ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML.Acute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, arrayCGH, SNP arrays, direct sequencing of all coding exons and pyrosequencing of the 5′UTR CpG islands of ETV6 and CDKN1B were performed. In total, 39 of 79 patients (49%) showed monoallelic deletions of 12p13 according to karyotypic data and 20 of 43 patients (47%) according to genomic profiling. Genomic profiling led to the minimal deleted region covering the 3′-UTR of ETV6 and CDKN1B. Direct sequencing revealed one novel monoallelic frameshift mutation in ETV6 while no mutations in CDKN1B were identified. Furthermore, methylation levels of ETV6 and CDKN1B did not indicate transcriptional silencing of any of these genes. ETV6 and CDKN1B had reduced expression levels in CK-AML patients with deletion in 12p13 as compared to CK-AML without deletion in 12p13, while the other genes  located within the minimal deleted region in 12p13 had very low or missing expression in CK-AML irrespective of their copy number status.To decipher deletions, mutations and methylation of ETV6 and CDKN1B are mainly affected by small monoallelic deletions, whereas mutations and hypermethylation play a minor role in CK-AML. Reduced gene dosage led to reduced gene expression levels, pointing to haploinsufficiency as the relevant mechanism of inactivation of ETV6 and CDKN1B in CK-AML.The online version of this article (doi:10.1186/1471-2164-15-784) contains supplementary material, which is available to authorized users. TP53 to determine the allelic status of 12p13 and delineate the breakpoints of 12p more closely. In 26 of these patients we used arrayCGH methods , five patients were analyzed by using the SNP 250 k array and 12 patients were analyzed by using the SNP 6.0 array. In summary, in 20 of 43 patients (47%), losses of 12p13 could be identified by genomic profiling encompassing the ETV6 and CDKN1B from different studies also comprising our data is shown in Figure ETV6 or CDKN1B. However, in the majority of studies, the minimal deleted region covered both ETV6 and CDKN1B.A graphic overview of the 12p13 deletion breakpoints including or excluding ETV6 [NM_001987] and CDKN1B [NM_004064], we performed mutation analyses of all coding exons of ETV6 in 56 patients and of CDKN1B in 67 patients with and without 12p13 deletions for which DNA was available  in one patient (#21)  [ETV6 mutation was present, indicating that this mutation emerged early in the course of the disease. In the following samples during the course of the disease, a complex karyotype with several balanced translocations and a terminal deletion of 14q was detected  and no typical cytogenetic aberrations of CK-AML like del(5q), -7/del(7q), or del(17p) were present in this patient. However an NPM1 mutation ) was identified (data not shown).To further determine the modes of inactivation of TV6 NM_00987 and Cdex.php) . The mutTV6 NM_00987 and CTP53 mutations in the subgroup with and without 12p13 deletions, we performed mutation analysis of all coding exons of TP53 in all 79 patients. In 44 of 79 patients (56%), TP53 mutations were identified .To determine the frequency of ETV6 and CDKN1B, quantitative methylation analysis of the 5′UTRs of both genes was performed by pyrosequencing. We examined 23 single CpG sites within the ETV6 5′UTR CpG island and 22 single CpG sites within the CDKN1B 5′UTR CpG island in 55 patients . The mean methylation levels of ETV6 and CDKN1B ranged from 0% to 1.43% and 0.09% to 1.77%, respectively  for CK-AML patients with and without deletion in 12p13. We compared eight cases of CK-AML with deletion in 12p13 and eight cases of CK-AML without deletion in 12p13. ETV6 and CDKN1B are expressed in CK-AML with and without deletion in 12p13 . Notably, the expression level of ETV6 and CDKN1B decreased significantly by 0.53-fold (p < 0.03) and 0.62-fold (p < 0.003), respectively, in cases with 12p13 deletions compared to those without 12p13 deletions. The other candidate genes located within the minimal deleted region  showed no or very low expression levels irrespective of their copy number status (0.13 – 3.72 RPKM)  with monosomy 7  and CK-Aet al, that these genes do not show any expression or very low expression in blood or bone marrow cells of AML patients [http://servers.binf.ku.dk/hemaexplorer/), they show a low expression in normal hematopoietic stem and progenitor cells, compared to the expression level of ETV6 (5- to 40-fold higher) and CDKN1B (3- to 20-fold higher) in these cells [TP53, PTEN, NPM1, NF1 and RPS14[APC, ATM, BRCA1/2 and RB haploinsufficiency contributes to tumorigenesis [CDKN1B is strongly implicated in numerous cancer types, the vast majority of lymphatic origin [CDKN1B heterozygous mice are predisposed to tumors in multiple tissues [ETV6 and CDKN1B are known to be inactivated mostly by monoallelic deletions. Yet, the mode of inactivation has not been investigated in detail in CK-AML with a high frequency of 12p13 deletions.However, we now demonstrate according to the expression data provided by TCGA in confirmation of data published by Haferlach patients . Likewisse cells . It seemse cells –29. Popund RPS14, 29. Likigenesis . Haploinc origin , 17. CDK tissues . ETV6 anCDKN1B and the 3′UTR of ETV6  of the analyzed CK-AML patients according to karyotype. In 20 of 43 patients (47%), losses of 12p13 could be identified by genomic profiling, of those 13 small interstitial deletions , -7/del(7q), or del(17p) present in this patient, which could provide a proliferative advantage or even initiate leukemogenesis. This might strengthen the role of this ETV6 mutation as a driver rather than a passenger mutation. Unfortunately, no fibroblast DNA was available to prove whether the ETV6 mutation was of germline origin. However, this seems to be unlikely as ETV6 is a critical regulator in the survival of multiple cell types during early embryonic development and ETV6 knockout mice are embryonically lethal [osed AML , AML-M0 osed AML  and MDS osed AML . We detey lethal .CDKN1B mutations have been previously reported in childhood leukemia [CDKN1B gene in our analyzed patient cohort .As expected, in more than half of the analyzed patients, ETV6, increased methylation was suggested as a possible mechanism since decreased ETV6 protein expression was reported in AML patients [CDKN1B was excluded in not further defined AML and MDS, but found in the lymphoblast-like cell line Raji [ETV6 and CDKN1B 5′UTRs are not hypermethylated in CK-AML  show no or very low expression levels irrespective of their copy number. In contrast, ETV6 and CDKN1B are expressed in CK-AML and their expression levels significantly decreased in cases with 12p13 deletions . The research has been conducted in compliance with the Helsinki declaration.et al[ArrayCGH using the 2.8 k and/or the 8.0 k platform and SNP analyses using Affymetrix GeneChip Human Mapping 250 K Array  and/or Genome-Wide Human SNP 6.0 Array  were performed as previously described by Rücker et al. Resultset al[http://www.ncbi.nlm.nih.gov/geo/). Aberration summaries are archived under GSE55535. GEO accession numbers are given in Additional file For arrayCGH using the Agilent Human Genome Microarray Kit 244A , a high resolution 60-mer oligonucleotide-based microarray, the procedures for DNA labeling, hybridization and washing were performed according to the manufacturer’s instructions (protocol version 6.1.) with some modifications as previously described by Praulich et al. The sliETV6 (ENSG00000139083), exons 1-2 of CDKN1B (ENSG00000111276), and exons 2-11 of TP53 (ENSG00000141510) were PCR-amplified from genomic DNA using FastStart Taq DNA Polymerase . After purification with the magnetic bead-based CleanSEQ® system , PCR fragments were sequenced in both directions using the GenomeLab™ DTCS Quick Start Kit and CEQTM 8000 Genetic Analysis System . Cloning of PCR products was performed in patients with complex mutations to describe the mutations properly using the TOPO TA Cloning® Kit . All mutations were described according to the nomenclature for the description of sequence variations of the Human Genome Variation Society .Exons 1-8 of http://cpgislands.usc.edu). We used the following settings: a G + C content of at least 55% GC, an observed CpG/expected CpG in excess of 0.65, and a minimum length of 500 bp. The pyrosequenced regions were also selected in the context of primer design, optimal PCR conditions and stringency.CpG islands were identified using CpG island searcher  and extension at 72°C for 1 min, followed by 25 cycles at 96°C for 30 s, annealing at 53°C for 30 s and 72°C for 1 min, finished with 72°C for 7 min; for fragment CDKN1B denaturation at 97°C for 7 min, touchdown for 10 cycles including denaturation at 96°C for 30 s, annealing at 53°C for 30 seconds (which was decreased by 0.5°C in each cycle) and extension at 72°C for 1 min, followed by 25 cycles at 96°C for 30 s, annealing at 48°C for 30 s and 72°C for 1 min, finished with 72°C for 7 min. Primer sequences were as follows: fragETV6-fw: 5′-GYGGGTGGGAGGAGAG-3′; fragETV6-rev-biot: 5′-biotin-TTCTTCCAACATCTCTCCC-3′; fragCDKN1B-fw: 5′-GTAGGTTTGTTGGTAGTAG-3′; fragCDKN1B-rev-biot: 5′-biotin-AAAAAAAATCCATTAATTAC-3′.Sodium bisulfite treatment of genomic DNA was performed using the EZ DNA Methylation Direct Kit™ . Fragments for pyrosequencing were generated by PCR using the FastStart Taq™ DNA Polymerase Kit  with the following protocol: 10pmol of forward and reverse PCR primers , and 0.78 units of FastStart Taq™ DNA Polymerase as well as different amounts of dNTPs and MgCl2. Cycle conditions were as follows: for fragment For purification of biotinylated fragments, 5 to 13 μL of PCR products were added to a mixture consisting of 3 μL Streptavidin Sepharose HP™ Beads  and 47 μL binding buffer . Single-stranded fragments were purified using the Vacuum Prep Tool™ . Sepharose beads with the single-stranded templates attached were added to a PSQ 96 Plate Low™  containing a mix of 11 mL annealing buffer  and 800 nmmol/L of the corresponding sequencing primers  with following sequences: fragETV6-seq: 5′-GATTTG TAGATTT-3′; fragCDKN1B-seq: 5′-GATTAGTTAATTTTT-3′.Pyrosequencing was performed in a PyroMark MD™ System  with the PyroGold SQA™ Reagent Kit  containing nucleotides and prepared mixtures with enzymes or substrates. For pyrogram exposure including CpG-site methylation calculation, the Pyro Q-CpG™ Software  was applied. Only pyrograms including sharp peaks with satisfactory heights for each injected nucleotide of interest and without peaks for unsuccessful bisulfite treatment or background controls were considered.Based on our previously published gene expression profiling , 24, we http://cancergenome.nih.gov/). Visualization and selection of the SNP data was done using the Integrative Genomics Viewer [t-test program of the statistical program R, version 3.1.1 (http://www.R-project.org/) [SNP level3 datasets (archive: broad.mit.edu_LAML.Genome_Wide_SNP_6.Level_3.25.2004.0) and RNAseq level3 datasets (archive: bcgsc.ca_LAML.IlluminaGA_RNASeq. Level_3.1.7.0) of AML patients were obtained from the TCGA Research Network  . Accordit-test and the ANOVA one-way analysis of variance. An effect was considered significant if the p value was <0.05.We used the two-tailed Fisher’s exact test, Kruskal-Wallis one-way analysis of variance, the unpaired ETV6 and CDKN1B (mutations are named according to HGVS (http://www.hgvs.org/)), and mean methylation levels. (XLS 43 KB)Additional file 1: Table S1: Karyotypes, genomic profiling, sequencing results and methylation analyses of the patient cohort. Karyotypes of the 79 patients with CK-AML investigated in this study (classified according to ISCN recommendations), results obtained by genomic profiling, mutation analyses of TP53 mutations. TP53 mutation status of the 79 patients investigated in this study, mutations are named according to HGVS (http://www.hgvs.org/). FISH results with a probe for the locus 17p13 (TP53) are also shown if available. (XLS 26 KB)Additional file 2: Table S2: Overview of Additional file 3: Table S3: Normalized log2 fluorescence ratio of six CK-AML samples obtained from 2.8/8.0 k BAC/PAC arrays. Cut-off levels for gains and losses were determined for each individual experiment. After computing the ratios from dye-swap hybridization and subsequent normalization, an individual set of balanced clones for each experiment was used to calculate the mean and standard deviations. The cutoff levels were defined as mean plus/minus three times the standard deviation. Frequently affected regions recently detected as copy number polymorphisms  were excluded from data analysis. (XLSX 1 MB)Additional file 4: Table S4: List of GEO accession numbers. List of GEO accession numbers for all samples run on the Agilent/Affymetrix platform. (DOC 56 KB)


In the process, supercritical ethanol acted as a reductant to reduce graphene oxide and MnO2 gels simultaneously. The synthesized aerogels consisted of 10–20 nm Mn3O4 nanocrystallites, with BET-specific surface areas around 60 m2/g. The performance of the aerogels as anode materials for lithium-ion batteries was also evaluated in this study. The results showed that Mn3O4 aerogels as anode materials exhibited a reversible capacity of 498.7 mAh/g after 60 charge/discharge cycles while the reversible capacity for Mn3O4/GN composite aerogels could further increase to 665 mAh/g. The mechanisms for the enhanced capacity retention could be attributed to their porous structures and improved electronic contact with GN addition. The process should also offer an effective and facile method to fabricate many other porous metal oxide/GN nanocomposites for low-cost, high-capacity, environmentally benign material for lithium-ion batteries.MnThe online version of this article (doi:10.1186/s11671-015-0960-x) contains supplementary material, which is available to authorized users. Mn3O4 is an attractive anode material for lithium-ion batteries due to high abundance of Mn element in natural resource, low cost, and environmental benignity. Its theoretical capacity can reach as high as ~936 mAh/g. However, previous studies reported that Mn3O4 showed poor performance as anode materials for lithium batteries. At a current density of 40 mA/g, the pure solid Mn3O4 powder showed a capacity lower than 300 mAh/g, which further decreased to ~200 mAh/g after ten cycles. Even for Co-doping Mn3O4, the first discharge capacity of ~900 mAh/g could be reached; however, it also further decreased to ~400 mAh/g after ten cycles [3O4 nanoparticles even showed worse performance. After 10 cycles, only a capacity of ~115 mAh/g was retained. Only recently, have the capacity and rate capability of Mn3O4 been greatly improved by wiring up the Mn3O4 nanoparticles through a two-step solution-phase method [Nanostructured materials such as nanowires, nanotubes, nanosheets, and porous nanomaterials have attracted great interest in the recent years because of the novel properties from their reduced dimensionality. They are becoming increasingly important for electrochemical energy-storage applications, especially for lithium-ion batteries –5. The a powders . Thereafstigated –12. Howen cycles . Mn3O4 ne method .3O4 aerogels and its graphene composite aerogels for the first time by a straightforward method, a supercritical-ethanol process. The results indicate that the supercritical-ethanol process can not only serve as a drying method to obtain the porous structure of aerogels but also reduce high-valence manganese oxide and graphene oxide (GO) simultaneously. The lithium-storage abilities of Mn3O4 aerogels were also investigated. The anode based on pure Mn3O4 aerogels exhibited an initial capacity of 1274.3 mAh/g. After 60 discharge/charge cycles, the capacity of 498.7 mAh/g was retained while the capacity increased to 665 mAh/g when graphene nanosheets (GNs) were incorporated into aerogel structure to improve their electronic contact.In this paper, we have synthesized Mn3 were placed in a flask. 75 mL of H2SO4 (98 wt %) was then added with mechanical stirring in an ice-water bath. After 10 min, 4.5 g of KMnO4 was added gradually in the flask in 1 h. After the mixture was stirred vigorously for 5 days at room temperature, 3 mL of H2O2 (30 wt % aqueous solution) was added, and the mixture was stirred for 2 h at room temperature. The mixture was washed thoroughly with a mixed aqueous solution of 3 wt % H2SO4/0.5 wt % H2O2 to remove the excess manganate and the sulfate. Then, the solution was subjected to dialysis for 3–4 days to completely remove residual metal ions and acids. A typical AFM of GO is shown in Additional file GO was prepared by a modified Hummers method , 16. Bri2 wet gels were prepared by the previously reported method [4 was dissolved in 12.5-ml de-ion water. 0.232 fumaric acid was then added into the vigorously stirred KMnO4 solution. The resulting brown sol was then poured into polypropylene molds for gelation and aging for 24 h. The gel then was rinsed in 1-M sulfuric acid, followed by multiple rinses with water to remove impurity. MnO2/GO composite was prepared by adding GO into MnO2 sol first. The rest of the gelation and aging procedures were the same. Wet gels were then rinsed with ethanol for several times. The rinsed MnO2 gels and its graphene nanosheet (GN) hybrid were subjected to a supercritical-ethanol process at 260 °C at 10 MPa for 6 h to form Mn3O4 aerogels and Mn3O4/GN composite aerogels.MnOd method . Briefly3O4/GN composite was determined by a thermogravimeter , and the measurements were carried out in air over a temperature range of 30–500 °C with a ramp rate of 10 °C /min. X-ray photoelectron spectroscopy (XPS) experiments were carried out on a RBD-upgraded PHI-5000C ESCA system (Perkin Elmer) with Al Kα radiation (1486.6 eV). XPS Peak Version 4.1 software was used to perform curve fitting. The N2 absorption-desorption analysis was conducted on an Autosorb-1 instrument. The distribution of pore size was calculated from the desorption data using the Barret-Joyner-Halenda (BJH) method.X-ray diffraction (XRD) patterns were obtained by an X-ray diffractometer (Rigaku D/Max-RB) with high intensity Cu Kα radiation .The morphology of samples was observed by a scanning electron microscopy . Transmission electron microscopy (TEM) was conducted with a JEOL JEM-2010 electron microscope operating at 200 keV. The GN content in the Mn3O4 aerogels and Mn3O4/GN composite aerogels as active materials, Super P carbon black and polyvinyldifluoride (weight ratio 80:10:10), were mixed in N-Methylpyrrolidone (NMP) solvent to produce an electrode slurry. The slurry was coated onto a copper foil using the doctor-blading method and then dried to form the working electrode. The electrochemical tests were performed using two-electrode coin-type cells with lithium as both the counter and reference electrode. 1 M of LiPF6 in a 1:1:1 (volume ratio) mixture of ethylene carbonate and diethyl carbonate and dimethyl carbonate was used as the electrolyte. Cell assembling was carried out in an argon-filled glove box. Galvanostatic charge-discharge cycling was conducted using a battery tester  with a voltage window of 3–0.01 V at the current density of 50 mA/g.The powder of Mn3O4 and Mn3O4/GN aerogels is illustrated schematically in Fig. 2 gels were prepared by adding fumaric acid to a stirred KMnO4 solution, following the reaction below [The process for preparing Mnon below , 19:\doc3O4 gels and Mn3O4/GN composite gels after the supercritical-ethanol process are brown and gray, respectively . After the supercritical-ethanol process , plenty of sharp peaks appear , indicating formation of Hausmannite-type Mn3O4. No peaks of any other phases or impurities are observed, demonstrating that high-purity Mn3O4 can be obtained using the above method. According to Scherrier analysis, the size of Mn3O4 is estimated to be ~10 nm. The above results indicate that MnO2 was reduced into Mn3O4. The following chemical reaction was suggested to happen in the supercritical-ethanol process,as synthesized monoliths of Mnear Fig.  and c an2 into Mn3O4 during the process [3O4.Supercritical ethanol acted as the reducing agent to reduce MnO process , 21. Mea2 gels into GN. Here, XPS  pattern of the final products is shown in the insert of Fig. 3O4, consistent with the relevant XRD analyses. Surface area, pore size, and pore volume are important characteristics for aerogel materials. The data related with Mn3O4 aerogels and Mn3O4/GN aerogels are summarized in Table 3O4 aerogels and Mn3O4/GN composite aerogels are type IV isotherms with H1 hysteresis loops, which are characteristic of an interconnected mesoporous system with cylindrical pores. BET-specific surface areas are 69 and 67 m2/g, respectively. The BET-specific areas are not as high as the reported value (~200 m2/g) of MnO2 aerogels [3O4 in the supercritical-ethanol process at relative high temperature and pressure.The bright field TEM image Fig.  of the raerogels , which m3O4-based aerogels, the samples were used as the anodes for Li-ion battery. Figure 3O4 and Mn3O4/GN aerogels, respectively. In the first discharge curve, a sloping plateau in the range of ~1.5– 0.3 V is observed. It might be due to the formation of solid-electrolyte interface (SEI) film on the active materials. The well-defined voltage plateau around 0.35 V reflected the reduction reaction of Mn3O4 as follows: Mn3O4 + 8Li+ + 8e− → 3Mn(0) + 4Li2O [3O4 electrode and Mn3O4/GN electrode at a current of 50 mA/g. The Mn3O4 electrode shows an initial discharge capacity of 1274.3 mAh/g with an initial Coulombic efficiency of 51.7 %. With GN addition, the initial discharge capacity for Mn3O4/GN was measured to be 1134.2 mAh/g with an initial Coulombic efficiency of 58.7 %. The capacity loss results from the formation of SEI film, some other irreversible side-reactions, and the disengagement of the Mn3O4 particles from the electrodes. After 60 cycles, the Mn3O4/GN composite electrode retained a discharge capacity of 665 mAh/g with a Coulombic efficiency of 97.7 %, compared with the Mn3O4 electrode with a discharge capacity of 498.7 mAh/g with a Coulombic efficiency of 97.2 %. The specific capacities of Mn3O4 and Mn3O4/GN are both much higher than the reported values of the anode based on Mn3O4 nanoparticles, 115 mAh/g after ten cycles [3O4 aerogels could be attributed to the structure of aerogels. The high porosity from mesopores of aerogel structures facilitates the transportation of lithium ions in the aerogel particle, and the large surface area of aerogel enhances the utilization of active materials [3O4/GN can be attributed to the wiring effect of GN, that is, that the large-sized GNs can build an excellent conductive network which facilitates the electron transfer in the structure of aerogels. This was supported by electrochemical impedance spectroscopy (EIS) measurements performed after the fifth cycle. The Nyquist plots of the Mn3O4 and Mn3O4/GN electrodes are presented in Fig. 3O4/GN electrode is smaller than that of pure a Mn3O4 electrode, indicating the enhanced electron conductivity of the Mn3O4/GN [To evaluate the electrochemical performance of Mn + 4Li2O . The cha + 4Li2O . Figure n cycles . The impMn3O4/GN .Fig. 6Ga3O4 aerogels and its GN composite aerogels were synthesized by the simple supercritical-ethanol process on MnO2 and MnO2/GO gels, respectively. Supercritical ethanol served as both drying and reducing agents of MnO2 and GO in the process. The samples were characterized by X-ray, SEM, XPS, and TEM. The electrochemical measurements demonstrated that Mn3O4 aerogels possessed better capacity retention compared with Mn3O4 nanoparticles. The enhanced performance of the synthesized Mn3O4 was ascribed to its porous aerogel structure. With GN addition to Mn3O4 aerogels, their electrochemical property was improved further. The higher capacity retention was attributed to the fact that GNs enhanced the electron transfer and reduced the resistance within Mn3O4. The process should also offer an effective and convenient technique to fabricate many other metal oxide/graphene porous nanocomposites for low-cost, high-capacity, environmentally benign material for lithium-ion batteries.Mn


For neurons to function correctly in neuronal circuitry they must utilize appropriate neurotransmitters. However, even though neurotransmitter specificity is one of the most important and defining properties of a neuron we still do not fully understand how neurotransmitter fates are specified during development. Most neuronal properties are determined by the transcription factors that neurons express as they start to differentiate. While we know a few transcription factors that specify the neurotransmitter fates of particular neurons, there are still many spinal neurons for which the transcription factors specifying this critical phenotype are unknown. Strikingly, all of the transcription factors that have been identified so far as specifying inhibitory fates in the spinal cord act through Pax2. Even Tlx1 and Tlx3, which specify the excitatory fates of dI3 and dI5 spinal neurons work at least in part by down-regulating Pax2.In this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2.We demonstrate that Evx1 and Evx2 are expressed by spinal cord V0v cells and we show that these cells develop into excitatory (glutamatergic) Commissural Ascending (CoSA) interneurons. In the absence of both Evx1 and Evx2, V0v cells still form and develop a CoSA morphology. However, they lose their excitatory fate and instead express markers of a glycinergic fate. Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism.Evx1 and Evx2 are required, partially redundantly, for spinal cord V0v cells to become excitatory (glutamatergic) interneurons. These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes.The online version of this article (doi:10.1186/s13064-016-0059-9) contains supplementary material, which is available to authorized users. Hundreds of millions of people across the world are affected by neurological diseases and injuries. Understanding how functional neuronal circuitry is established in the vertebrate central nervous system (CNS) is essential for developing better treatments for these conditions. How neuronal circuitry develops is also a fundamental question in developmental neuroscience. To answer this question, we need to identify how the functional properties of distinct neurons are specified; since these properties determine which circuits the neurons participate in, the functional roles that the neurons have within those circuits and the resulting outputs of the circuitry. The spinal cord is a powerful system for establishing fundamental principles of neuronal fate specification, function and circuit assembly, as it is relatively simple and experimentally tractable compared to the brain. This has enabled considerable progress in establishing the functions of different ventral spinal cord interneurons in locomotor circuitry e.g. –9). Howe. Howe9])For neurons to function correctly they must synthesize and utilize correct neurotransmitters. Within neuronal circuitry, if they inhibit rather than excite their synaptic partners, or vice versa, then the behaviors and functional outputs of those circuits will be dramatically disturbed, and may give rise to pathological conditions. For example, disruptions in the balance of excitatory and inhibitory neurons in the CNS have been implicated in epilepsy, autism, Alzheimer’s and many other neurological disorders e.g. –13). How. How13])B cells )ls e.g. –34). Evx. Evxv ce mutants . However mutants .D interneurons are crucial for correct left-right alternation during locomotion, with V0v cells in particular being required for hindlimb left-right alternation during fast locomotion [In mammals, both V0v and V0comotion , 34. Whicomotion , 35, 36;evx1 and evx2 are expressed in a similar intermediate dorsal-ventral spinal cord position to that observed in other vertebrates [evx1 and evx2 are co-expressed by V0v cells and we show that V0v cells are glutamatergic and have a Commissural Ascending  morphology. We also provide the first analysis of evx1;evx2 double mutants in any vertebrate and the first analysis of the spinal cord phenotype of evx2 mutants. Significantly, we demonstrate that Evx1 and Evx2 are required, partially redundantly, to specify the glutamatergic fates of V0v cells. Given that we know so little about how excitatory fates are specified in the spinal cord and particularly the ventral spinal cord, these findings add considerably to our understanding of CNS circuit development.In zebrafish, tebrates , 32, 33,Evx1 mutant [evx1;evx2 double mutants V0v cells become inhibitory, but they do not express Pax2, suggesting that they are acquiring their inhibitory fates through a novel Pax2-independent mechanism.In the absence of both Evx1 and Evx2, V0v cells lose their glutamatergic fates but other functional characteristics like soma/cell body morphology and axon trajectory are unchanged. In addition, and in contrast to a previously described mouse 1 mutant , these cAll zebrafish experiments in this research were approved either by the UK Home Office or by the Syracuse University IACUC committee.Danio rerio) were maintained on a 14 h light/10 h dark cycle at 28.5 °C and embryos were obtained from natural, paired and/or grouped spawnings of wild-type (WT) adults , identified heterozygous or homozygous Tg(slc17a6:EGFP) ; [Tg(evx1:EGFP)SU1 or Tg(evx1:EGFP)SU2 adults, double heterozygous evx1i232;evx2sa140 mutants or double heterozygous evx1i232;evx2sa140 mutants that also carried one of the Tg(evx1:EGFP) lines (see below). Embryos were reared at 28.5 °C and staged by hours post fertilization (h), days post or prim staging/or prim staging [Zebrafish (a:EGFP); , 37), Tg staging .evx1i232 mutation has been described before [evx2sa140 mutant was received from the Wellcome Trust Sanger Centre, (https://www.sanger.ac.uk/sanger/Zebrafish_Zmpbrowse). Both mutations produce a single base pair change (a C to a T in the case of evx2sa140) that results in a premature stop codon before the homeobox  evx1 genomic sequence (ENSDARG00000099365) and orthologous sequences from human (ENSG00000106038) and mouse (ENSMUSG00000005503) were obtained from Ensembl (http://www.ensembl.org). Zebrafish sequence was used as the baseline and annotated using exon/intron information from Ensembl. The alignment was performed using a 100 bp window and a cutoff score of 70 % identity. A multi-species comparison of approximately 23Kb of Danio rerio genomic DNA sequence containing evx1 and extending 5 Kb into flanking regions revealed high conservation in both coding and non-coding sequences among compared species. We identified three Conserved Non-coding Elements (CNEs) located 5’ and 3’ to evx1. The first is located 79 bp upstream of zebrafish evx1 coding sequence and extends over 100 bp. The other two are located 3' to evx1. One is 2354 bp downstream of the stop codon and is 184 bp long whereas the other is 2979 bp downstream of the stop codon and extends over 140 bp SU1 line was generated by cloning this 3’ CNE amplicon into the pDONRTMP4-P1R vector from Invitrogen using Gateway technology [basegfp plasmid and the pCSDest2 vector [Tol2:1.3Kb 3’ zfish evx1:ßcarp minimal promoter:EGFP:Tol2.The chnology , 43. The2 vector . This reTg(evx1:EGFP)SU2 line. In this case, the GAL4VP16;UAS-EGFP cassette was taken from the pBGAL4VP16;UAS-EGFP plasmid [cfos minimal promoter sequence [GAL4 was synthesized and used with a RVeGFPAttb2 primer to PCR amplify the GAL4VP16;UAS-EGFP cassette.The same 3’ CNE amplicon was used to generate the  plasmid  and clon plasmid , 43. An sequence  plus 17 FWcfosGAL4VP16 primer: CACTCATTCATAAAACGCTTGTTATAAAAGCAGTGGCTGCGGCGCCTCGTACTCCAACCGCATCTGCAGCGAGCAACTGAGAAGCCAAGACTGAGCCGGCGGCCTTTGTACAAAAAAGCAGRVeGFPAttb2 primer: GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA.This PCR product was used to generate a second PCR product using the primer FWattB1cfos: GGGGACAAGTTTGTACAAAAAAGCAGGCTCACTCATTCATAAAATCGCTT and the RVeGFPAttb2 primer.Tol2:1.3Kb 3’ zfish evx1:cfos minimal promoter:GAL4VP16;UAS-EGFP:Tol2.The final amplicon was cloned into pDONR™221 using gateway technology. This middle entry vector was used to generate a final vector containing Tg(evx1:EGFP)SU2 or Tg(1.3 kb evx1:cfos:GAL4-UAS:EGFP) line has the advantage that it contains a GAL4-UAS cassette to amplify EGFP expression. This facilitates visualization of axons. However, this line has a slightly more variegated expression than the Tg(evx1:EGFP)SU1 or Tg(1.3Kb evx1:ßcarp:EGFP) line, presumably because of stochastic silencing of the construct due to the GAL4-UAS sequences [Tg(evx1:EGFP)SU1 line labels all V0v cells more consistently, but the EGFP expression is slightly weaker and we were never able to obtain evx1-/-;evx2-/- double mutant embryos that contained this transgene, even though we could obtain evx1-/-;evx2+/+ and evx1-/-;evx2+/- embryos. This suggests that the Tg(1.3Kb evx1:ßcarp:EGFP) construct integrated close to the WT evx2 allele.Each of these plasmids was separately co-injected with transposase mRNA into 1-2 cell embryos as described by . Embryosequences . In contApproximately 5 nl of a 1:1 combination of two Evx2 ATG Morpholino antisense oligonucleotides (MOs) at 1.25 mg/ml each were injected into 1-2 cell embryos . In all cases, we confirmed that the MOs had completely knocked down Evx2 using immunohistochemistry for zebrafish Evx2.Tg(elav13:EGFP), Tg(evx1:EGFP)SU1, Tg(pax2a:GFP), Tg(Xla.Tubb:DsRed (formerly Tg(NBT:DsRed)), Tg(vsx2:DsRed) and Tg(gata1:GFP) [Tg(elav13:EGFP) trunks. V0v neurons are EGFP-positive cells from Tg(evx1:EGFP)SU1 trunks. V1 neurons are double-positive EGFP-positive, DsRed-positive cells from Tg(pax2a:GFP);Tg(Xla.Tubb:DsRed) trunks. V2a neurons are double-positive DsRed-positive, EGFP-positive cells from Tg(vsx2:DsRed);Tg(elavl3:EGFP) trunks. V2b + KA neurons are double-positive EGFP-positive, DsRed-positive cells from Tg(gata1:GFP);Tg(Xla.Tubb:DsRed) trunks. Total RNA was extracted using an RNeasy Micro Kit . RNA quality and quantity was assayed on an Agilent 2100 Bioanalyser , before converting to fluorescently-labelled cDNA  and hybridizing to a custom-designed Agilent microarray . Details of this microarray will be described elsewhere, along with the characterization of additional genes identified from these analyses. Data pre-processing and normalization was performed using Bioconductor software (https://www.bioconductor.org/). Two-class eBayes and three-class ANOVA analyses were performed using GEPAS software ). All reported statistics were corrected for multiple testing (Benjamini and Hochberg (1995)).To determine which neurotransmitters V0v cells express and to identify additional transcription factors expressed by these cells, different combinations of spinal cord and trunk cells were extracted from live transgenic zebrafish embryos at 27 h using fluorescence activated cell-sorting (FACS). Prior to FACS, embryos were prim-staged, deyolked, dissected and dissociated as in , 50. In ta1:GFP) , 51–54. in situ hybridizations were performed as previously described [dbx1a and dbx1b [evx1 [evx2 [eve1 [pax2a, pax2b, pax8 [eng1b [glyt2a and glyt2b) for slc6a5  was used to label glycinergic cells [slc6a5 encodes for a glycine transporter necessary for glycine reuptake and transport across the plasma membrane. A mixture of two probes to gad1b  and one probe to gad2  was used to label GABAergic cells [gad1b and gad2 encode for glutamic acid decarboxylases, necessary for the synthesis of GABA from glutamate. A mixture of slc17a6b  and slc17a6a  probes was used to label glutamatergic cells [slc32a1 , which encodes for a vesicular inhibitory amino acid transporter, to label all inhibitory cells [Embryos were fixed in 4 % paraformaldehyde and single and double escribed , 56. RNAnd dbx1b , evx1 [51b [evx1 , evx2 [3x1 [evx2 , eve1 [5x2 [eve1 , pax2a, 2b, pax8  and eng18 [eng1b . To deteic cells , 62. slcic cells , 62. gadic cells , 62. Theic cells , 62. We kor2 (ZDB-GENE-060825-57) probe was generated by PCR-amplifying the 3’ region of skor2 from cDNA using a reverse primer containing a T3 promoter sequence at the 5’ end . Total RNA was extracted by homogenizing 50-100 mg of 27hpf wild-type zebrafish embryos in 1 mL of TRIzol reagent . cDNA was synthesized using Bio-Rad iScript Reverse Transcription Supermix kit . A 50 μL PCR was assembled containing 5 μL cDNA and 1 unit of Phusion High-Fidelity DNA Polymerase . PCR conditions were: 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 56.5 °C for 30 s, 72 °C for 1.5 min and then a final extension step of 72 °C for 10 min. PCR product was purified by phenol:chloroform extraction. Forward primer: CGCAAGACGCTTTTTATCCThe DNA template for the sAATTAACCCTCACTAAAGGGAAAATGGAGAGCTGCCTTTCAG.Reverse primer: ZFIN Identification numbers are provided for all genes in Additional file in situ hybridization were mouse anti-Dig  and rabbit anti-Flu . These were detected with Invitrogen Tyramide kits #12 and #5. Secondary antibodies used were Alexa Fluor 568 goat anti-rabbit , Alexa Fluor 488 goat anti-rabbit  and Alexa Fluor 488 goat anti-mouse .Primary antibodies used were rabbit anti-Evx2 , mouse anti-GFP , rabbit anti-GFP  and mouse anti-Pax2 (Covance PRB-276P 1:300). The Pax2 antibody recognizes both Pax2a and Pax2b in zebrafish [Embryos for immunohistochemistry were treated with acetone for 15 min (24 h embryos) or 20 min (30 h embryos) to permeabilize them, washed for 5 min in distilled water, then washed 2 x 10 min in PBS. Embryos were treated with Image-iT Signal Enhancer  for 30 min, then incubated in block solution  for 1 h at room temperature followed by incubation in primary antibody in fresh block solution at 4 °C overnight. Embryos were washed with PBT (PBS + 0.1 % Triton) for 2 h at room temperature and incubated with secondary antibody in block solution at 4 °C overnight. Embryos were then washed with PBT for at 2 h at room temperature and stored in 2 % DABCO ., after incubation with primary antibody, samples were incubated in fresh blocking solution with goat anti-rabbit IgG  at 4 °C overnight. Embryos were then washed with PBT for 2 h and incubated with rabbit PAP  in block solution at 4 °C overnight. Embryos were then washed in PBT for 2 h. Staining was performed using Sigma Fast 3,3’- diaminobenzidine tablets .For 3,3’- diaminobenzidine (DAB) stainingEmbryos were mounted in 70 % glycerol, 30 % PBS and DIC pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. Fluorescent images were taken on a Zeiss LSM 710 confocal microscope. Images were processed using Adobe Photoshop software  and Image J software . In some cases different focal planes were merged to show labeled cells at different medial lateral positions in the spinal cord.skor2 + Tg(evx1:EGFP) double-labeling experiments, the skor2 + Tg(slc17a6:EGFP) double-labeling experiments and the pax2a and eng1b in situ hybridization results. In all of these cases 4 embryos were counted. Results were analyzed using the student’s t-test; Error bars indicate standard deviation.In all cases, cells counts are for both sides of a five-somite length of the spinal cord adjacent to somites 6-10. Most values are an average of at least 5 embryos. Exceptions are the Evx1 is expressed in V0v cells and while double-labeling experiments have not yet been performed, the data suggest that Evx2 is probably co-expressed by these same cells [evx1 and evx2 expression in a similar region of zebrafish spinal cord [In mouse, me cells , 27, 28.nal cord , 33 but in situ hybridization experiments and found that zebrafish evx1 and evx2 are co-expressed in an intermediate region of the dorso-ventral axis of the spinal cord SU1. We constructed this line using enhancer sequences identified downstream of evx1   Fig. , howevervx2 Fig. . This ishan evx2 , 33, altdbx1 [evx1/2-expressing cells are V0v cells we performed EGFP immunohistochemistry and in situ hybridization for dbx1a and dbx1b in Tg(evx1:EGFP)SU1 embryos. We found that zebrafish evx genes are expressed lateral to cells expressing both of these dbx1 genes, as would be predicted for cells developing from the p0 domain. In addition, dbx1a and dbx1b expression persists in some EGFP-positive cells SU1 transgenic line and an additional transgenic line that has stronger expression in V0v cell axons, Tg(evx1:EGFP)SU2 (see methods), to examine the morphology of zebrafish V0v cells. We found that by 27-30 h, almost all of the cells have extended their axons ventrally and have at least started to cross the midline to the other side of the spinal cord  for one to four somite segments . We usedcord Fig  & b. By cord Fig  & f.Fig.Tg(slc17a6:EGFP) line which labels glutamatergic interneurons in the zebrafish spinal cord [in situ hybridization for slc17a6 (vglut) genes, markers of glutamatergic cells (see methods for more details) combined with EGFP immunohistochemistry in Tg(evx1:EGFP)SU1 embryos also confirmed that evx1-expressing cells are glutamatergic . We also observed no double-labeled cells when we performed EGFP immunohistochemistry and in situ hybridization for slc32a1  which labels all inhibitory neurons [Tg(evx1:EGFP)SU2 embryos  and excitatory (glutamatergic) interneurons , 28, 36,nal cord , 67 revegic Fig. . We also neurons , 69 in Tyos Fig. .Fig. 5NeTg(evx1:EGFP)SU1 line we found that these cells express markers of glutamatergic fates  and slc17a6b ) and do not express either glycinergic markers  or slc6a5 ) or GABAergic markers (gad1b or gad2)  Fig.  & e.evx1 and evx2 are co-expressed with pax2 in the spinal cord [evx1 and pax2 using several complementary approaches. These included in situ hybridization for evx1 and immunohistochemistry for Pax2, EGFP immunohistochemistry and in situ hybridization for pax2a, pax2b and pax8 SU2 embryos and double immunohistochemistry for EGFP and Pax2 in Tg(evx1:EGFP)SU2 embryos. In all cases, we observed no double-labeled cells, suggesting that evx1 and evx2 are not co-expressed with pax2/ pax8 genes in zebrafish spinal cord  in Tg. We found that all of the WT (21/51) and heterozygous embryos (20/51) had Evx2 antibody staining but all of the homozygous mutants (10/51) did not . However, we did obtain fish homozygous for evx1 and heterozygous for evx2 . Our analyses of incrosses from identified evx2 heterozygous fish suggest that evx2 mutant embryos have no obvious morphological defects for the first few days of development but that most of them die by larval stages SU2 embryos . However, strikingly, most V0v cells are still labeled with EGFP and these cells have a normal commissural ascending CoSA morphology SU1 transgene, suggesting that this transgene probably integrated in the vicinity of the WT evx2 allele (see methods). However, consistent with our results using the Tg(evx1:EGFP)SU2 transgenic line, when we injected evx2 ATG morpholinos, at a concentration that eliminates Evx2 protein, into evx1 single mutant Tg(evx1:EGFP)SU1 embryos, we also observed EGFP-labeled V0v cells with normal CoSA axon trajectories SU1 embryos (73/75 cells counted in 4 embryos). Given this high number of double positive cells and the fact that there is usually a delay in EGFP expression it is possible that all of the ventral skor2-expressing cells are V0v cells. Interestingly, double labeling experiments with skor2 and Tg(slc17a6:EGFP) demonstrated that both the ventral and dorsal skor2-expressing cells are excitatory cells . From tain Fig. . Double lls Fig. . On averlls Fig. , suggest in Tgevx:EGFPSU1 skor2 is lost, we decided to test if V0v cell neurotransmitter phenotypes were changed. When we examined the expression of slc17a6 (vglut) genes in embryos from a cross of evx1;evx2 heterozygous parents, we saw a significant reduction of glutamatergic cells in both of the single mutants when compared to WT embryos and an even more severe reduction in double mutants SU2 transgene. In WT embryos we see no co-expression of slc32a1 and EGFP ; Fig. To further confirm that V0v cells are switching to an inhibitory fate, we performed GFP Fig. . Howeverslc32a1 (viaat) result, we see a slight increase in both single mutants as well as a more pronounced increase in double mutants SU2 transgene, using either the Pax2 antibody or in situ hybridization with a mix of pax2a, pax2b and pax8 probes. In each case we examined at least two WT embryos and two double homozygous mutants and we did not observe any double-labeled cells  and teleosts, including zebrafish, have three , although only evx1 and evx2 are expressed in the spinal cord ([evx genes (adjacent to specific Hox clusters) along with phylogenetic analyses strongly suggest that the third evx gene in teleosts (eve1) is not the result of the extra genome duplication in the teleost lineage . In addition, we confirm and extend previous reports that suggested that V0v neurons extend commissural axons [Here we provide the first comprehensive analysis of the functions of Evx1 and Evx2 in spinal cord interneuron development in any vertebrate. We demonstrate that, within the spinal cord, both of these transcription factors are expressed exclusively by V0v cells. We also show that V0v cells are glutamatergic. These findings complement and extend those of Satou and colleagues , who replleagues , who shoal axons , 35, 36 Evx1 mutants, most V0v cells completely change their fate and acquire characteristics of V1 cells, the cell-type that normally forms ventral to V0v cells. Cells that would have formed V0v interneurons lose expression of Evx1 and Evx2 and instead express the V1 marker Engrailed1 (En1) and develop axon trajectories and migration patterns characteristic of V1 interneurons [Evx2 mutants have not been described in mouse and before this study, Evx1;Evx2 double mutants had not been described in any vertebrate.In mouse rneurons . Most norneurons . In addirneurons . The rolrneurons , but spiNkx2.2 mutant mice, cells that would have formed V3 interneurons change their fates (transfate) and become motoneurons instead [Dbx1 mutant mice, cells that would have become V0v cells assume the characteristics of V1 cells [These amniote data suggest that Evx1 is required to inhibit the V1 fate in post-mitotic V0v cells. This global cell fate change is unusual for a transcription factor expressed in post-mitotic cells: it is more commonly seen with transcription factors expressed in spinal progenitor domains e.g. , 81, 82), 8281, 8 instead . SimilarV1 cells , 28.evx1 and evx2 single or double mutants. Notably, there is no increase in the number of cells expressing eng1b, which is specifically expressed in V1 cells, or Pax2, which is expressed by V1 and V0D cells. Instead most V0v cells continue to express evx1 mRNA and Tg(evx1:EGFP) and these EGFP-labelled V0v neurons have what appear to be normal CoSA axon morphologies. These data strongly suggest that V0v cells still form in zebrafish evx1;evx2 double mutants and that they do not become a different class of neuron. One possible explanation for the differences between our results and the previously reported analyses in mouse might be evolutionary changes in the functions of Evx1 and Evx2. However, it is also possible that the consequences of removing these transcription factors are different in mouse and zebrafish because of variations in the expression of Dbx1 and/or the timing of V0v development. Interestingly, in this paper, we have shown that expression of dbx1a and dbx1b persists in at least some V0v cells in zebrafish. Therefore, it is possible that in zebrafish evx1;evx2 mutants, Dbx might be able to inhibit post-mitotic V0v cells from becoming V1 cells  CoSA interneurons. We also show that Evx1 and Evx2 are required, partially redundantly for expression of skor2 and glutamatergic markers and inhibition of glycinergic markers in V0v cells and that in the absence of Evx1 and Evx2 function V0v cells become glycinergic through a novel Pax2-independent mechanism. Taken together, our data significantly increase our understanding of how neurotransmitter fates are specified and the genetic networks involved in these processes.In conclusion, in this paper we demonstrate that zebrafish V0v cells express


Data from the phase III registration trial comparing tumour-treating alternating electric fields (TTFields) Using an unsupervised binary partitioning algorithm, we segregated both cohorts of the trial based on the dexamethasone dose that yielded the greatest statistical difference in overall survival (OS). The results were validated in a separate cohort treated in a single institution with TTFields and their T lymphocytes were correlated with OS.vs 11.0 months respectively  in the TTField-treated cohort and 6.0 vs 8.9 months respectively  in the chemotherapy-treated cohort. In a single institution validation cohort treated with TTFields, the median OS of patients who used dexamethasone >4.1 mg per day was 3.2 months compared with those who used ⩽4.1 mg per day was 8.7 months . There was a significant correlation between OS and T-lymphocyte counts.Patients who used dexamethasone doses >4.1 mg per day had a significant reduction in OS when compared with those who used ⩽4.1 mg per day, 4.8 Dexamethasone exerted profound effects on both TTFields and chemotherapy efficacy resulting in lower patient OS. Therefore, global immunosuppression by dexamethasone likely interferes with immune functions that are necessary for the treatment of glioblastoma. Two-tailed Wilcoxon's rank-sum test with continuity correction was used to determine whether two independent groups of data were statistically different from each other. A modified binary search algorithm . We therefore investigated further whether there was a threshold dose of dexamethasone that affected outcome within the entire trial population. Using an unsupervised binary partitioning algorithm (n=64) exhibited a significantly shortened median OS of 4.8 months : 3.9–6.0) vs those who used ⩽4.1 mg per day (n=56), with a median OS of 11.0 months (95% CI: 8.8–16.6)  vs 8.9 months (95% CI: 7.2–16.1) (n=63)  vs 3.9 (range 0.0–8.6) months for the BPC chemotherapy-treated subgroup (n=40)  vs 16.8 (range 8.9–36.7) months for the BPC chemotherapy-treated subgroup (n=23)  vs 8.7 (range 6.0–29.6) months (n=22)  as compared with the latter having a median OS of 2.8 (range 0.2–5.8) months (n=32)  in our validation set. This sub-population exhibited a median OS of 8.0 months (95% CI: 3.8–13.8) compared with 3.2 months (95% CI: 1.4–NA) for the remaining patients with a KPS <70 (n=12)  vs 3.2 months (95% CI: 1.2–NA) (n=4), respectively  to the phase III TTField therapy cohort who used dexamethasone ⩽4.1 mg per day , respectively  cm2 in the TTField-treated phase III cohort  by the strong correlation between the size of the gadolinium-enhanced T1-weighted and FLAIR measured bidimensional tumour size among those not on bevacizumab  had a worse outcome compared with the corresponding cohort in the phase III trial (n=64), with a median OS of 3.2 months (95% CI: 1.2–NA) vs 4.8 months (95% CI: 3.9–6.0), respectively  (n=7). In contrast, the median OS of those with CD3+ >382 cells per mm3 was 7.6 months (95% CI: 4.3–13.9) (n=22)  (n=9) as compared with those with CD4+ >236 cells per mm3 with a median OS of 8.0 months (95% CI: 4.6–NA) (n=20)  (n=5) as compared with 6.8 months (95% CI: 3.9–13.8) (n=24) for those with CD8+ >144 cells per mm3  and between CD3+ and CD8+ cells (r2=0.5001) but not between CD4+ and CD8+ cells (r2=0.0733). However, there was no correlation between white blood cells (WBC) and CD3+ cells (r2=0.0053), WBC and CD4+ cells (r2=0.0023), and WBC and CD8+ cells (r2=0.0032). No correlation was also detected between platelets and CD3+ cells (r2=0.2576), platelets and CD4+ (r2=0.2746), and platelets and CD8+ (r2=0.0887). Similarly, there was no correlation between the daily dexamethasone dose and CD3+ cells (r2=0.1888), dexamethasone and CD4+ cells (r2=0.1531), and dexamethasone and CD8+ cells (r2=0.0451). Taken together, CD3+, CD4+, and CD8+ lymphocyte counts appear to be independent of the peripheral blood counts and dexamethasone dose effect. Therefore, T-lymphocyte counts may serve as an independent measure of immunocompetence in our patients and predict treatment outcome when using NovoTTF-100A.We next asked whether CD3post hoc analysis of responders in the phase III trial comparing NovoTTF-100A monotherapy and BPC chemotherapy for recurrent glioblastoma revealed that dexamethasone and prior low-grade glioma histology were predictors of response . Traditionally, oncologists view dexamethasone's influence on glioblastoma patients from the perspective of its antioedema effect from the tumour  subjects taking ⩽4.1 mg per day of dexamethasone is essentially negated by the hindrance caused by the 29 (24%) patients taking >4.1 mg per day of dexamethasone when the populations were not segregated based on dexamethasone burden. This dexamethasone interference with TTField efficacy may explained the improved outcome seen in the trial for newly diagnosed glioblastoma patients (+ >382 cells per mm3, CD4+ >235 cells per mm3, and CD8+ >144 cells per mm3 in an unsupervised analysis. + lymphocyte count, which predisposes glioblastoma patients to infectious complications, and a CD4+ count <200 cells per mm3 is associated with poor survival. However, we also noted that dexamethasone's immunosuppressive effect also blunted the therapeutic efficacy of TTField therapy and chemotherapy, probably as a result of its global interference with the patient's immune system. This notion is supported by our in vitro experiments, which demonstrated that cells attempting to divide in the presence of the TTFields are disrupted in mitosis during the metaphase-to-anaphase transition and experienced aberrant mitotic exit (+ cytotoxic T-lymphocytes (Our data also indicate that T-lymphocyte subsets may have an important role in the outcome of our validation cohort of patients treated with TTField therapy, with prolonged OS associated with absolute CD3tic exit . Because+, CD4+, and CD8+ lymphocytes could also negatively influence the activation of other cytotoxic subsets such as NK cells (There are a number of limitations in the interpretation of our findings. First, our data only allowed us to examine global immunosuppression in our patients but provide no means to assess local immunosuppression within the tumour microenvironment. This local suppression of immune surveillance is thought to be mediated by arginase, regulatory T cells, and myeloid-derived immunosuppressive cells (+ >382 cells per mm3, CD4+ >236 cells per mm3, and CD8+ >144 cells per mm3. Taken together, these data strongly suggest that the stimulation of immunity against the tumour operates in both of these therapeutic approaches. Future clinical trials for recurrent glioblastoma, as well as other types of brain tumours, may need to take into account the influence of dexamethasone on therapeutic outcome.In conclusion, dexamethasone exerted a profound interference on the therapeutic effects of both TTField therapy and BPC chemotherapies. The threshold dose at which dexamethasone was able to be used with minimal interference on these treatments was 4.1 mg per day or lower. In our validation set of TTField-treated patients, the cluster that had the longest OS had CD3


Fraxinus mandshurica Rupr. to dehydration  was investigated under four different conditions viz. 20 °C; 20 °C with silica gel; 1 °C; and 1 °C after pretreatment with Ca2+. Proteins from seeds dehydrated under different conditions were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2919 protein spots were detected, and high-resolution 2D-DIGE indicated there were 27 differentially expressed. Seven of these were identified using MALDI TOF/TOF mass spectrometry. Inferences from bioinformatics annotations of these proteins established the possible involvement of detoxifying enzymes, transport proteins, and nucleotide metabolism enzymes in response to dehydration. Of the seven differentially abundant proteins, the amounts of six were down-regulated and one was up-regulated. Also, a putative acyl-coenzyme A oxidase of the glyoxylate cycle increased in abundance. In particular, the presence of kinesin-1, a protein important for regulation and cargo interaction, was up-regulated in seeds exposed to low temperature dehydration. Kinesin-1 is present in all major lineages, but it is rarely detected in seed desiccation tolerance of woody species. These observations provide new insight into the proteome of seeds in deep dormancy under different desiccation conditions.Desiccation tolerance is the ability of orthodox seeds to achieve equilibrium with atmospheric relative humidity and to survive in this state. Understanding how orthodox seeds respond to dehydration is important for improving quality and long-term storage of seeds under low temperature and drought stress conditions. Long-term storage of seeds is an artificial situation, because in most natural situations a seed that has been shed may not remain in a desiccated state for very long, and if dormant it may undergo repeated cycles of hydration. Different types of seeds are differentially sensitive to desiccation and this directly affects long-term storage. For these reasons, many researchers are investigating loss of desiccation tolerance during orthodox seed development to understand how it is acquired. In this study, the orthodox seed proteome response of  Fraxinus mandshurica Rupr. (fam. Oleaceae) is an important broadleaf timber tree species in the northeastern forest region of China, and it is classified as an orthodox seed species. Seed propagation is its main mechanism of breeding, and the seeds exhibit characteristics of deep dormancy. As the seeds require 8–20 months to break dormancy, this causes difficulties for nurseries to produce seedlings of this species. In a previous study, seeds were treated with various temperatures using indoor naked stratification, and a reliable method was developed to break seed dormancy by controlling relative water content (RWC). Previously, the effects of dehydration under different conditions on the germination physiology of dormancy-released F. mandshurica seeds were studied -1-propanesulfonate (CHAPS) and 30 mM Tris-HCl, pH 8.5) and this was stored at −80 °C. Prior to quantification, the pH of each protein sample was adjusted to 8.5 with concentrated HCl. Protein concentration was determined with a 2-D Quant kit  using bovine serum albumin (2 mg/mL) as the standard. The optimal concentration of the protein sample was 1–10 mg/mL.Approximately 300 mg of seeds were ground in N2+ replicate 1; gel 2, Cy3 1 °C after pretreatment with Ca2+ replicate 2 plus Cy5 20 °C replicate 1; gel 3, Cy3 20 °C replicate 2 plus Cy5 20 °C with silica gel replicate 1; gel 4, Cy3 20 °C with silica gel replicate 2 plus Cy5 1 °C replicate 2; gel 5, Cy3 20 °C replicate 3 plus Cy5 1 °C replicate 3; gel 6, Cy3 20 °C with silica gel replicate 3 plus Cy5 1 °C after pretreatment with Ca2+ replicate 3. Each sample contained an equal amount of a Cy2-labeled internal standard protein mix derived from pooling an equal amount of protein from each of the four extracts and this allowed for normalization of each gel. The labeling procedure was performed for the CyDye DIGE fluors  and Ettan DIGE according to the manufacturer’s instructions . Prior to isoelectric focusing (IEF), labeled samples were mixed with an equal volume of 2× sample buffer  containing 2% dithiothreitol (DTT) and 1% immobilized pH gradient (IPG) buffer  to bring the final volume to 450 µL. IEF was carried out using 24-cm Immobiline Dry Strip gels (pH 3–11) at 25 °C. The electrophoresis consisted of six steps: 30 V for 12 h, step-n-hold; 100 V for 1 h, step-n-hold; 1000 V for 1 h, gradient; 8000 V for 3 h, gradient; 8000 V for 55,000 V·h, step-n-hold; and 500 V for hold, step-n-hold. After IEF, strip gels were removed and equilibrated for 15 min in 1% DTT, 6 M urea, 75 mM Tris-HCl (pH 8.8), 29.3% glycerol, 2% sodium dodecylsulfate (SDS) and 0.002% bromophenol blue. After removing from the equilibrium buffer, the strips were allowed a second equilibration for 15 min in the same equilibration buffer without DDT but this time supplemented with 2.5% iodoacetamide. The equilibrated strips were then placed on a top of an SDS-polyacrylamide gel (12.5%) on low fluorescent glass plates. Separation was performed at 25 °C on an Ettan™ DALT-six apparatus at 3 W/gel for 45 min, and then 17 W/gel until the bromophenol blue front had reached the bottom of the gel.Each protein sample was labeled with Cy3 or Cy5 protein minimal dye (100 pMol for each 50 µg of protein), according to the manufacturer’s directions . An internal standard sample was prepared by pooling an equal protein quantity from each of the samples. Each protein sample was labeled with Cy3 or Cy5 fluorescent dye separately according to the experiment design. Thus protein mixes were prepared in the following sequence for 2D-DIGE analysis: gel 1, Cy3 1 °C replicate 1 plus Cy5 1 °C after pretreatment with CaCyDye-labeled gels were scanned using a Typhoon™ 9400 Trio imager  choosing parameters appropriate for the excitation and emission wavelengths of each dye . Gels were scanned individually at 100 µM resolution.t-test were used, with p < 0.05 considered to be significant. Gels were stained with Coomassie Brilliant Blue (CBB) and spots of interest were excised from the preparative gels.The voltage of the photo multiplier tube was adjusted for fluorescence intensity between 60,000–100,000. Images were analyzed using DeCyder Difference Analysis software version 7.0 . Scanned images were imported by Image Loder using the differential in-gel analysis module of this software, and inter-gel matching was performed using the Biological Variation Analysis module. To calculate the abundance change of each matched spot pair, one-way analysis of variance tests and paired student’s 4HCO3 and 30% CH3CN-H2O  for 15 min. De-stained spots were centrifuged briefly and the supernatant was removed before placing in CH3CN for 5 min. Gel pieces were dried at room temperature for 5 min so that the CH3CN had completely evaporated, and 2 µL of 50 ng/µL trypsin (adjusted to pH 7.5–8.5 with 50 mM NH4HCO3) was added. Gel pieces were placed for 60 min at 4 °C, and then 2 µL of 50 mM NH4HCO3 (pH 7.8–8.0) were added to cover the gel pieces completely. Digestion was performed overnight at 37 °C.Spots of interest were picked from preparative CBB-stained gels and de-stained in a solution containing 100 mM NHm/z 800–2000 in the positive-ion mode and accumulated at a laser intensity of 3700 with an acceleration of 20 kV. Spectra were calibrated using PeptideCalibStandard before survey scans were prepared giving mass outlier errors of 5 ppm. Peaks were detected with a minimum signal/noise ratio >20. Peptide precursor ions corresponding to contaminants (including trypsin and keratin autolytic products) were excluded with a mass tolerance of ±0.5 m/z. In total, 15 precursor ions with the strongest signals were selected for the MS/MS scan. The MS/MS spectra were accumulated at a laser intensity of 4100, an acceleration of 1 kV and a pressure of 5 × 10−8 Torr. Mass spectra were processed using Global Server Workstation software. A database search for protein identifications was performed using SwissProt Viridiplantae in MS and MS/MS combination mode  was mixed with matrix solution ((1 µL) 5 mg/mL α-cyano-4-hydroxycinnamic acid) and deposited onto the MALDI target, which was dried at room temperature. Tryptic peptides were analyzed by ABI 4800 plus MALDI TOF/TOF Analyzer. MS survey scans were acquired for the mass range mode see .F. mandshurica seeds dehydrated at a critical stage to 10% RWC and low temperature, thereby providing insight into the processes that lead to desiccation-induced damage. Kinesin-1 has been rarely found in wood seed plants, and this protein may play a role in the establishment of cell morphology and environmental adaptation strategies. Changes in proteins associated with ROS also indicate that defense mechanisms in response to low temperature stress and dehydration undergo important alterations. These results indicate that differentially abundant proteins change cellular functions in dormancy-released F. mandshurica seeds during low temperature and dehydration conditions, and highlight some important processes involved in cellular protection processes, transport processes and regulation. These results give a unique insight into the processes of cellular protection and transport that may be explored for improving desiccation tolerance in dormancy-released seeds of this species.The sensitivity of 2D-DIGE, coupled with protein identification using MALDI TOF/TOF, has permitted the study of the dynamic proteome of dormancy-released


Clostridium difficile infection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients. C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. Some C. difficile strains encode two Agr loci in their genomes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date, whereas the agr2 locus is present in a few strains. The functional roles of agr1 and agr2 in C. difficile toxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of both agr loci and examined the mutants for toxin production and virulence. The results showed that the agr1 mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-type agr1. Furthermore, the agr1 mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease. C. difficile infections has been increasing exponentially in the United States, resulting in about 4.8 billion U.S. dollars in health care costs annually. As a multidrug-resistant, spore-forming, anaerobic pathogen, C. difficile overpopulates the colon after the gut microbiota has been altered by antibiotic therapy. With increasing resistance to antibiotic treatment of C. difficile infections, patients are experiencing higher costs of health care and a lower quality of life as treatment options decrease. During infection, C. difficile produces toxins A and B, which directly cause disease. As a result, the toxins have become promising nonantibiotic treatment targets. Here, we have identified a pathway responsible for activating the production of the toxins. This important finding opens up a unique therapeutic target for the development of a novel nonantibiotic therapy for C. difficile infections.Within the last decade, the number of cases of  Clostridium difficile infections (CDIs) account for most of the cases of antibiotic- and hospital-associated diarrhea with defined etiology worldwide and are a frequent cause of morbidity and mortality in older hospitalized patients  and released into the extracellular milieu. The AgrC histidine kinase protein senses and binds extracellular AIP, which activates its ATPase activity, resulting in autophosphorylation and phosphoryl group transfer to AgrA. Phosphorylated AgrA forms a dimer, which enables the C-terminal DNA binding domain to bind either to the promoter region of the agr operon to produce more transcripts of the agr system components or at the RNAIII promoter to regulate the transcription of target genes (The accessory gene regulator (Agr) quorum signaling system regulates virulence and colonization factors in several Gram-positive bacteria , 10, 11., and -D –14. The et genes .agr operon can commonly be divided into two pathways: (i) a signal generation pathway that generates the AIP and (ii) a response pathway that senses and transduces the AIP to activate target gene expression. Some C. difficile strains possess two loci of the agr component genes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date  and contains only the AIP generation pathway genes (agrB1 and agrD1). On the other hand, the agr2 locus, which is mostly present in a small number of strains, including the hypervirulent NAP1/027 R20291 strain, contains both AIP generation  and AIP response  genes. The main roles of these two accessory gene loci in C. difficile pathogenesis have not been clear until now. We previously demonstrated that C. difficile toxins A and B are regulated by an Agr-like quorum signaling system and purified a peptide thiolactone AIP  from the stationary-phase culture supernatant that activates toxin production  for strain R20291  PCR product. To confirm the locations of the deletions in the genome, the PCR products were sequenced and the DNA sequence results demonstrated complete deletion of the agrB1D1 locus from the genomes of both strains and of the agrB2D2 locus from that of strain R20291.To examine the roles of the two accessory gene regulator loci in loci see . Followin R20291  and B. DagrB1D1 and agrB2D2 deletions on toxin production, 48-h culture supernatant fluids collected from the mutant and wild-type strains were concentrated and analyzed by Western blotting with monoclonal antibodies that specifically recognize toxins A and B. No toxin bands were detected in the culture fluids collected from the R20291 agrB1D1 mutant (agrB1D1 mutant (see agrB2D2 mutant. The culture fluids were further tested for the presence of toxins by enzyme-linked immunosorbent assay (ELISA), and the results corroborated those obtained by Western blotting  medium or TY medium and quantitative PCR was performed with cDNA converted from total RNA as the template. No significant mRNA transcripts corresponding to the toxin genes (tcdA and tcdB) were detected from the 630 and R20291 agrB1D1 mutants cultured in either BHI or TY medium  in the mRNA transcripts detected in the mutants cultured in both media. These results demonstrate that the agrB1D1 mutant is unable to transcribe significant amount of the toxin genes and that the agrB1D1 locus plays a central role in toxin production.To examine whether the Y medium  to D. OnagrB1D1 genes, the mutants were transformed with a plasmid bearing the wild-type agrB1D1 locus. This restored toxin production in both 630 and R20291 agrB1D1 mutants at levels comparable to those of the wild-type strains  from strain 630 and R20291 stationary-phase culture supernatant fluids that induced toxin production in all strains . Bioinfo mutants  and B. RagrB1D1 mutants can produce the TI signal, culture supernatant fluids from the mutants were examined for the presence of the TI signal by a previously described high-performance liquid chromatography (HPLC)-based purification method  between the amount of formazan produced by fibroblast cells incubated with culture fluids from the 630 and R20291 agrB1D1 mutants and that produced by untreated control cells . However, the amount of formazan produced by fibroblast cells treated with culture fluids collected from the agrB1D1 mutants and untreated controls was significantly higher (P = 0.001) than that produced by the wild-type, complemented agrB1D1 mutant, and R20291 agrB2D2 mutant strains. These results demonstrate that the culture fluid from the agrB1D1 mutant is not cytotoxic and this is likely due to the absence of toxin.To assess the viability of the treated fibroblast cells, the inherent ability of living cells to convert tetrazolium salt into a blue formazan product was measured using the CellTiter 96 Non-Radioactive Cell Proliferation Assay . In this assay, the amount of tetrazolium salt conversion to formazan corresponds to the number of viable cells. The results showed no significant difference  diarrhea, hunched posture, and physical appearance; (ii) movement and response to external stimulus; and (iii) weight loss. Mice (n = 12 in each group) infected with the wild-type strain became moribund from day 2, and only 8% survived for 14 days . However, when the mice were infected with the complemented agrB1D1 mutant, 42% survived and the survival rate was not statistically significantly different from that of mice treated with the wild-type strain (P = 0.2206).The hypervirulent tients 2023. To ev 14 days . Of the agrB1D1 mutant to colonize, collected stool pellets were diluted and cultured on selective plates for CFU counting. There was no significant difference (P = 0.335) between the CFU counts of mice infected with the wild-type strain, mice infected with the agrB1D1 mutant, and mice infected with the complemented agrB1D1 mutant . However, the AgrB1 protein is required to process and cleave the AgrD1 autoinducing prepeptide to generate the TI signal, and hence, deletion of the agrB1D1 locus was the most plausible alternative. Also, Martin et al.  was purchased from the American Type Culture Collection , and the R20291 stock  was from a previous study . The BHI medium used in all of the experiments was reduced by incubation overnight in the anaerobic chamber prior to use. The substrate for the Cdifftox activity assay .Toxigenic us study . BacteriBacterial stocks were stored short-term in chopped meat broth  at room temperature or long-term in autoclaved 10% dimethyl sulfoxide at −80°C.C. difficile toxins A and B in culture supernatant fluid. Briefly, the culture was centrifuged for 15 min at 10,000 × g at 4°C; 250 µl of the supernatant fluids was incubated with 30 µl of 30 mM p-nitrophenyl-β-d-glucopyranoside and incubated either for 4 h at 37°C or overnight at room temperature. The assay was quantitated spectrophotometrically by reading the absorbance of 410 nm. Total protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.). A molar extinction coefficient (ε) of 17,700 M−1 cm−1 for p-nitrophenol . This assay was performed by the protocol provided by the manufacturer.The presence of toxins A and B in the culture supernatants was also tested with the Wampole Clostridium difficile cytotoxicity assay kit . The protocol used was based on the instructions provided by the manufacturer. Briefly, human foreskin fibroblast cells were incubated for 24 to 48 h at 37°C with 48-h culture supernatant fluids collected from the wild-type 630 and R20291 strains and the respective agrB1D1 and agrB2D2 mutants. Cells were observed with an EVOS XL microscope .The effect of the culture supernatant on cytotoxicity was tested with the Bartels agr mutants were tested for their effects on foreskin fibroblast cell viability with the CellTiter 96 Non-Radioactive Cell Proliferation Assay . This assay measures the ability of living cells to convert tetrazolium salt into a blue formazan product, which can be measured with a spectrophotometer. Ready-made human foreskin fibroblast cells  in 150 µl of medium were treated with 50 µl of culture supernatant fluids from the wild-type and agr mutant strains and incubated for 48 h at 37°C. Following the incubation period, 100 µl of the supernatant was removed and replaced with 15 µl of the dye solution and the cells were incubated for 4 h at 37°C. Solubilization Stop Mix Solution (100 µl) was then added and mixed thoroughly to obtain a uniformly colored solution. Absorbance at 570 nm was measured. A reference wavelength of 650 nm was used to reduce background noise contributed by cell debris and other nonspecific absorbance.The culture supernatants from the wild-type 630 and R20291 strains and the respective agrB1D1 and agrB2D2 loci from the 630 and R20291 strains were constructed in pUC57. To create the cassette for the agrB1D1 locus, 1,527- and 923-bp sequences homologous to the DNA sequences flanking the upstream and downstream regions of the locus, respectively, were amplified with primers RH-AGRB1D1-F, RH-AGRB1D1-R, LH-AGRB1D1-F, and LH-AGRB1D1-R (see agrB1D1 locus were inserted into replication-defective C. difficile vector pMTL82151 (kindly provided by Alexandra Faulds-Pain) by using the Sac1 and BamHI sites, resulting in allelic-exchange cassette pAgrB1D1. For the agrB2D2 cassette, 1,118- and 939-bp regions upstream and downstream of the agrB2D2 locus were amplified with primers LH-AgrA2-F, LH-AgrA2-R, RH-AgrB2D2-F, and RH-AgrB2D2-R containing Sac1, Kas1, and BamHI restriction sites. The amplified fragments were assembled in the pBSK(+) Simple-Amp vector and subsequently cloned into pMTL82151, resulting in allelic-exchange cassette pAgrB2D2. Both the pAgrB1D1 and pAgrB2D2 assembled allelic-exchange cassettes were sequenced to confirm the absence of unintended mutations.The allele exchange cassette used for deletion of the C. difficile R20291 and 630 cells were incubated with conjugative Escherichia coli CA434 cells bearing either the pAgrB1D1 or the pAgrB2D2 allelic-exchange vector. The conjugants were plated on Cdifftox agar plates  under the following conditions: initial denaturation at 94°C for 30 s and 36 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 120 s. The PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.To generate the deletion mutants, agrB1D1 and agrB2D2 loci were PCR amplified from wild-type R20291 and 630 DNA templates with oligonucleotides containing EcoRI and XbaI restriction sites .For complementation studies, the entire regions of the agr mutants and the wild type were concentrated with the Pierce 150-kDa Concentrator , and total protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.). The concentrated supernatant fluids (100-µg protein each) were separated by 6% polyacrylamide gel electrophoresis (PAGE) and transferred onto a 0.45-µm nitrocellulose membrane (Fisher Scientific Inc.) with a Trans-Blot cell (Bio-Rad) transfer apparatus. The membrane was blocked overnight in 10 mM Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% skim milk. Following blocking, the membrane was incubated with mouse monoclonal antibodies specific for C. difficile toxins A and B . Blots were washed three times (5 min each) in TBST between incubations. The Protein Detector Western blot BCIP/NBT kit  was then used to probe the membrane for the presence of each toxin with an alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody, followed by incubation with the BCIP/NBT substrate, according to the manufacturer’s instructions.Culture supernatant fluids from 48-h cultures of the agr mutants at an optical density at 600 nm of 0.6 were diluted 1:100 in 30 ml of reduced BHI or TY medium and incubated anaerobically at 37°C for 16 h. Total RNA was isolated with the RNeasy kit (Qiagen) according to the manufacturer’s directions. The total RNA (1 µg of each) was converted to cDNA by reverse transcription with the ProtoScript AMV First Strand cDNA synthesis kit  according to the manufacturer’s instructions. Relative expression levels of tcdA and tcdB transcripts were determined with SYBR green JumpStart Taq Ready Mix (Sigma), gene-specific primers  by oral gavage. The animals were observed twice daily for 14 days postinfection. An endpoint scoring system was devised and used to evaluate the animals. A scoring chart based on three main parameters , was used to determine the endpoint. Diarrhea, hunched posture, and physical appearance were scored as follows: 0, normal ; 1, hunched posture and infrequent grooming; 2, hunched posture, no grooming (ruffled hair), distended stomach, and diarrhea (noticeable dehydration). Movement and response to external stimuli were scored as follows: 0, normal ; 1, reduced movement, reduced alertness, reduced response to stimuli, or uncoordinated movement; 2, no movement, no activity, no eating, no drinking or alertness. Body weight changes were scored as follows: 0, normal ; 1, 5 to 15% loss of body weight; 2, 15 to 20% loss of body weight. Animals that were assessed an endpoint score of 2 for any of the above three parameters during the 14-day observation period were euthanized.Seven-week-old C57BL/6 mice  were given a cocktail of antibiotics  for 5 daC. difficile TOX A/B II assay . Briefly, one to three stool pellets or watery diarrheal stool samples were collected from the anal region of each mouse, weighed, suspended in 250 µl of sterile PBS, and centrifuged at 10,000 × g. ELISA of the supernatant was performed, whereas the pellet was evaluated for the number of CFU by resuspension of the pellet in BHI, dilution, and plating on Cdifftox agar plates.Pellets collected from the mice on day 5 postinfection were tested for the presence of toxins A and B with the Wampole t test was used to compare differences between samples. A Kaplan-Meier curve was used to analyze the survival of the animals. In all cases, statistical significance was defined as a P value of <0.05.All of the data were analyzed and plotted with GraphPad Prism version 6.07 for Windows . The Student Figure S1 C. difficile accessory gene regulator loci showing the location of the region deleted by allelic exchange. The agr1 locus in strains 630 (A) and R20291 (B) and the agr2 locus in strain R20291 (C) are shown. Download The Figure S1, TIF file, 0.4 MBFigure S2 agrB1D1 mutant. Supernatant from a 48-h culture was concentrated with the Pierce 150-kDa concentrator  and subjected to 6% PAGE. The protein bands were transferred onto a 0.45-µm nitrocellulose membrane and probed with monoclonal antibodies specific for toxins A and B. The toxin bands on the transferred membrane were detected with the Protein Detector Western blot BCIP/NBT kit . I, wild type; II, agrB1D1 mutant. (B) The agr1 deletion abolishes toxin production in both the 630 (B) and R20291 (C) strains. The agrB1D1 mutants of both strains and a agrB2D2 mutant of the R20291 strain were incubated in BHI medium for 48 h anaerobically at 37°C. Toxin production was tested by ELISA with the Wampole C. difficile TOX A/B II assay . agrB1D1 Mut, agrB1D1 deletion mutant; agrB1D1 Mut+Comp, agrB1D1 mutant complemented with a plasmid bearing the wild-type agrB1D1 locus; agrB2D2 Mut, agrB2D2 deletion mutant. There were significant differences (P = 0.0023 for 630 and 0.0001 for R20291) between the amounts of toxins produced by the wild-type and agrB1D1 mutant strains. Error bars represent the standard deviations of three independent experiments. Download (A) Western blot analysis of 48-h culture supernatant fluid from the 630 Figure S2, TIF file, 0.5 MBFigure S3 agr1 mutants do not produce toxin in TY medium. agrB1D1 mutants of both strains and the R20291 agrB2D2 mutant strain were incubated in TY medium for 48 h anaerobically at 37°C. Toxin production was detected with the Cdifftox activity assay. agrB2D2 Mut, R20291 agrB2D2 mutant; agrB1D1 Mut, agrB1D1 mutant. There was a significant difference (P = 0.003 for 630 and 0.0001 for R20291) between the levels of toxin activity produced by the wild-type and agrB1D1 mutant strains. Error bars represent the standard deviations of three independent experiments. Download The Figure S3, TIF file, 0.3 MBFigure S4 agrB2D2 mutant produces an active TI signal that restores toxin production in 630 and R20291 agrB1D1 mutants unable to make toxins. 630 and R20291 agrB1D1 mutant strains were incubated in BHI medium anaerobically for 24 h in the presence of the TI signal purified from the R20291 agrB2D2 mutant. Toxin activity was detected with the Cdifftox activity assay (A), and toxin production was tested by nonquantitative ELISA (B) with the Wampole C. difficile TOX A/B II assay . agrB1D1 Mut, agrB1D1 deletion mutant; TI, TI signal. There were significant differences (P = 0.0041 for 630 and 0.0001 for R20291) between the amounts of toxins produced by the wild-type and agrB1D1 mutant strains in the absence of the TI signal. Error bars represent the standard deviations of three independent experiments. Download The R20291 Figure S4, TIF file, 0.5 MBTable S1 Primers used in this study.Table S1, TIF file, 0.4 MB


N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.Coxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3) Nancy strain. Using HeLa cell plaque assays with agar overlays, we noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. We identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. We determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. Our data reinforce the idea that culture-adapted laboratory virus strains can have reduced fitness  Coxsackieviruses frequently infect humans, and although many infections are mild or asymptomatic, there can be severe outcomes, including heart inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. We used a cell culture-adapted strain of CVB3, Nancy, to examine viral replication and pathogenesis in orally inoculated mice. We found that mice shed viruses distinct from input viruses because they formed extremely large plaques in cell culture. We identified a single mutation, VP3 N63Y, that was sufficient for large-plaque formation. N63Y mutant viruses have reduced glycan binding and replication in cell culture; however, they have enhanced replication and virulence in mice. We are now using N63Y mutant CVB3 as an improved system for viral pathogenesis studies. Picornaviridae family. CVB3 is implicated in a range of diseases, including myocarditis (2Coxsackievirus B3 (CVB3) is a nonenveloped RNA virus in the carditis , 2, typearditis 24, and asarditis 2, causingarditis 2. HoweverCVB3 capsids are formed by viral proteins VP1, VP2, VP3, and VP4, and binding to the coxsackievirus adenovirus receptor (CAR) is required for infection –9. In po–12–in vivo , the immune deficiency of these mice is not ideal for studies seeking to understand innate and adaptive responses to viral infection. Therefore, a better model system for oral CVB3 infections is needed since some effects, such as those imparted by intestinal microbiota, can only be uncovered by using the natural oral infection route –34.–in vitro and in vivo and found that N63Y mutant CVB3 has a growth defect in tissue culture but is more pathogenic than wild-type (WT) CVB3 in mice. We also explored what factors promote the emergence of the large-plaque variant, the basis for large-plaque formation, and mechanisms underlying differences in viral replication and pathogenesis.In this study, we identified a large-plaque variant of CVB3 that emerged after oral inoculation of mice. We found that a single mutation, N63Y, in the VP3 protein was sufficient for the formation of large plaques. We characterized this variant both −/− here) mice with 5 × 107 PFU of neutral-red-labeled WT CVB3. Because mice shed unreplicated inoculum virus in their feces, it is difficult to quantify viral replication in the intestine in the first few days after oral inoculation. To avoid this problem, we examined viral replication by using light-sensitive, neutral-red-labeled virus. Virus propagated in the presence of neutral red dye is sensitive to light-induced inactivation by RNA cross-linking but loses light sensitivity upon replication in the dark inside mice, facilitating the assessment of replication and differentiating the inoculum virus from replicated virus in feces. Fecal samples were collected at 24, 48, and 72 h postinfection (hpi), and virus titers were determined by plaque assay using agar overlays , or intramuscularly (i.m.) inoculated with 5 × 107, 5 × 107, or 2 × 106 PFU of CVB3, respectively. Fecal samples were collected from orally inoculated mice at 72 hpi; livers, hearts, and spleens were harvested from i.p. inoculated mice at 48 hpi; and muscles and livers were harvested from i.m. inoculated mice at 48 hpi. We measured the virus plaque sizes and quantified the percentages of large plaques in different tissues  responsible for the large-plaque phenotype, we isolated six individual large-plaque viruses from mouse feces, performed plaque purification to generate large-plaque “fecal isolate” samples, extracted RNA, performed reverse transcription (RT)-PCR, and sequenced products by consensus sequencing. We sequenced the capsid-coding region with the rationale that major changes in plaque size frequently map to capsid proteins , 36. SeqTo characterize N63Y mutant CVB3, we first compared the growth of N63Y and WT CVB3 in cell culture by using single-cycle growth curve assays. Three human cell lines, HeLa, Huh7, and RD, and two rodent cell lines, CHO-K1 and L929, were infected at a multiplicity of infection (MOI) of 0.1, and viral titers were determined over time. N63Y mutant CVB3 demonstrated a growth defect in most of the cell lines –41. Ther–35S-labeled CVB3 was incubated with HeLa, Huh7, RD, L929, and CHO-K1 cell layers at 4°C to facilitate binding but not internalization. After washing, we quantified the cell-associated 35S label. N63Y mutant CVB3 showed significantly reduced attachment to HeLa and Huh7 cells  or human or murine DAF . After washing, the amount of virus bound was quantified by phosphorimager analysis  and least susceptible to oral infection (5 × 107 PFU). Third, we found that N63Y mutant CVB3-infected mice had a lower survival rate than WT CVB3-infected mice. In summary, N63Y mutant CVB3 replicated and disseminated more efficiently in both IFNAR+/+ and IFNAR−/− mice and was more pathogenic than WT CVB3 in IFNAR−/− mice.To examine the virulence of WT versus N63Y mutant CVB3, we monitored the survival of mice following infection via different inoculation routes. We infected IFNAR of CVB3 . First, −/− mice and understand why N63Y mutant CVB3 is more pathogenic than WT CVB3, we examined liver pathology. Wessely et al. demonstrated that CVB3-infected IFNAR−/− mice develop liver pathology , a marker of liver damage, in serum. ALT levels were low at 24 hpi in all of the mice  supplemented with 10% calf serum. CHO-K1 cells and mutant CHO cells  were obtained from Jeffrey Esko  and maintained in F12/DMEM supplemented with 5% fetal bovine serum. Human rhabdomyosarcoma (RD) cells, Huh7 cells, 293 cells, and murine L929 cells were maintained in DMEM supplemented with 5% fetal bovine serum.35S-labeled CVB3 was prepared as previously described , who received it from Reinhard Kandolf. This version of CVB3 Nancy binds HS . Stocks escribed . The spe1cov) . RT-PCR was performed with SuperScript II reverse transcriptase (Invitrogen) by using primer 5′ CGCCACGGACGAGGTA 3′ to generate cDNA. PCRs were performed with primer pairs that cover most of the VP1-VP3 coding region, i.e., sense primer 5′ GCTGCCCGATGCTTTGT 3′, antisense primer 5′ GGCCGAACCACAGAACATAAAC 3′, sense primer 5′ GCAATATGACGTCACACCAGAGAT 3′, antisense primer 5′ AGGGCTGTTGAGTACTTGTTATGA 3′, sense primer 5′ ACTACCGGTTTGTTGCTTCAGATC 3′, and antisense primer 5′ CTACTAGACCTGGTCCTTCAAACGA 3′. PCR products were sequenced by the University of Texas (UT) Southwestern Sequencing Core. All six isolates contained two amino acid changes, VP3 N63Y (an A-to-T mutation at position 1956) and VP2 T151S (a C-to-G mutation at position 1432 and a G-to-C mutation at position 1433), as well as other silent point mutations. Because position 151 of VP2 is highly variable in CVB3 sequences, we focused our efforts on VP3 N63Y. A 377-bp fragment containing the N63Y mutation was generated by BsiWI/BglII digestion of an RT-PCR product from one fecal isolate, and this fragment was cloned into a new CVB3 Nancy plasmid from nucleotide 1695 (BsiWI) to nucleotide 2072 (BglII). The new plasmid was then sequenced, and N63Y was confirmed as the only mutation in the PCR-generated region. The large-plaque phenotype of this new clone was confirmed by plaque assay. The structure in 1cov) .6 HeLa, Huh7, RD, CHO, or L929 cells. Each plate was inoculated with WT or N63Y mutant CVB3 at an MOI of 0.1. After 30 min of incubation at 37°C, the inoculum was aspirated from the cells. The cell monolayers were washed, and 3 ml of fresh medium was added. At 2, 4, 6, 8, 10, and 12 h after infection, cells were trypsinized and pelleted. To release intracellular virus, cell pellets were freeze-thawed three times before the virus titer was determined by plaque assay with HeLa cells. All data were compared to input stock titers, which were set to 100%.Six-centimeter plates were each seeded with 1 × 1035S-labeled CVB3  was incubated with 8.8 × 106 HeLa, Huh7, RD, CHO, or L929 cells at 4°C for 40 min to facilitate viral binding. Cells were washed three times with cold phosphate-buffered saline (PBS) and trypsinized, and 35S was quantified in a scintillation counter. For certain experiments, cells were treated with 10 U/ml heparinase I (Sigma Aldrich) in 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl, 3 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 0.1% glucose, 1% fetal calf serum, and 0.5% bovine serum albumin for 90 min prior to the viral attachment assay  was mixed with heparin agarose resin (Sigma Aldrich) or streptavidin agarose resin (Sigma Aldrich). Samples were incubated for 3 h at 37°C. Following incubation, resin was pelleted by centrifugation at 2,600 × g for 2 min. Resin was washed three times with PBS+ (PBS supplemented with 100 μg/ml CaCl2 and 100 μg/ml MgCl2), and resin-associated radioactivity was quantified by scintillation counting. Data are expressed as a percentage of the input counts per minute ([resin-associated counts per minute/input counts per minute] × 100). For CAR and DAF binding assays, human (R&D Systems) or murine CAR  or human (R&D Systems) or murine DAF  was immobilized on nitrocellulose membranes and the membranes were incubated with 35S-labeled CVB3  at 37°C for 12 h   were harvested at 48 hpi, and fecal samples were collected from orally inoculated mice at 72 hpi. In the experiments shown in 7 PFU of CVB3 or inoculated i.p. or i.m. with 1 × 104 PFU and 1 × 102 PFU of CVB3, respectively. Tissue samples were processed as previously described  staining was performed by the UT Southwestern Molecular Pathology Core. For ALT quantification, serum was separated by centrifugation and ALT concentrations were determined by enzyme-linked immunosorbent assay  according to the manufacturer’s instructions. In the experiments shown in 35S-labeled WT or N63Y mutant CVB3 at 2 × 107 PFU/20,000 cpm and tissues were collected 1 hpi for quantification in a scintillation counter. In all cases, feces and tissue samples were processed prior to viral titer assay or scintillation counting as previously described  by using protocols approved by the local Institutional Animal Care and Use Committee in a manner designed to minimize pain, and any animals that exhibited severe disease were euthanized immediately with isoflurane. C57BL/6 PVR IFNAR, Japan) , and allescribed . In the escribed . In the escribed  prior tot tests. P < 0.05 was considered statistically significant. All analyses were performed with Graph Pad Prism 6.03 software .Data throughout are shown as the mean ± the standard error of the mean. The differences between groups were examined by unpaired two-tailed Student During the course of this study, we saw a large-plaque CVB3 variant emerge in mice and unexpectedly found that this mutant virus had increased replication and virulence in mice. Upon observing the enhanced virulence of this virus, we consulted with the UT Southwestern Biological and Chemical Safety Office. It was determined that our existing Animal Biosafety Level 2+ protocols, the same used for our poliovirus work, were appropriate for these experiments.Figure S1 N63Y mutant CVB3 has a growth defect in several cell lines. Single-cycle assays of viral replication in RD (A), CHO (B), and L929 (C) cells. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. Download Figure S1, TIF file, 0.6 MBFigure S2 35S-labeled WT or N63Y mutant CVB3 was incubated with nitrocellulose membranes containing immobilized hCAR or mCAR protein. After washing, the bound viral counts per minute were determined to quantify binding. n = 3. Download N63Y mutant CVB3 does not show altered binding to human or murine CAR. Figure S2, TIF file, 2.4 MBFigure S3 2). Download Sulfated glycans/GAGs inhibit WT CVB3 plaque formation. Plaque assays were performed with WT or N63Y mutant CVB3 by using agar and agarose overlays in the presence or absence of 0.1 mg/ml HS or heparin. Plaque sizes were quantified with ImageJ. For ease of comparison, data are displayed as fold WT plaque size, which is the size of each plaque divided by the average size of all WT plaques from plates with agar overlays (average = 0.2473 mmFigure S3, TIF file, 0.7 MBFigure S4 −/− mice were orally infected with 5 × 107 PFU of WT or N63Y mutant CVB3. At 72 hpi, liver tissue was collected and H&E staining was performed. Tissue samples from two representative mice per condition are shown. Download Histopathology of liver tissue. IFNARFigure S4, TIF file, 2.6 MB