and when other factors are co-expressed. Upstream factors are essential to regulate Atoh1 level of expression duration while downstream, co-activated by other factors, will define the context of Atoh1 action. We suggest that these insights need to be taken into consideration and approaches beyond the simple Atoh1 expression need to be designed able to generate the radial and longitudinal variations in hair cell types for normal function of the organ of Corti. Pou4f3  were completely deaf, “owing to a failure of HCs to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia”  development and thus could be used to regenerate HCs and restore hearing was born in the late 1990s: Mice with a deletion of the Pou domain gene Pou4f3 relevant for the discussion of the role of Atoh1 (aka Math1) for HC differentiation and maintenance? In the following we will explore that Atoh1 has much in common with Pou4f3 in terms of claims raised as a gene that is “necessary and sufficient” for HC differentiation  of the cerebellum that are unable to migrate and differentiate and eventually die  such as Ascl1, Neurog1 and, rarely, Atoh1  (Ono et al., In the original paper describing absence of HC differentiation in Atoh1 null mutant mice, some supporting cells stain for the LacZ used to replace Atoh1 (Bermingham et al., Atoh1 expression in inner pillar cells may be counterbalanced by Hes and Hey factors (Doetzlhofer et al., Neurod1 suffices to turn some neurons into HCs expressing Atoh1 and Myo7a (Jahan et al., Using the same LacZ knockin model as previous papers (Bermingham et al., Atoh1 generates a “self-terminating” system that results in loss of Atoh1 after a transient presence of Atoh1 protein (Pan et al., Atoh1 and how long residual Atoh1 protein remains in the cell. Thus, while all cells will see recombination of the LoxP flanked Atoh1, this varies between HCs and thus results in different delay lines of HC precursor apoptosis (Pan et al., Atoh1 is replaced by Neurog1 (Jahan et al., Atoh1. A recently available hypomorph mutant of Atoh1 shows a somewhat similar picture of longitudinal and a less clear radial HC loss (Sheykholeslami et al., More recent data provide yet a more complicated picture of lack of Atoh1 expression on HC and OC differentiation. Using an Atoh1 enhancer to drive Cre that activates the Cre only upon presence of Atoh1 protein combined with floxed Data using inducible Cre expression have complicated this picture even further by showing a rapid and complete loss of all HCs when Cre is induced at different stages of late development (Cai et al., In summary, Atoh1 is, much like Pou4f3, a critical factor for HC differentiation and long term maintenance. Atoh1 is involved in regulating Pou4f3 whereas and its long term expression may be dependent on Atoh1 expression. Further work combining the recently reported hypomorphic allele (Sheykholeslami et al., Why is it important to go beyond the idea of “necessary and sufficient” for Atoh1 function in the ear? First, while unregulated expression of Atoh1 can convert most ear cells into hair cells (Kelly et al., Second, most HCs generated with Atoh1 treatment alone have limited long term viability. In part this may relate to the progressive loss of Atoh1 in these experiments that may needed to maintain long term Pou4f3 expression (Masuda et al., Finally, while the single gene approach to HC regeneration has been extremely influential to catapult much research forward, it is now time to reflect why this approach has not lived up to its promise. We therefore suggest more complex procedures that recapitulate steps in development of the OC in addition to Atoh1. For example, expressing Eya1, Pax2, Sox2, Jag1, Foxg1, Neurod1, Neurog1, and Gata3 prior to Atoh1 expression may “prime” remaining cells of the OC to respond to Atoh1. Alternatively, combining Atoh1 with downstream essential genes for HC maintenance that are only partially regulated by Atoh1 (Ahmed et al., 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.


TPVM is a promising technique for measuring myocardial mechanics  but longK-space is fully sampled with 8 spiral interleaves . Data is acquired at 2 levels of acceleration, acquiring 4 (R2) and 3 (R3) out of the 8 spirals in order to assess the effect of acceleration on the measured velocities. Velocity compensated and encoded data are acquired in consecutive heartbeats, with an initial heartbeat used to collect coil sensitivity information (breath-hold durations are 17 and 13 heartbeats for R2 and R3). Acquired spatial resolution is 1.7 × 1.7 mm (reconstructed pixel size 0.85 × 0.85 mm). Retrospective cardiac gating is used to cover the entire cardiac cycle (50 phases reconstructed). Images are passed to the Gadgetron for reconstruction and returned to the scanner for viewing within 1 min 20 s. Basal, mid and apical short-axis slices were acquired in 10 healthy volunteers on a Siemens Skyra 3T scanner.Example images and velocity maps for R2 and R3 are shown in Figure The use of spiral trajectories, non-Cartesian SENSE and the Gadgetron GPU reconstruction framework has allowed the acquisition of high temporal resolution TPVM images within a breath-hold time that is easily achievable in the clinical environment (13 heartbeats), while reconstruction time is short enough to allow viewing at the time of scanning. The acceleration is not affecting peak velocity measurements (comparisons with previous unaccelerated spiral data [NHIR CBRU, Royal Brompton Hospital. HRUK grant RG2584.


Efficient right ventricular (RV) pumping function requires optimal blood flow dynamics. In the left ventricle (LV), diastolic vortex ring formation distal to the mitral valve (MV) has been reported to be an important mechanism for such blood flow optimization. Earlier work based on computational fluid dynamics (CFD) simulations using simplified RV geometry modeling have reported vortex ring formation in the RV during the early filling phase and its breakdown at the late diastolic phase. However, neither those CFD studies have characterized vortex rings nor have they been confirmed by 4D flow MRI. The purpose of this study was to investigate and characterize the formation of vortex rings during diastolic filling in the RV and to compare them with those of LV in healthy volunteers.Ten healthy volunteers (age: 20 ± 7 years) underwent three-dimensional (3D), time resolved, three-directional velocity-encoded MRI at 3T (Philips). MRI was performed in a 3D isotropic dataset of 4.2 × 4.2 × 4.2 mm 3 with whole heart coverage. Retrospective gating with 30 phases reconstructed and velocity sensitivity of 150 cm/s in all directions was used. The Lambda2 (λ2) method was used to extract the 3D vortex structures inside the RV at the phases of early (E) and late (A) filling. The most circular and compact ring was extracted from each phase. The location of a vortex ring was characterized by its longitudinal position (L) and its orientation : project number 11626.


Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame ( Sesamum indicum L.) is one of the first oilseed plants to be used for many different purposes [Sesame  is a powerful alternative method for phytoplasma detection and quantification because it offers elevated detection sensitivity, short analysis time, and high automation capability . Real-tiSesame phyllody, an economically important disease of sesame plants, is a serious threat in regions where peanut witches’ broom (16SrII) ,12,38,39Permissions from the owners of sesame fields for sampling were obtained. No endangered or protected species were involved in the study.Orosius orientalis was identified as the only insect vector of phyllody phytoplasmas in our previous study [Plant and insect samples were collected from naturally infected sesame fields in the Aksu, Aspendos, Batem, Belkıs, Beşkonak, Boğazkent, Bozova, Cumalı, Denizyaka, Gündoğdu, Kadriye, Kocayatak, Kovanlık, and Taşağıl locations of Antalya province from June to September 2011 through 2014 . Additious study , the qPCus study ,44,45.Total DNA from samples of both sesame plant and vector insects was extracted by the CTAB method . A 100 m® qPCR assay. Representative sequences of 16Sr groups I through XIV [® were designed from the18S ribosomal DNA sequence (AJ236041) to detect sesame plant DNA in qPCR. This sesame DNA specific primer pair and probe were developed to serve as an endogenous control to normalize the DNA quantities and to allow for relative quantification of the phytoplasmas in infected plant tissue. The plant primer pair and probe combination were also used to verify the quality of extracted DNA and the absence of PCR inhibitors. Primer Express software  was used to design qPCR primers and probes. They were synthesized by Metabion International AG .Primers and probes designed in this study are listed in ough XIV ,48,49 , MA , ASHY2 , and AP15  were kindly provided by Dr. Assunta Bertaccini of the University of Bologna in Italy. Extracted DNA from phytoplasma samples was amplified with universal phytoplasma primers by nested PCR [Specificity of qPCR assay for 16SrII and 16SrIX group phytoplasmas was determined by testing against DNA from 4 phytoplasma samples representing 4 different 16Sr groups. The samples of phytoplasma strains PPT . qPCR was performed in 20 μl reaction volumes, containing 2XTaqMan PCR master mix , 300 nM forward and reverse primers, 150 nM 16SrII LC Red, 16SrIX Texas Red, and Sesame 18S-Hex labeled probes, and 2 μl of template DNA (10 ng per reaction). The thermal cycling protocol consisted of an initial denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec and at 60°C for 1 min. Cycle threshold (Ct) values were calculated and analyzed with the Rotorgene Q series software version 1.7 .® PCR purification kit  and quantified using a spectrophotometer. The copy number of PCR amplicons was calculated using the following formula: Copy number = (Amplicon amount x Avogadro's number) / (Amplicon size in bp × 650 x 1 x 109) [8 to 1.8 × 102 and 1.6 × 108 to 1.6 × 102 copies per reaction, respectively, was qPCR-amplified in triplicate to generate a standard curve for each phytoplasma group. qPCR assays were also performed to determine whether PCR inhibitors were present in DNA extracted from sesame plant tissues. Each dilution series of 16SrII and 16SrIX group DNA used for construction of phytoplasma standard curves was added separately to 2 μl (10 ng per reaction) sesame DNA from healthy tissue to produce the same final concentrations in each reaction and simulate proportions of naturally infected sesame tissue. Standard curves were also created from amplification of these samples containing 16SrII or 16SrIX group DNA and sesame DNA. Absolute quantities of 16SrII and 16SrIX phytoplasma DNA in infected sesame plant and insect tissues were calculated based on standard curves using comparative cycle threshold (Ct) values of sesame field test samples.qPCR assays were used to determine both absolute and relative quantities of 16SrII and 16SrIX group phytoplasmas in sesame plant samples and absolute quantities only in insect samples. DNA extracted from sesame phyllody plant and insect samples was added to a multiplex qPCR reaction mix and amplified to determine the amounts of each phytoplasma in field samples. Negative control (uninfected sesame checked by nested PCR), and non-template control (PCR water) were included in each independent run. Standards were prepared by amplifying 16S rRNA sequences of 16SrII and 16SrIX group sesame phyllody phytoplasmas directly with the primer pair Fu5/Ru3 from DNA extracted from sesame samples infected with only the respective 16Sr group phyllody phytoplasmas in conventional PCR ,45. AmplTo measure relative quantities of 16SrII and 16SrIX group phytoplasma DNA in sesame tissues, relative qPCR using the comparative Ct method was used as described in Rotor-Gene Q series user bulletins . Ten ng of total DNA extracted from field collected sesame tissues was simultaneously amplified with both phytoplasma specific and endogenous control primers (18S rDNA) in the same tubes. The lowest detectable dilutions of 16SrII and 16SrIX determined by inhibition assays were designated as calibrators for phytoplasma group quantification experiments. The quantities of calibrators were regarded as 1 and the quantities of all the other field samples were expressed as n-fold difference relative to the calibrators. Absolute quantities calculated from standard curves were omitted in relative quantification because sample quantities were divided by the calibrator quantity. Quantities of 16SrII and 16SrIX group DNA normalized to the endogenous control sesame DNA and relative to the calibrators were either calculated automatically by the software or manually .® Green  revealed that the primer pair SPHY-16SrII-IX-F/SPHY-16SrII-IX-R amplified a 136 bp PCR product from all 16Sr group phytoplasma sequences tested  of 16SrII and 16SrIX purified PCR products, and sesame DNA to detect sensitivity limits of the qPCR assay . The inibination . Linear bination .4 pg of DNA. Addition of sesame SEPL-18SDNA-F/R primers and the SESAME probe into phytoplasma primers and probes did not affect specificity. The slope, intercept, correlation coefficient, and efficiency values of standard curves for all assays are given in Internal control primer pair efficiently amplified sesame plant DNA through at least five orders of magnitude of 10-fold dilutions beginning with 1.0 × 10A qPCR reaction combination of phytoplasmas and sesame primers and probes was used to amplify serial DNA dilutions of the target phytoplasma group (16SrII or 16SrIX) DNA in the presence of sesame plant DNA in inhibition tests. The multiplex assays successfully amplified the same seven 10-fold target 16SrII and/or 16SrIX DNA dilutions and resulted in consistent Ct values through 30 cycles . The sta2 and 1.6 × 102 copies per reaction, respectively. Amplification efficiencies of the 16SrII (0.98) and 16SrIX (1.0) assay in the presence of sesame DNA (Detection limits of the multiplex qPCR assay for 16SrII and 16SrIX group phytoplasmas in the mixed phytoplasmas and sesame DNA were found to be 1.8 × 10same DNA  were fousame DNA .DNA extracts from 109 plant and 92 insect samples collected from 15 different locations were tested for the presence of sesame phytoplasmas in multiplex qPCR developed in this study . Amplifi7), had the highest and Bogazkent (1.12 × 106) lowest absolute quantities (copies) of 16SrII DNA per mg of plant tissue. In relative quantification, the highest and lowest amounts of 16SrII phytoplasma DNA were from the Gundogdu and Belkıs district samples (6) contained the highest amounts of 16SrIX phytoplasma per mg of sesame tissue.Absolute and relative quantities of 16SrII and 16SrIX phytoplasma DNAs were determined for the 94 samples from which sesame phytoplasmas were detected . The sam samples . Absolut5) per mg. Samples from other districts contained 16SrII DNA ranging from 4.26 × 104 to 4.97 × 103 copies of DNA per mg insect tissue. Insect samples from the campus greenhouse had high 16SrII DNA quantities (1.63 × 107 copies per mg of insect tissue) because these insects were continuously fed on sesame plants with phyllody disease in cages for transmission assays.Quantities of 16SrII DNA were also found to be quite low in insect samples. Of all districts sampled, insect samples from Kovanlık had the highest quantities of 16SrII DNA Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigSpecific detection of 16SrII (A) and 16SrIX (B) group phytoplasmas and housekeeping gene (C).(DOCX)Click here for additional data file.


Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane–cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in  Arabidopsis thaliana mutational analysis, CESAs are not assumed to work alone. Some proteins were predicted to be associated with the cellulose synthase complex, although their direct interaction has never been demonstrated. One of these proteins is KORRIGAN (KOR1), a membrane-bound endo-1,4-β-D-glucanase with a single transmembrane domain and two putative polarized targeting signals in the cytosolic tail Pichia pastoris cleaves non substituted but non-crystalline 1,4-β–linked glucan chains and shows no activity against xyloglucans Agrobacterium tumefaciens and Acetobacter xylinumCellulose is synthesized by a large rosette terminal complex, which comprises at least three different cellulose synthases (CESAs). On the basis of KOR1, two additional genes encoding membrane-anchored endoglucanases (EGase) have also been characterized in A. thalianaKOR1 gene is the most widely expressed membrane-anchored EGase throughout various plant tissue while KOR2 and KOR3 are active in specific cell types. Expression of a GUS reporter gene driven by the endogenous promoters of KOR2 and KOR3 have shown that KOR2 is active in trichomes and floral organs while KOR3 is active in developing root hairs. Microsomal fractioning demonstrated that KOR1 is present in the tonoplast, the Golgi apparatus Besides korrigan (kor1-1) showed defects in some aspect of cell wall loosening in primary cell wall biosynthesis KOR mutations (kor1–2) have shown to cause the formation of aberrant cell plates and incomplete cell walls acw1 altered cell wall and rsw2 root swelling) showed abnormal plant morphology, defects in primary cell wall formation, reduced cellulose content, increased pectin synthesis, and aberrant cell division similar to that found in the CESA1 mutant rsw1acw1 mutant grown at 31°C and shows a 40% reduction in crystalline cellulose content compared to the wild type rsw1) and KOR (rsw2) in comparison to either of the single mutants which demonstrates that cellulose biosynthesis in the primary cell walls of plants requires both a glycosyl transferase and glycosyl hydrolase providing further evidence that KOR has a key role in cellulose deposition in the primary cell wall The dwarf mutant KOR homolog was observed during secondary cell wall deposition in cotton irx2-1 and irx2-2) in the KOR1 genes showed similar phenotypes as the irx mutants, i.e. collapsed xylem cell walls due to reduced cellulose synthesis in the secondary cell wall in the base of mature stems kor1-1 mutants that were reported to be primary cell wall mutants also had severely collapsed xylem cells similar to the irx2 mutants KOR genes from hybrid poplar led to moderate to severe defects in plant growth, an irregular xylem (irx) phenotype commonly associated with KOR and other secondary cellulose-specific mutants in A. thalianaReports have indicated that KOR also plays a role in secondary cell wall development. An accumulation of a Since KOR appears to be associated with cellulose synthesis and direct association has been detected with the primary cellulose synthase protein complex The constructs for the MbYTH system concerning the primary and secondary CESAs were generated as described previously Saccharomyces cerevisiae in the Split Ubiquitin System kit . Interactions were performed according to supplier instructions (DUAL membrane Kit 1) and were tested with KOR1 fused to the C-terminal part of the ubiquitin (Cub) and the transcription factor (bait), whereas the CESA1, 3 and 6 proteins were fused to the N-terminal part of the ubiquitin . The bait and prey constructs were co-transformed into the yeast strain NMY51  according to the provided transformation procedure (DUAL membrane Kit 1). Upon interaction between the bait and the prey the transcription factor (TF) is released into the nucleus where it activates reporter genes allowing the yeast to grow on selective medium lacking leucine and tryptophan (SD med. -L-T), and subsequently grown at 30°C for three days. To quantify the interactions between different preys 100 colonies of each combination were spotted onto selection medium containing the appropriate amount of 3-ammonium-triazole (3-AT) and grown at 30°C for three days. The number of spots grown was then counted.The interactions between the CESAs and KORRIGAN1 were assayed with the split-ubiquitin membrane-based yeast two-hybrid CESA genes, KORRIGAN1 as well as the truncated forms of KOR1 were generated through Phusion DNA Polymerase  with suitable primers  have been demonstrated irx) mutants also link KOR1 to cellulose synthesis in the secondary cell wall. To test a possible interaction between KORRIGAN and the secondary CESA proteins  the KOR1 was expressed as bait in combination with the secondary CESA as prey. As a negative control, we tested KOR1 as bait with an unrelated protein AGL5 as prey . The yeast strains failed to grow in multiple repetitions of this experiment, demonstrating that a specific interaction between the bait and prey is required to activate the system , and an extracellular catalytic domain . In ordein vitro . Vice vein vitro .in planta, truncated forms of KOR1 were tested for interaction with the primary  and secondary  CESAs using the BiFC assay. All the primary CESAs, CESA1, 3 and 6, were able to interact with KOR1N and KOR1C truncated proteins  and also with CESA4 and CESA8 from the secondary cell wall  and secondary  CESAs using the BiFC assay. Confirming the in vitro assay results, the primary CESAs, CESA1, 3 were able to interact with the truncated protein KOR1TMD  .in planta. Our results showed that fluorescence was restored when two different fusion proteins (YFP/C-KOR1 and YFP/N-KOR1) were expressed in Nicotiana benthamiana, indicating the formation of homodimers or higher oligomers of KOR1 proteins  only two of the secondary CESAs (CESA4 and 8), suggesting possible specificity for the different CESAs from the primary and secondary cell wall.KORRIGAN1 has shown to play an important role in cellulose biosynthesis as the knockouts out of this gene results in reduced cellulose content. Despite previous studies indicating that some primary CESAs do not co-immunoprecipitate with KOR1 kor1-1 dwarf mutant korrigan, (kor1-1) phenotype results in a severe reduction in crystalline cellulose both in the primary and secondary cell wall kor1-3 and kor1-1 mutants than in controls acw1 mutation effects cellulose accumulation in the cell wall and microfibril formation resulting in 40% reduction in crystalline cellulose content compared to wild type plants.The alterations in cellulose content in the kor1-1 mutant that showed distinct primary cell wall defects also showed signs of severely collapsed xylem similar to the irx2 mutants in the secondary cell wall in vitro and in planta we demonstrate that there is indeed a physical interaction between KOR1 and the CESAs in the secondary cell wall except for CESA7. Other reports have also indicated that KOR plays a role in secondary cell wall development. Co-expression of Populus KOR orthologue with the three secondary cell wall CESA proteins has also been reported Atkor1 orthologue in hybrid aspen (PttCel9A1) causes a decrease in cellulose crystallinity irx2-1 and irx2-2 KOR1 mutants showed reduced cellulose synthesis (30% of the WT) in the secondary cell wall irx mutants. PtKOR1 is also shown to be required for secondary cell wall cellulose formation. RNAi suppression of PtKOR1 revealed a significant decrease in crystalline cellulose content and a reduced secondary cell wall thickness It must also be mentioned that the Interestingly, not all the CESAs in the primary and secondary cell wall have a similar interaction pattern with KOR1. The different reaction between the primary and the secondary cell wall proteins is difficult to explain as the specific functions of the different CESA proteins are not known. As previously mentioned CESA1 was the most efficient interactor in the primary cell wall whereas CESA7 from the secondary cell wall did not interact with KOR1 suggesting that specific CESA isoforms may have unique roles in recruiting KOR1. In other words the binding of KOR1 to the different CESA proteins is specific, as KORRIGAN1 does not bind to all of them. Not only does this imply that the methods used are sensitive enough to specifically determine interactions between these highly homologous proteins, it also indicates that the KOR1 protein has a specific position within the rosette.in vitro, CESA6 and CESA4 show a very weak to no interaction with KOR1TMD in planta suggesting that at least for the two mentioned CESAs the presence of TMD is essential but not sufficient for interaction and would require more than just the TMD. It is interesting to note that some proteins required for cellulose synthesis are found in sterol-rich lipid rafts A more detailed view on the interaction between KOR1 and the CESA proteins using truncated versions of KOR1 revealed that all portions which contain the TMD were able to interact and this led to the conclusion that KOR1 transmembrane domain is required for the interaction with CESAs. These data suggest that KOR1 and CESA associate in the CSC in the plasma membrane, between the TMD of KORRIGAN and the TMDs of the CESA proteins. Surprisingly, despite showing interaction in vitro and in planta supporting the hypothesis that KOR1 makes up a part of the rosette structure. The physical interaction also indicates that KOR1 is directly involved in cellulose biosynthesis, and probably does so in the form of a homodimer or a higher order oligomer. Furthermore, our study showed that the TMD of KOR1 is essential not only in the interaction with the different CESA proteins, but also for KOR1 homodimerization.In conclusion, we have determined that the KOR1 protein interacts with secondary cellulose synthase complex proteins both Figure S1Interactions between the different KOR1 domains and the different CESA proteins using the Membrane-based Yeast Two Hybrid. The bars represent the percentage of yeast colonies grown for 3 days on selective medium at 30°C. The different CESA proteins were expressed in yeast as prey and the different KOR1 protein domains as bait.(PDF)Click here for additional data file.Figure S2Bimolecular Fluorescence Complementation (BiFC) experiments in tobacco leaf epidermis. Confocal images are presented, showing YFP fluorescence indicating interaction. Tests for interactions between the CESAs and truncated versions of KOR1 are shown (A) KOR1N/CESA1, (B) KOR1C/CESA1, (C) KOR1TMD/CESA1, (D) KOR1N/CESA3, (E) KOR1C/CESA3, (F) KOR1TMD/CESA3, (G) KOR1N/CESA6, (H) KOR1C/CESA6, (I) KOR1TMD/CESA6, (J) KOR1N/CESA4, (K) KOR1C/CESA4, (L) KOR1TMD/CESA4, (M) KOR1N/CESA8, (N) KOR1C/CESA8, (O) KOR1TMD/CESA8, (P) KOR1C/CESA7, (Q) KOR1N/CESA7, (R) KOR1TMD/CESA7. Scale bars = 100 µm.(PDF)Click here for additional data file.Table S1Primers used for the MbYTH system.(PDF)Click here for additional data file.


YHWAE with NUTM2B was identified. Integrated analysis of expression and methylation data identified promoter hypermethylation and low expression of the tumor suppressor gene TCF21 (Pod-1/capsulin/epicardin) in all CCSKs except the case with t. TARID, the long noncoding RNA responsible for demethylating TCF21, was virtually undetectable in most CCSKs. TCF21 hypermethylation and decreased TARID expression were validated in an independent set of CCSK tumor samples. The presence of significant hypermethylation of TCF21, a transcription factor known to be active early in renal development, supports the hypothesis that hypermethylation of TCF21 and/or decreased TARID expression lies within the pathogenic pathway of most CCSKs. Future studies are needed to functionally verify a tumorigenic role of TCF21 down-regulation and to tie this to the unique gene expression pattern of CCSK.Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants  were identified. One tumor with t involving fusion of  Clear Cell Sarcoma of the Kidney (CCSK) comprises approximately 5% of all renal malignancies in children, and is observed most often below 3 years of age , 2. CCSKYWHAE intron 5 (17p13) and NUTM2 intron 1 (10q22), resulting in fusion of YWHAE exon 5 to NUTM2 exon 2  . cD. cDTARIDYHWAE exon 5 and NUTM2 exon 2 [RT-PCR analysis was performed using previously reported primers for 2 exon 2 . Briefly


Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube  Genetic reprogramming offers unprecedented opportunities for regenerative medicine . Geneticex vivo; clonal analysis of independent amniocentesis samples indicate that the vast majority of cells do not proliferate , 1% L-glutamine and 1% penicillin/streptomycin solution). Cells were routinely maintained on culture wares pretreated with 1:100 dilution of growth factor reduced matrigel (BD Biosciences). All media components in this work were obtained from Life Technologies unless stated otherwise.5 cells/cm2 for immediate use or cryropreserved with standard methods after 24 h recovery. Conditioned hESC media was prepared by culture of inactivated MEFs in hESC media without bFGF for 24 h, supplemented with 4 ng/ml bFGF and filtered sterilized before use. Feeder-free cultures were maintained in MEF-conditioned hESC media, mTeSR-1 (StemCell Technologies) or Essential 8 (Life Technologies) media. Passaging of PSCs cultured on MEF feeders or in MEF-conditioned media was done by manual microdissection of optimal undifferentiated colonies with a fire-polished glass pipette using a dissecting microscope. Feeder-free cultures were passaged with EDTA as described (The H9 (WA09) line of human embryonic stem cells (hESCs)  and iPSCescribed . The ROC5 target cells were seeded at subconfluent densities ∼1.4 × 103 cells/cm2 and transfected the following day with pooled plasmid combinations in equimolar ratios (∼0.2 µg DNA/cm2) with Fugene HD  0.15 µl/µg DNA at 8 to 12 h intervals for a total of 3 transfections. Transfected cells were maintained in DMEM 15% for ∼4 days and then switched to MEF conditioned hESC media supplemented with 2.5 mM valproic acid (Sigma-Aldrich) for ∼2 weeks after colonies appeared. Independent populations of ChMRC.B1p3 cells were transfected with the 3-vector combination and 7 to 9 colonies were recovered from each population. A single representative colony was selected from each and maintained separately as iChMRC.B1A, iChMRC.B1C, and iChMRC.B1E candidate populations. A population of ChM5p10 cells was transfected with the 2-vector combination, but the population became highly confluent in hESC media within 2 weeks and potential colonies were difficult to identify. The transfected population was passaged with Accutase and replated on MEF feeders. hESC-like colonies emerged within 2 weeks, optimal colonies were pooled and maintained as the iChM5A candidate population. Transfected ChM5p12 cells were maintained for 4 days in growth media, treated with Accutase and passaged to MEF feeders as separate populations; a single hESC-like colony was recovered from one population of transfected cells and maintained as the iChM5B candidate population. Optimal hESC-like candidate colonies and control H9 hESC colonies were passaged as needed to maintain healthy cultures.The episomal vectors  that were used in this work are described in Following the first manual passage of candidate colonies from MEF feeders, residual colony fragments in the primary culture plate were maintained in conditioned hESC media without bFGF for 3 to 5 days to allow colony expansion and then switched to regular hESC media to encourage spontaneous differentiation as the MEF feeders age and pluripotency of the expanding population by the absence of bFGF in hESC media. Rosettes were manually isolated as they emerged and passaged in hESC media to matrigel-treated cover slips for immunostaining. Long term cultures of neural progenitors/stem cells (NSPs) were established as described ; neural Total cellular DNA was isolated with GenePure (Qiagen) or QiaAmp DNA Mini (Qiagen) kits and treated with RNAse to remove RNA. Transgenes or endogenous genes were amplified in reactions containing 100 ng genomic DNA or <1 ng plasmid DNA with GC-rich polymerase (Life Technologies) in 1X Buffer A, 3 µl of Enhancer and 250 nM of oligonucleotide primers  with tou® Gene Expression Master Mix (Life Technologies). Amplification rates  of cDNA were assayed in more 2 replicates for each gene. The mean (AVG) and standard error (SE) was calculated with the Descriptive Statistics tool in Excel and normalized to β-glucuronidase (GUSB) with the ΔΔCt method described in Applied Biosystems/Life Technologies technical resources.Total cellular RNA was isolated with RNAeasy kits (Qiagen) and contaminating DNA was removed by DNAse treatment. RNA was converted to cDNA using SuperScript First-Strand Synthesis System (Life Technologies) and 1 µl of 1:4 dilution of cDNA in water was amplified in each reaction. Transcript levels in http://dx.doi.org/10.6084/m9.figshare.1153969.Genomic DNA was processed with an Epitect kit (Qiagen) as directed by the vendor. Amplification products were generated with primers that were specific to converted DNA , purifieCells were cultured in multiwell tissue culture plates on cover glass or in multiwall chamber slides that were pretreated with 1:100 dilution of growth factor reduced matrigel. Samples were fixed and immunostained as described  with antAmniotic cell populations were derived from amniocentesis samples  that werReprogramming targets were selected to reflect the range of cell types in amniocentesis samples and proliferation characteristics that we considered to be important to the efficiency of reprogramming. The ChM5 mixed cell population was highly enriched for fibroblast-like stromal cells and cell proliferation continues in confluent cultures, generating dense stratified cell layers . The ChMReprogramming used combinations of 2 or 3 first generation episomal vectors  that colImmunostaining of transfected populations showed a high frequency of Oct4 positive cells following transfection, but the loss of virtually all immunopositive cells by passage 5 . Haemacy5 cells were serially transfected every 8 to 12 h for 3 transfections in order to increase the number of transfected cells. ChMRC.B1p6 cells were transfected with the 3-vector combination . Cells are fixed and prepared for digital analysis of chromosomes in mitotic figures and recognition of chromosomal abnormalities with suitable software. The results of karyotype analysis here indicated that early passage iChM5Ap14 and iChM5Bp14 cells had a normal female  karyotype without chromosomal abnormalities at a detection limit of 5 Mb  expressed Nanog and Sox2 as well as Oct4. Dual labeling showed that nuclear localized Nanog was correlated with nuclear localized Oct4 (n > 500), with and without colocalized Oct4 and Nanog expression, expressed Sox2  in iChM5A and iChM5B cultures  as a stromal cell control  of the parental ChM5 mixed cell pool is unknown and cannot be tested directly, amniotic stromal and epithelial cells alike show stromal cell traits . Stromal control . TranscrThe promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources, in part because immunologically compatible iPSCs from allogenic sources is the more likely path for clinical applications . Particiin vivo  and can provide critical internal controls in the same culture and within the same field of view. Immunostaining in this case is superior to flow cytometry that cannot discriminate between nuclear and cytoplasmic localization of transcription factors or easily correlate gene expression and changes in cell morphology in differentiating cells.The value of neural rosettes in candidate selection was substantiated by subsequent validation of pluripotency of iChM5 derivatives, including evidence for epigenetic modification of chromatin structure  as expecin vivo  iChM5Ap4-derived rosettes and NSPs. Dissociated rosettes from candidate colonies were immunostained as indicated. Rosette immunostained for nestin is indicated by asterisk (*).  Scale bar, in microns as indicated.(A). Click here for additional data file.10.7717/peerj.668/supp-2Figure S2Phase images of NSPB6p12 showing early stage differentiation by withdrawal of mitogens in confluent culture in top image. Middle and bottom images show induced differentiation of NSPB6p12 cells and control hVMNSPs, respectively, at day 7. Representative of presumptive axonal extensions are indicated by arrows. Scale bar, in microns as indicated.Click here for additional data file.10.7717/peerj.668/supp-3Figure S3This image show immunodetection of Sox2 in a subset of HEK293 cells that were transfected with the 2 vector combination of episomal plasmids. This image shows the vast majority of cells did not show SOX2 expression, likely because they lacked an episomal vector and did not express an endogenous genes.Click here for additional data file.


Due to concerns raised about Fig 4 and Fig 6, the authors would like to publish the raw, uncropped film for Fig 6 as well as an alternate experiment for Fig 4 which verifies the conclusion of the published image.The authors and editors confirm that these changes do not alter their findings.S1 FileThis file includes the alternate experiment for Figure 4.(PDF)Click here for additional data file.S2 FileThis file includes the original autorad for Figure 6.(JPG)Click here for additional data file.


Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of  Blood flow is an important biological regulator and initiates and maintains many events during embryonic development. Shear stress, a mechanical force created by blood flow, is an important factor regulating many physiological functions. Although there is expression of arterial and venous specific genes in the vasculature before the onset of flow , 2, therNotch and Dll4 expression and initiate arterial differentiation. In regions of low VEGF concentrations, however, venous identity prevails [Dll4 and Hey1 are expressed in an arterial specific manner before the onset of flow [Genetic predetermination of arterial identity has been shown to occur through the Notch signaling pathway. Notch receptors and ligands are involved in a plethora of developmental processes including somite coordination , 8, cardprevails , 16. In  of flow . Dll4 isNotch1 just after the onset of circulation in mouse embryos. We found Notch1 expression begins with the onset of flow but is pan-endothelial at the stage where erythroblasts enter circulation. Only by E9.5, after vascular remodeling has occurred, is Notch1 restricted to arteries. Using a technique we previously developed [Notch1 expression was arterial after culture while no expression in either arteries or veins was observed in the embryos with ablated flow, indicating that Notch1 requires flow to be expressed. We investigated the patterns of flow that could regulate Notch1 expression and found the largest increase occurred at low levels of laminar shear stress (1–5 dynes/cm2) and with oscillatory flow types (either 0 ± 3 or 2 ± 3 dynes/cm2). Not only could Notch1 expression be upregulated by flow, but the expression of Notch ligands and transcription factors were also induced by exposure to shear stress. We therefore knocked down Notch1 and investigated the effect on flow-induced expression of Notch signaling molecules as well as typical shear-induced genes and downstream targets in arterial-venous differentiation. Flow could induce the expression of approximately half the genes investigated, even in the absence of Notch1. Overall, our results indicate that Notch1 expression occurs after the onset of flow and does not become arterial-specific until remodeling has occurred. While Notch1 is required for the mechanotransduction of some genes, our results indicate that it is not an essential component of mechanotransduction since the flow-induced expression of many genes is not affected by Notch1 knockdown.In this work, we have investigated the expression of eveloped , we ablaIn situ hybridization has been described previously [Notch1 [Notch4 [Hey1 [All procedures were approved by the Animal Care Committees of McGill University and we have followed recommendations of the Canadian Council on Animal Care. eviously  with pro [Notch1 , Notch4  [Notch4 , and Heyh4 [Hey1 . Antibodh4 [Hey1 . BrieflyHuman abdominal aortic endothelial cells  were propagated through passage 5 in complete endothelial cell growth medium MV (PromoCell) supplemented with 1% Penicillin Streptomycin. Cells were expanded on 0.1% gelatin. For flow experiments, HAAEC were seeded at a density of 150 000 cells/mL on a culture slide, coated with 4% rat tail collagen type 1 and grown to confluence (3 days).2 and oscillatory shear stress of 0 to 5 ± 3 dynes/cm2 as indicated. Static slides were cultured in parallel. After 1 hour, total RNA or protein was isolated.A parallel plate flow chamber was designed in-house. The parallel plate flow chamber was connected to a closed-loop perfusion system consisting of a vented media reservoir, a flow dampener, a peristaltic pump and/or a computer-driven syringe pump. The viscosity of the endothelial cell media was measured using a Bohlin CVO 120 HRNF Viscometer  to be 1.02334 cP. Laminar flow was verified by seeding the perfusate with fluorescent microparticles and imaging with a high-speed camera on a fluorescent microscope. HAAEC were exposed to laminar flow at a calculated wall shear stress of 1 to 15 dynes/cm16 primers (Applied Biosystems), and M-MLV reverse transcriptase (Invitrogen). Gene expression was analyzed on an ABI Prism 7900HT Sequence Detector with SA Biosciences RT2 Real-Time SYBR Green master mix (Qiagen). Quantitect Primer Assays (Qiagen) were used for HPRT (QT00059066), 18S (QT00248682), Dll1 (QT00057631), Nrp1 (QT00023009), VEGFR2 (QT00069818) and Notch1 (QT01005109). All other primers were designed in-house . RNA concentration and purity was quantified on a NanoDrop. 1 μg of RNA was reverse transcribed using Oligo-d(T)in-house . The datNotch1 siRNA (SI00119035) and AllStars Negative Control siRNA (1027280) were obtained from Qiagen. 10nM siRNA diluted in 100μl OptiMem serum-free medium (Invitrogen) with HiPerfect Transfection Reagent (Qiagen) was added to 150 000 cells pre-seeded on a collagen-coated culture slide in 1mL of OptiMem without Penicillin Streptomycin. After 1h of transfection, 1.3mL complete endothelial cell growth medium was added to each slide and incubated for 48 hours. Transfection media was then replaced with 2mL of complete endothelial cell media and the samples incubated for another 24 hours before performing experiments. qPCR and Western Blot was used to measure knockdown. For western blot, primary antibodies for Notch1 were obtained from EMD Millipore , glyceraldehyde phosphate dehydrogenase (GAPDH) from Santa Cruz Biotechnologies .Data are presented as the mean ± standard error of the mean (SEM). Results were analyzed using ANOVA with Tukey’s post hoc test.Notch1 expression is absent at 5 somites, before the onset of blood flow , expression is the same as static control (2) in which a complete reversal of flow direction is present; oscillatory flow with a slight reversal present (2 ± 3 dynes/cm2) and purely pulsatile flow with no retrograde component (5 ± 3 dynes/cm2). We find that both flow types in which flow reversal is present (0 ± 3 and 2 ± 3 dynes/cm2) induce an upregulation of Notch1, but that pulsatility without flow reversal does not , which corresponds to the average level of shear stress in the embryo at the onset of circulation but is also a level of shear stress in which we observed increased Notch1 expression. Endothelial cells were exposed to flow for one hour. In the presence of control siRNA, exposing endothelial cells to laminar shear stress led to an upregulation of the genes Klf2 and VEGFR2. The knockdown of Notch1 alone had no effect on the expression of these genes in static conditions, nor did it prevent the shear stress-dependent increase in Klf2 expression. However, shear stress failed to upregulate VEGFR2 by shear stress when Notch1 was knocked down.The role of Notch1 in mechanotransduction has been studied surprisingly little. Shear stress induces Notch cleavage in embryonic stem cell-derived VEGFR2+ cells . In zebr+ cells . Other tDll1, in response to flow. A positive feedback loop for amplification of Dll1 by Notch signaling has not been shown unlike the other ligands tested. We also investigated whether Notch4 would compensate for the loss of Notch1. No changes in Notch4 expression are observed.Masumura et al. reported that Notch intracellular domain (NICD) is cleaved by shear stress . NICD inNrp1, EphrinB2) and two venous genes (EphB4 and CoupTFII) gave the clearest results  and 5 dynes/cm2  induced Notch1 expression to the same extent, explaining why expression is present in both vessels at the onset of flow. Our results also show that the onset of circulation is necessary for this expression to occur. The results are, however, contrary to what has been reported in the zebrafish where ablation of flow resulted in an increase in Notch signaling due to an upregulation of Dll4 [VEGFR2 (KDR) and ephrinB2 expression are not affected by the ablation of flow and that VEGFa expression is inhibited by flow. This is contrary to what is observed in mammalian systems, by us and by many others, where VEGFR2, VEGF and EphrinB2 are all upregulated by flow [Notch1 expression by flow is dependent on the magnitude and type of flow that is present. The levels and patterns of hemodynamic stress present in the zebrafish when circulation begins are likely to be very different than in mammalian embryos.We found that flow was required for the expression of Notch1 during development, but that adult artery physiological levels of flow do not induce Notch1 expression. Embryonic shear stress levels can go as high as 5 dynes/cmand E9.5 , 27), anand E9.5 . Given tand E9.5 , we woul of Dll4 . The res by flow , 42–44. in vitro results show that adult artery physiological shear stress levels do not induce Notch1 expression, implying that the continued expression in arteries is not dependent on Notch1 induction by shear stress. Activation of Notch receptors induces a positive feedback loop resulting in the expression of Notch ligands [Notch1 but sustained signaling from flow may not be required once expression has been initiated.Our  ligands , 31. Simin vivo work shows a decrease in Nrp1 expression in the dorsal aorta of Notch1 knockout mice [Nrp1 expression with Notch1 knockdown in vitro. We achieved a 70% knockdown of Notch1 in HAAEC, and residual expression of Notch1 in the endothelial cells might account for this variation. We find that Nrp1 expression remains unchanged after cells with Notch1 siRNA are exposed to laminar flow. This indicates that although Nrp1 and VEGFR2 are upstream of Notch1 in the VEGF-signaling pathway [Notch signaling plays a crucial role in arterial and venous differentiation during development. Although earlier out mice , we obse pathway , there mDll1, Jag1 and possibly Cx40) as well as venous-specific genes (CoupTFII), and others (KLF2). Hence, our results show that Notch signaling can modulate how flow affects endothelial cells, but it does not appear to be an indispensable protein in the process of mechanotransduction.Though many genes involved in arterial-venous differentiation failed to be upregulated by flow after the knockdown of Notch1, there are exceptions both within the set of arterial specific genes (