FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER–resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the  SCS3 and YFT2, the yeast homologs of mammalian FIT2, are part of a large genetic interaction network connecting lipid metabolism, vesicle trafficking, transcription, and protein synthesis. From these interactions we determined that yeast strains lacking SCS3 and YFT2 are defective in their response to chronic ER stress and cannot induce the unfolded protein response pathway or transcription of phospholipid biosynthetic genes in low inositol. Our findings suggest that the mammalian FIT genes may play an important role in ER stress pathways, which are linked to obesity and type 2 diabetes.The ability to form lipid droplets is a conserved property of eukaryotic cells that allows the storage of excess metabolic energy in a form that can be readily accessed. In adipose tissue, the storage of excess calories in lipid droplets normally protects other tissues from lipotoxicity and insulin resistance, but this protection is lost with chronic over-nutrition. The FAT storage-inducing transmembrane (FIT) proteins were recently identified as a conserved family of proteins that reside in the lipid bilayer of the endoplasmic reticulum and are implicated in lipid droplet formation. In this work we show that specific functions of the FIT proteins are conserved between yeast and humans and that  Eukaryotic cells store neutral lipids  in cytoplasmic lipid droplets (LDs) surrounded by a monolayer of phospholipids and associated proteins FIT gene expression decreases LD production in an adipocyte differentiation cell culture model and in zebrafish Fat storage-Inducing Transmembrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were identified as unique, evolutionarily conserved ER-resident proteins that affect the partitioning of TG into LDs FIT2 gene homologs, SCS3 and YFT2, that are readily identified as the only homologous genes in S. cerevisiae in BLAST searches using the full-length human protein as a query  Budding yeast contains two The metabolic pathways leading to the formation of neutral lipids and the hydrolytic reactions catalyzing their mobilization are conserved between yeast and mammalian cells FIT gene homologs, SCS3 and YFT2. Despite the distant evolutionary relationship between these genes in fungi, the data show that SCS3 and YFT2 have shared as well as unique functions. Common functions are also demonstrated between the proteins in yeast and human systems. However, in contrast to FIT gene knockdown experiments in higher eukaryotes, deletion of SCS3 and/or YFT2 does not noticeably impact the number or size of LDs. A genetic network centered on SCS3 and YFT2 is presented that identifies a multitude of aggravating and alleviating interactions that connect major biosynthetic processes critical for cell growth. We find that the functions of SCS3 and YFT2 relate lipid metabolism and signaling with transcription of phospholipid biosynthetic genes and protein synthesis. More specifically, the data show that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and suggest that the function of Scs3 and Yft2 impacts a regulatory response that serves to coordinate these processes.We have initiated studies in yeast to identify chemical and synthetic genetic phenotypes associated with deletions of the mammalian FIT genes in mammals YFT2 was an uncharacterized open reading frame (YDR319C) whose relationship  to SCS3 was not appreciated. Consequently, none of the studies conducted to date on SCS3 have taken into account its potential functional redundancy with YFT2. Phylogenetic evidence from 42 sequenced fungal genomes YFT2 arose by a segmental duplication of SCS3  elements and drives the expression of phospholipid biosynthetic enzymes and other lipid metabolic proteins SCS3 or YFT2 or both genes were hypersensitive to cerulenin as indicated by the reduced cell density at saturation  and sterol-14 reductase (Erg24) to inhibit ergosterol biosynthesis  but relates SCS3 function to increased translational fidelity based on the properties of a single base substitution within yeast 18S rRNA that also confers this phenotype SCS3 increased the toxicity of cadmium ions. Although the mechanism of this toxicity is not thoroughly understood, cadmium is known to cause lipid peroxidation and like many metals, affects membrane fluidity scs3Δ strains results from an accumulation of metal ion, potentially due to altered membrane composition and/or transporter function (see below). Inositol auxotrophy has been associated with deletion of SCS3 since the gene was first cloned scs3Δ strain was attributed to decreased levels of inositol-3-phosphate synthase (Ino1), the rate-limiting enzyme in the synthesis of phosphatidylinositol (PI) YFT2  YFT2 . Howeverof scs3Δ  as well FIT2 in yeast, we used the fluorescent LD-specific dye BODIPY 493/503 to determine whether either SCS3 or YFT2 could drive the production of LDs in human embryonic kidney (HEK293) cells. Remarkably, transient expression of either SCS3 or YFT2 under the control of the CMV promoter induced the appearance of LDs in HEK293 cells similar to overexpression of the human and mouse FIT genes , suggest that more complex interpretations should be considered: Yeast may possess alternative mechanisms for storing neutral lipids in droplets. Thus, the absence of one mechanism due to deletion of SCS3 and YFT2 may be compensated by the presence of another. Differences in the size of LDs between S. cerevisiae and organisms where FIT gene-dependent changes have been observed could also be important: The average diameter of LDs in yeast ranges between 0.35–0.45 µm compared to 1.1 µm in C. parapsilosis and up to 100 µm in higher eukaryotes Given the complementation of mutant phenotypes by human IT genes  [10]. NoIT genes . Recentlhenotype . Taken aan cells  and the SCS3 and YFT2 and to uncover functional relationships with other genes and processes, we conducted synthetic genetic array (SGA) analysis using query strains deleted for either or both genes , we found that the total number of interactions increased significantly using the double mutant query  in at least one of the three screens and then compared their ε values for all three queries with the corresponding values for the overlapping verified genes. The scores were highly correlated  among the 221 “true positives” revealed a sharp cutoff for aggravating and alleviating interactions at −0.1 and 0.2, respectively, in the single mutant query screens . The samSCS3 and YFT2. Indeed, we identified many interactions with one or the other gene as well as interactions that required deletion of both genes  phosphatase that is localized to the ER and Golgi and functions in protein trafficking, secretion and cell wall maintenance. Deletion of SAC1 dramatically and selectively increases the level of PtdIns(4)P (8–12 fold) and causes missorting of Chs3 to the vacuole SAC1 mutant compromises cell wall maintenance SAC1 substantially reduces strain fitness (to 0.48 relative to wild-type) and this is further aggravated by deletion of either SCS3 or YFT2. However, the fitness of the triple mutant strain is significantly improved (to 0.78 relative to wild-type) indicating the strong alleviating effect of deleting both SCS3 and YFT2. On the other hand, deletion of CHS3 does not have a significant impact on strain fitness by itself but exhibits reduced fitness in combination with either SCS3 or YFT2. These effects are suppressed in the triple mutant strain. The converse of this pattern of genetic interactions was also seen  where each query gene-deletion had alleviating interactions with a particular array gene deletion that was reversed when both SCS3 and YFT2 were deleted. These types of genetic interactions indicate that the functions of SCS3 and YFT2 (or the processes that they impact) sometimes antagonize one another.Synthetic genetic interactions reveal functional relationships between genes th genes . These ge mutant . In otheSCS3 and/or YFT2. Five GO bioprocess terms were significantly over-represented in our data (SCS3 and YFT2 in the cell: They linked together processes (e.g. chromatin-transcription and secretion) that individually are among the most highly connected in the global genetic landscape SCS3 and/or YFT2 is very similar to the genome as a whole  followed by the Golgi and the mitochondrial envelope  that convert PE to PC in the terminal steps of the CDP-DAG pathway had the opposite phenotype  in combination with deletions of SCS3 and/or YFT2  since deletion of either one or both genes rescues the poor growth of a strain lacking Arg82, the inositol polyphosphate multikinase responsible for synthesizing IP4 and IP5  which function as controlling ligands for different biochemical activities P (noted above), suppresses defects associated with a temperature-sensitive mutant of the opposing PI 4-kinase, Stt4, localized at the plasma membrane sac1Δ strain by deletion of both SCS3 and YFT2 could involve or impact functions at the plasma membrane.A role for  and IP5 . DeletioSCS3 and YFT2 to the synthesis of phospholipids, sphingolipids and inositol phosphates are reflected in the cross-talk between these pathways involving lipid-derived second messengers such as phosphatidic acid (PA) and diacylglycerol (DAG) PAH1 and DGK1 which encode PA phosphatase (lipin) and DAG kinase, respectively. Deletions of PAH1 and DGK1, which catalyze the interconversion of PA and DAG SCS3 and YFT2  elevates the concentration of PA which up-regulates the transcription of phospholipid biosynthetic genes and drives a dramatic expansion of the nuclear/ER membrane  and restores normal levels of Ino1 protein SCS3, can be suppressed by deleting the Opi1 repressor INO-regulated genes, increased inositol synthesis and altered lipid composition scs3Δ strains  which is recruited to the INO1 promoter via its interaction with the DNA binding factor Ume6 SCS3 and YFT2 and this presumably coincides with reduced transcription of INO1 and other target genes in the triple mutants relative to the Rpd3(L) single mutants  and in Sgf73 which tethers this module to the rest of the SAGA complex INO-regulated genes SET2 and specific subunits of the Rpd3(S) complex (RCO1 and EAF3) exhibit aggravating interactions with SCS3 and YFT2 , deletions of multiple INO80 subunits led to alleviating rather than aggravating phenotypes in the scs3Δ yft2Δ strain (INO1 gene activation SCS3 and YFT2 rescuing the poor fitness of INO80 complex mutants that are deficient in a repressive function in transcription. This interpretation is supported by strong negative genetic interactions between the INO80 complex and the Rpd3(L) HDAC complex indicating that they function in redundant parallel pathways  pathway, namely the Ire1 sensor kinase/endoribonuclease and the Hac1 transcription factor INO1 gene transcription requires Ire1 and Hac1 and starvation for inositol  and ribosome assembly (e.g. Rei1)  . These rand YFT2 . These iSCS3 and YFT2 resulted in strong aggravating phenotypes with deletions such as SEY1 and ICE2 (proteins linked to ER morphology) and components of the secretory pathway  that causes secretory stress and activates the UPR as a result of defects in COPII-mediated vesicle formation YFT2, SCS3 or both genes in the sec13-1 strain caused an increasingly strong synthetic sick growth phenotype at the permissive temperature of 22°C  induce transcription of phospholipid biosynthetic genes (e.g. INO1) and activate the UPR (increase HAC1 pre-mRNA splicing) SCS3 and YFT2 deletion strains were significantly compromised for both of these responses  and 0.2% Tween 80 SCS3, YFT2 and OPI1 were generated de novo in BY4742 using standard one step gene replacement and dominant drug selectable markers Synthetic complete media contained 400 µM myo-inositol unless otherwise stated. Low or no inositol media was prepared from inositol-free YNB (Difco) and amino acid dropout mixes. YPO media contained 0.3% yeast extract, 0.5% peptone, 0.1% glucose, 0.5% KHSCS3 (scs3Δ::natMX), YFT2 (yft2Δ::natMX) or both genes (scs3Δ::natMX yft2Δ::URA3) and screened by standard SGA methods against an array of 4292 strains representing a healthy subset  of the viable gene-deletion collection hda robot. Digital images of the double or triple mutant colonies were captured and analyzed using ColonyImager software ura3Δ::natMX), against the same deletion array.Query strains were derived from Y7092 his3Δ::kanMX strain his3Δ::kanMX colonies on the inner perimeter of each plate (the plate median) and of all 98 plates  was determined and used to normalize the colony sizes on each plate as follows: Normalized size = unnormalized size×343/plate median. Following normalization, the mean colony size (± standard deviation) was calculated for each mutant (represented by 10 colonies in the control screens and six colonies in each query screen) and the data were filtered to remove outliers (>2 standard deviations from each mean). After recalculation of the mean and standard deviation for each colony, the difference in the pixel size of each mutant on the array was computed between the control and query screens. The significance of this difference was assessed by performing a two-tailed t-test  of the null hypothesis that the sizes of the colonies in the control and query screens are statistical the same. The fitness of the query strains relative to wild-type was determined by comparing the median his3Δ::kanMX colony sizes on the inner perimeter for all plates in each query screen versus the control screen (i.e. 3108 colonies for each query and 5180 colonies for the control). The resulting relative fitness values  were used to calculate genetic interaction scores (ε) as described below. The fitness of each array deletion strain as a single mutant or as a double or triple mutant (combining deletions of SCS3 or YFT2 or both genes) relative to wild-type was determined from the ratio of the mean colony size of the mutant to the median his3Δ::kanMX colony size from the 5180 control colonies. Genetic interaction scores for digenic pairs were calculated as the difference between the observed and expected fitness values of the double mutants where expected fitness was calculated as the product of the fitness of the corresponding single mutants . For trigenic interactions, a similar multiplicative model was employed, ε = Wxyz – (Wx * Wyz), where Wxyz is the observed fitness of the triple mutant, Wx is the fitness of the array deletion strain and Wyz is Wscs3Δ yft2Δ (see above). The general applicability of this solution in the current study is satisfied by the modest 11% fitness defect (Wscs3Δ yft2Δ = 0.89) of the scs3Δ yft2Δ query strain relative to wild type. Unless otherwise indicated, subsequent analyses employed stringent criteria to select interactions with a high probability of representing true positives. These criteria included (i) ≥40 pixel size difference between single mutant and double or triple mutant colonies; (ii) a p value ≤0.01 representing the probability that the colony sizes of the strains being compared are significantly different from one another and (iii) ε scores ≤−0.12 or ≥0.2. Similar criteria for high stringency cutoffs were applied in the large scale study of Costanzo et al SCS3. In an E-MAP of the early secretory pathway SCS3 were reported that had an S score <−1.5. We scored 22 of these and found 14 (64%) that met our stringent criteria . In addition, many genetic interactions with SCS3 were identified by Constanzo et al., p value (IεI>0.08 and p<0.05). From this group we determined that 140 unique interactions were scored in our dataset. Forty-eight of these met our stringent criteria and a total of 64 interactions (46%) were found when interactions were compared at the same p value limit (p<0.05).Each plate of the deletion array contained a two-colony perimeter of the same SCS3 and/or YFT2 and then calculated enrichment by hypergeometric distribution.Biological process annotations for genes on the viable gene-deletion array were obtained from supplementary data file S6 of Costanzo et al 600 readings from 100-well plates every 15 minutes. A single colony was inoculated at a starting OD600 of 0.1 into SD/MSG media that contained 0.1% Nonidet-P40. Doubling time and growth curve data were derived from three independent colonies per strain or condition as per established protocols Growth at 30°C with shaking was measured at using a Bioscreen C Microbiology Reader (Growth Curves USA), which recorded ODSI Materials and Methods: “Microscopy” section. For yeast imaging an upright Olympus BX61 microscope with a sensicam QE cooled CCD camera (Black/White) and IP Lab 4.0.8 software was used at the Analytical Imaging Facility at the Albert Einstein College of Medicine. Cells were viewed with a 100X NA = 1.4 oil objective and FITC filter. Sections (0.2 µm) were collected for each image and combined into a single projection using Image J (http://imagej.nih.gov/ij/). Cells, either early log phase (between OD600 nm 0.2 and 0.8), freshly saturated overnight or 5 day stationary phase cultures, were stained with 0.5 µg/µl BODIPY 493/503 (Invitrogen), for 5 to 10 min. prior to fluorescence microscopy. Cells were prepared as indicated from either SD/MSG, YPD, YPO or SC-Ino media and either imaged immediately or fixed in paraformaldehyde and permeablized in 0.1% Triton as described in HEK293 cells were imaged as described in Methods for RNA preparation and Northern analysis have been reported elsewhere SCS3 and YFT2 deletion strains was achieved by growing strains for 2 hours in 1 µCi/ml of [1-14C]acetate (57 mCi/mmol) or for 6–8 generations in 10 µCi/ml 32P-orthophosphate. Lipids were extracted by the two-step 4°C method 2 and dissolved in chloroform/methanol  for thin layer chromatography. Labeled neutral and phospholipids were separated by one-dimensional TLC in hexanes∶ethyl ether∶acetic acid (80∶20∶1) 20 (30∶35∶35∶7) 600 of log phase wild-type and SCS3 and YFT2 gene-deletion cells were prepared in triplicate and analyzed by mass spectrometry. Total lipid species are reported as nmol/108 cells. Mass spectrometry was performed at the Kansas Lipidomics Research Center Analytical Laboratory.Metabolic labeling of lipids in log phase wild type and Figure S1SCS3 and YFT2 in the genomes of sequenced fungi. Evolutionary relationships were constructed using the sequences of 153 genes that are universally present in the 42 genomes shown Saccharomyces complex, the group of species that share the whole-genome duplication (WGD) and those with the variant genetic code (CTG). YFT2 and SCS3 orthologs were identified by Blast searches of yeast genomes at NCBI or the Broad Institute databases using the complete amino acid sequences for SCS3 or YFT2 or the corresponding signature sequences from the fourth transmembrane domain of each protein. FIT gene orthologs were identified in all organisms except P. chrysosporium, A. terreus and C. globosum. Figure was adapted from ref. Phylogenetic distribution of (TIF)Click here for additional data file.Figure S2SCS3 YFT2 gene-deletion strains. A HsFit expression complements synthetic growth defects in scs3Δ yft2Δ xxxΔ strains. The HsFit2 gene, controlled by a TDH3 promoter and a CYC1 terminator, was substituted for YFT2 by one-step gene replacement in the Y7092 scs3Δ::URA3 query strain. SGA techniques were then used to recover wild-type, scs3Δ yft2Δ and HsFit2-expressing scs3Δ yft2Δ strains containing the indicated deletions from the viable gene-deletion array. Final haploid colonies generated in quadruplicate were printed onto SGA selection media and photographed at 48 hours. Each row of panels is deleted for a different array gene (indicated on the right). Wild-type , scs3Δ yft2Δ (ΔΔ) and HsFit2-expressing scs3Δ yft2Δ (ΔΔ HsFit2) strains are annotated across the top. Note that only the ice2Δ strain is an inositol auxotroph and the growth phenotypes were assayed on SGA medium which contains excess inositol. B HsFit expression in the scs3Δ yft2Δ strain does not induce the UPR. Northern analysis of the distribution of HAC1 mRNA into unspliced Hac1u and spliced Hac1i forms and stable U3 snRNA was performed on RNA from early log phase cultures of wild-type, scs3Δ yft2Δ (ΔΔ) and HsFit2-expressing scs3Δ yft2Δ (ΔΔ HsFit2) strains grown in the presence of excess inositol. The extent of Hac1 splicing is expressed as % of total Hac1 mRNA and is indicated under each lane. ER stress in untreated and DTT-treated cells is reported by the accumulation of spliced HAC1 mRNA.Expression of HsFit2 in (TIF)Click here for additional data file.Figure S3SCS3 YFT2 gene-deletion strains exhibit comparable lipid droplet phenotypes under various stress conditions. A. The indicated strains were grown in inositol-free media supplemented with 10 µM or 100 µM inositol . Early log phase and saturated overnight cultures were analyzed for lipid droplets by direct staining of live cells with BODIPY 493/503 as described in sec13-1 mutation on lipid droplet production in wild-type and SCS3 YFT2 gene-deletion strains. SGA methodology was used to introduce the conditionally-viable sec13-1 mutation as in Wild-type and (TIF)Click here for additional data file.Figure S4SCS3 YFT2 double gene-deletion strain and the SCS3 or YFT2 single gene-deletion strains, respectively. The data are represented in p value<0.01.Comparisons of genetic interaction scores between genes identified in two or more screens. Epsilon scores are compared for 167 (A) and 72 (B) strains that yielded genetic interactions with the (TIF)Click here for additional data file.Figure S5SCS3 and/or YFT2 genetic interactions  were manually examined in GBrowse for overlap with the coding and likely promoter regions of verified genes. From the resulting 40 dubious ORFs and their neighboring/overlapping verified gene-deletions we performed pairwise comparisons of the ε values obtained in scs3Δ, yft2Δ and scs3Δ yft2Δ screens  and determined a Pearson correlation coefficient of 0.75.Correlation of genetic interaction scores between dubious ORFs and their genomic neighbors. Deletion strains corresponding to dubious ORFs in SGD that were contained in the set of 636 (TIF)Click here for additional data file.Figure S6SCS3/YFT2 gene-deletion strains. A. Neutral lipid content is comparable between a wild-type strain and strains deleted for SCS3 and/or YFT2. Log phase cells were metabolically labeled for 2 hours with 14C-acetate and lipids were extracted by the two-step 4°C method SCS3 and YFT2 does not affect total cellular lipid profiles. Lipids from log phase wild-type and scs3Δ yft2Δ cells were prepared from unlabeled log phase cells as described above and analyzed by quantitative mass spectrometry at the Kansas Lipidomics Research Center Analytical Laboratory. The abundance of lipid species is graphed as nmol/108 cells. Left panel: total polar lipids, right panel: total neutral lipid species. Error bars indicate the standard deviation from three biological replicate analyses. Lysophosphatidic acid, LPA; lysophosphatidylethanolamine, LPE; phosphatidic acid, PA; phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylglycerol, PG; phosphatidylinositol, PI; phosphatidylserine, PS; diacylglycerol, DAG; triaclyglycerol, TG.Lipid profiling of wild-type and (TIF)Click here for additional data file.Figure S7SCS3/YFT2 gene deletion strains. A CPY processing in wild-type and scs3Δ yft2Δ strains. Strains were transformed with a MET15 gene-containing plasmid prior to growth in methionine-minus SC media. Cells were pulse-labeled with Tran 35S-labeling reagent for 10 mins and chased with excess cold methionine and cysteine as depicted. Extract preparation and immuoprecipitation were as described p1) and Golgi-modified (p2) precursors and vacuolar mature form (mCPY) are indicated. B Tunicamycin hypersensitivity of SCS3 and YFT2 deletion strains. Two-fold serial dilutions of the indicated strains were spotted onto SC media containing 100 µM inositol with or without 1.0 µg/ml tunicamycin. Plates were photographed at 2 and 3 days respectively.CPY processing and tunicamycin sensitivity of (TIF)Click here for additional data file.Figure S8SCS3 YFT2 gene-deletion strains. Northern analysis of UPR induction after DTT treatment. Cells were grown to early log phase at 30°C in synthetic complete media, treated with 6 mM DTT for 1 hour and either left in DTT-containing media (panel A) or washed into fresh 30°C media lacking DTT (panel B). Cells were harvested over the time courses indicated. HAC1 mRNA (reporting both unspliced Hac1u and spliced Hac1i forms) was detected as described in Attenuation of the UPR in (TIF)Click here for additional data file.Table S1SCS3 and YFT2 Genetic Interactions.Stringent Set of (XLSX)Click here for additional data file.Table S2SCS3 and YFT2 Gene Interactions.GOSlim Component Analysis of (XLS)Click here for additional data file.


The dorsal cochlear nucleus (DCN) is a first relay of the central auditory system as well as a site for integration of multimodal information. Vesicular glutamate transporters VGLUT-1 and VGLUT-2 selectively package glutamate into synaptic vesicles and are found to have different patterns of organization in the DCN. Whereas auditory nerve fibers predominantly co-label with VGLUT-1, somatosensory inputs predominantly co-label with VGLUT-2. Here, we used retrograde and anterograde transport of fluorescent conjugated dextran amine (DA) to demonstrate that the lateral vestibular nucleus (LVN) exhibits ipsilateral projections to both fusiform and deep layers of the rat DCN. Stimulating the LVN induced glutamatergic synaptic currents in fusiform cells and granule cell interneurones. We combined the dextran amine neuronal tracing method with immunohistochemistry and showed that labeled projections from the LVN are co-labeled with VGLUT-2 by contrast to VGLUT-1. Wistar rats were exposed to a loud single tone  for 6 hours. Five days after acoustic overexposure, the level of expression of VGLUT-1 in the DCN was decreased whereas the level of expression of VGLUT-2 in the DCN was increased including terminals originating from the LVN. VGLUT-2 mediated projections from the LVN to the DCN are likely to play a role in the head position in response to sound. Amplification of VGLUT-2 expression after acoustic overexposure could be a compensatory mechanism from vestibular inputs in response to hearing loss and to a decrease of VGLUT-1 expression from auditory nerve fibers. The dorsal cochlear nucleus (DCN) in the auditory brainstem is a major termination point of the auditory nerve and auditory nuclei Synaptic projections can be specifically associated with vesicular glutamate transporters that package glutamate into synaptic vesicles The vestibular nuclear complex lies in the floor of the fourth ventricle and consists of four major nuclei, namely the medial, lateral, superior and inferior vestibular nuclei. The vestibular nuclear complex controls eye movements, reflex postural head and neck, movements and balance during stance and gait as well as modulation and modification of autonomic function to maintain homeostasis during changes in body posture Previous studies have shown that cochlear kanamycin injections which produce deafness, resulted in a significant reduction of VGLUT-1 in the regions of the ventral and dorsal cochlear nucleus receiving auditory nerve inputs as well as an increase of VGLUT-2 in regions receiving non auditory inputs Here we characterised VGLUT-2 mediated projections from the lateral vestibular nucleus (LVN) to the DCN by combining dextran amine neuronal tracing method with immunohistochemistry. We also showed that exposure to loud sound resulted in an increase in the number of VGLUT-2 positive terminals originating from the LVN and discuss its role within the context of cross modal compensation of hearing deficit.Experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the Home Office and Leicester University's Ethical Committee .2PO4, 26 NaHCO3, 0.5 ascorbic acid, 0.1 CaCl2 and 4 MgCl2 . The brainstem was dissected out of the brain, oriented in its coronal or its sagittal plane and glued onto a mounting plate that was placed in a slicing chamber of a VT1000S vibroslicer  containing the same ice cold solution as described above. Axonal tracing was performed on the isolated brainstem as described below.Forty six Wistar and Lister Hooded rats P18 to P27 day-old were culled by decapitation and their brains were immediately transferred to ice-cold low sodium artificial solution containing (in mM): 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 2 CaCl2, and 1 MgCl2 (pH 7.4 when gassed with 95% O2 ∶ 5% CO2). Dextran amine dye (see below for composition) was delivered under binocular control, using a custom made combined pressure and electroporation system coupled to a double-barrelled glass pipette  as described in Barker et al.2 and 2.5 CaCl2 at pH 7.3. Axonal tracing was performed on brainstems extracted from control animals and 5 days after initial exposure to loud sound using age matched rats from the same litters.One millimetre thick slices were cut under binocular control using a MZ75 stereomicroscope (Leica Microsystems Ltd). Slices were cut from the top of the brainstem until the DCN was reached. Injections were subsequently performed below the tissue surface to a depth of 500 µm ensuring that subsequent sections contain both the LVN and the DCN  2∶ 5% CO2 at 37°C for a period of 3 to 4 hours. After the incubation period, brainstems were either frozen down for immunohistochemistry (see below) or sliced before final observation. Slicing was performed by transferring the mounting plate with the glued brainstem to the vibroslicer slicing chamber and 120 µm thick slices were cut in the ice-cold low sodium artificial solution described above. Slices were performed up to a depth of 600 µm which contain both the DCN and the LVN. Slices were fixed in 4% paraformaldehyde for 10 minutes before being washed in 1× PBS for 10 minutes. Slices were finally mounted on slides with 1% agarose and viewed with an Olympus Fluoview FV300 confocal microscope  for final observation.Brainstems attached to their mounting plates were then transferred to an incubation chamber containing the artificial cerebrospinal solution detailed above and bubbled with 95% OAxonal tracing was performed on the isolated brainstem as described above. Serial sections were taken in slices containing the DCN and LVN . Transmitted light images were taken using a Nikon eclipse TE2000 microscope and fluorescently labelled cells were imaged using an Olympus Fluoview FV300 confocal microscope. Transmitted light images were then overlaid and the outlines of the sections including the DCN were traced over using Powerpoint. Labelled cell bodies within the LVN were also overlaid on the reconstruction and then represented by red dots with each dot representing a cell body.Immunohistochemistry was performed on tissue extracted from control animals (see below) and 5 days after initial exposure to loud sound using age matched rats from the same litters. Brainstems were rapidly frozen in Tissue Tek (Sakura) after their incubation with dextran amine dye, using hexane and dry ice. Frozen tissue was processed within a month and sectioned at 20 µm using a cryostat. Slices were mounted on Polysine coated slides and fixed in 4% paraformaldehyde for 10 minutes. Slides were washed for 3×5 min in 1× Dullbeco's PBS (Invitrogen) with 0.1% Triton X-100 (PBS-T solution). After washing, sections were incubated with 1% bovine serum albumin and 1% goat serum in 1× PBS with 0.1% Triton X-100 (blocking buffer) for 1 hour at 20°C. Sections were then incubated with anti-VGLUT antibodies  in blocking buffer overnight at 4°C. Slides were then washed with PBS-T for 6×10 min. The secondary antibody  was applied for 2 h at 20°C. Sections were washed in PBS-T solution for 6×10 min and then mounted with Prolong Gold antifade reagent (Molecular Probes).th, 50th and 75th percentile for each animal). The images were converted to 8 bit TIFF files in ImageJ (NIH software). Analysis was conducted as described in 2 using a 60× objective) were counted for alternate coronal and sagittal sections. The labelled terminals were classified into two different subtypes, mossy fiber like terminals which have large irregular endings (≥2 µm) and small boutons (<2 µm) Images were obtained using an Olympus Fluoview FV300 confocal microscope (Olympus Ltd). When imaging VGLUT immunoreactivity, confocal settings were determined using samples from animals aged P25 and the settings remained constant for all other imaging. To quantify VGLUT immunoreactivity sections were selected from an even distribution throughout the cochlear nucleus . Excitatory postsynaptic currents were elicited by stimulating the lateral vestibular nucleus with a concentric bipolar electrode  consisting of a platinum iridium inner pole of 50 µm and a stainless style outer diameter of 200 µm. Electrical pulses of 30–80 V (0.5–2 mA) and 0.1 ms duration were provided by a Digitimer DS2 A isolated stimulator  triggered by the pCLAMP software. Pharmacological blockers were bath applied using a peristaltic pump (Gilson Minipuls 3). Excitatory post synaptic currents were recorded in the presence of strychnine (10 µm) and gabazine (20 µm) and were blocked by NBQX disodium salt -quinoxalin) and D-AP5 . NBQX and D-AP5 were from Ascent Scientific , gabazine was from Tocris Bioscience . All the other drugs used in this study came from Sigma Aldrich . Once the whole cell recording was terminated, the pipette was gently removed from the cell by positive pressure and slices were fixed in 4% paraformaldehyde for 4 hours before being washed in 1× PBS for 15 minutes. Slices were finally mounted onto slides with 1% agarose with the cover-slip sealed with nail polish to prevent dehydration. Slices were kept overnight at 4°C before final observation with the Olympus Fluoview FV300 confocal microscope.Experiments were conducted in P18–25 day old rats as the use of juvenile rats allowed reliable and stable whole cell recordings. Patch clamp recordings were performed in control animals (see below) and 5 days after initial exposure to loud sound using age matched rats from the same litters. The dissection of the brainstem from 14 Wistar rats was performed as described above. Sagittal slices (160–180 µm thick) containing the LVN and the DCN were cut in low sodium artificial cerebrospinal solution before being transferred to the experimental chamber containing the artificial cerebrospinal solution (both solutions are described above). DCN cells were visualised with differential interference contrast (DIC) optics on an Axioskop microscope  with a 40× N.A. 0.75 water-immersion lens. Whole-cell patch-clamp recordings were made from fusiform and granule cells using thick-walled glass pipettes  with a Multiclamp 700A amplifier , connected to an analogue to digital converter  filtered at 6 kHz (8-pole Bessel filter) and sampled at 20 kHz. Currents were recorded with pCLAMP 9.2 software (Axon Instruments). Cells were maintained at a holding potential of −70 mV and whole-cell access resistances of less than 10 MΩ were not compensated. The intracellular solution contained 0.1% lucifer yellow and (in mM) 97.5 K gluconate; 32.5 KCl; 5 EGTA; 10 HEPES; 1 MgClWistar rats aged between 19 and 22 days were used (15 control and 15 acoustic over exposure) as this represents an age where the onset of hearing has already occurred Auditory brainstem response recordings were performed before AOE and 5 days after the first day of exposure to loud sound. Recordings were performed by inserting positive, negative and ground electrodes subcutaneously at the vertex, the mastoid and the back, respectively Means ± SEM were calculated using n samples which represented either the number of fields, terminals or grids as indicated in the text. N represents the number of animals for each condition. Statistical comparisons were performed by Mann-Whitney tests when comparing VGLUT-1 and VGLUT-2 intensities within regions of the cochlear nucleus and when comparing the effect of AOE on both VGLUT-1 and VGLUT-2 intensities in those regions. Although the test is non-parametric, it does assume that the two distributions are similar in shape and shape distributions were previously assessed as follows. Measurements of florescent intensity were taken along each layer of the DCN , in the MCD and in the shell region. Data were then compared using a one way ANOVA with Tukeys post hoc test. For all regions there was no significant difference in fluorescent intensity between all points. Unpaired T tests were used when comparing the size of labelled cell bodies in the LVN and when comparing terminal sizes under control conditions and after AOE; the percentage of VGLUT-2 labelled terminals originating from the LVN, the EPSC amplitude in control and exposed conditions. Significance was assessed for P<0.05. Data are represented as Mean ± SEM.The cochlear nucleus is divided into the ventral and the dorsal cochlear nucleus (DCN) When dextran amine was injected into the LVN , 2B synaTo assess the presence of functional synaptic projections between the LVN and the DCN, whole cell recordings were performed in sagittal slices containing both nuclei. Seven fusiform cells (N = 6) were characterised by their location in the fusiform layer, their morphology (based on Lucifer yellow filling) and their passive properties , the shell region and the DCN molecular layer . Labelled terminals originating from the LVN failed to co-localize with VGLUT-1  with onl2 area,  to 4.7±3.2 per 5000 µm2 area . The percentage of VGLUT-2 labelled terminals originating from the LVN increased from 1.6±0.1%  to 5.8±0.7%  P<0.001, unpaired t test). Examples of dextran amine labelled small boutons and mossy fiber terminals can be seen in Wistar rats were subjected to a loud 15 kHz tone for 2 times 3 hours . After 5 days, this procedure affected the auditory brainstem responses  and incrSo far, reported documentation of vestibular projections to the DCN remains scarce Our study shows that VGLUT-1 is strongly expressed in the MCD, the DCN molecular layer and the shell region. The MCD is primarily innervated by the axons of the type I spiral ganglion neurons The co-localization of VGLUT2 with LVN terminals in the DCN shown in this study confirms that the LVN pathway to the DCN is glutamatergic and demonstrates that projections from the LVN, like those from the spinal trigeminal nucleus and the cuneate nucleus Acoustic over exposure (AOE) can induce cochlear damage with resultant damage to the hair cells, spiral ganglion neurones and the myelin sheath of the auditory nerve An increase in the expression of VGLUT-2 could indicate a compensatory mechanism whereby specific synaptic inputs to the DCN are increased in response to the loss of synaptic activity from the auditory nerve. The inputs from the LVN would therefore consist of a compensatory mechanism originating from the vestibular system. Cross modal compensation has previously been demonstrated after deafferentation or sensory deprivation in other modalities It is of interest that vestibular conditions such as vertigo or vestibular schwannoma can result in tinnitus Figure S1Sagittal (top) and coronal (bottom) brainstem slice showing the site of injection of dextran amine in the dorsal cochlear nucleus DCN. Left, overlays of the brightfield and fluorescence photomicrographs at 3 hours post injection of dextran amine. Images on the right show the fluorescent micrographs. Scale bar 100 µm.(TIF)Click here for additional data file.Figure S2High magnification photomicrographs showing VGLUT-1 and VGLUT-2 positive puncta in the MCD (A), the shell region (B) and the layers of the DCN (C–E). VGLUT-1 positive puncta are most densely located in the MCD and molecular layer of the DCN (A and C) whereas VGLUT-2 puncta show a greater density than VGLUT-1 in the shell region, the fusiform and deep layers . Scale bar 2 µm.(TIF)Click here for additional data file.Figure S3Examples of mossy fiber terminals originating from the LVN labelled with VGLUT-2 in control condition (left) and after AOE (middle). Note the presence of multiple VGLUT-2 labelled mossy fiber terminals after AOE. Scale bar 5 µm.(TIF)Click here for additional data file.


Helicobacter pylori colonizes the human gastric mucosa, leading to a spectrum of gastric diseases in susceptible populations. Here we announce the draft genome sequences of strains HPARG8G and HPARG63. The data for both genome sequences provide insights regarding the diversity in gene content and rearrangement of the genomic islands commonly harbored by H. pylori. Helicobacter pylori strain HPARG8G was isolated from a patient with gastric ulcer disease and strain HPARG63 was recovered from a patient with chronic gastritis. Here we report the draft genome sequences of both strains. A total of 785,600 reads with an average length of 435.02 nucleotides, corresponding to 33-fold coverage for strain HPARG8G and 100-fold coverage for HPARG63, were obtained with a 454 GS titanium pyrosequencer. De novo assembly was done using 454 Newbler version 2.6, yielding 47 contigs for HPARG8G and 34 contigs for HPARG63. The genomes were annotated using the RAST server  as well as 36 tRNAs and 2 rRNAs. The average G+C content was 38.98%. The HPARG63 strain genome sequence contains approximately 1,668,716 bp and 1,623 coding sequences and also has 36 tRNAs and 2 rRNAs, with an average G+C content of 38.79%. The H. pylori P12 strain was predicted by RAST as one of the closest overall neighbors of HPARG8G and HPARG63. Both genomes displayed a conserved repertoire of housekeeping genes corresponding to various metabolic pathways. The two genomes each showed a complete cag pathogenicity island (cag PAI), with the complex rearrangement in the region delimited by dapB and murI genes described in the Mongolian gerbil-colonizing strain H. pylori B8  under accession numbers CBKZ010000001 through CBKZ010000047 and CBKY010000001 through CBKY010000034.The first versions of the HPARG8G and HPARG63 sequences are accessible at the European Nucleotide Archive (


Hox genes to direct homeotic transformations or thebreadth of their patterning potential. To begin delineating Hoxgene function in neural development we used a mouse ES cell based system thatcombines efficient neural differentiation with inducible Hoxb1 expression. Geneexpression profiling suggested that Hoxb1 acted as bothactivator and repressor in the short term but predominantly as a repressor inthe long run. Activated and repressed genes segregated in distinct processessuggesting that, in the context examined, Hoxb1 blockeddifferentiation while activating genes related to early developmental processes,wnt and cell surface receptor linked signal transduction and cell-to-cellcommunication. To further elucidate aspects of Hoxb1 functionwe used loss and gain of function approaches in the mouse and chick embryos. Weshow that Hoxb1 acts as an activator to establish the full expression domain ofCRABPI and II in rhombomere 4 and as arepressor to restrict expression of Lhx5 andLhx9. Thus the Hoxb1 patterning activityincludes the regulation of the cellular response to retinoic acid and the delayof the expression of genes that commit cells to neural differentiation. Theresults of this study show that ES neural differentiation and inducibleHox gene expression can be used as a sensitive model systemto systematically identify Hox novel target genes, delineatetheir interactions with signaling pathways in dictating cell fate and define theextent of functional overlap among different Hox genes.The evolutionarily conserved Hox family of homeodomain transcription factorsplays fundamental roles in regulating cell specification along the anteriorposterior axis during development of all bilaterian animals by controlling cellfate choices in a highly localized, extracellular signal and cell contextdependent manner. Some studies have established downstream target genes inspecific systems but their identification is insufficient to explain either theability of  Hox target genes have beenidentified. Some studies have established direct and downstream target genes inspecific systems but their identification is insufficient to explain either theability of Hox genes to direct homeotic transformations or thediversity of their patterning potential.The evolutionarily conserved Hox family of homeodomain transcription factors playsfundamental roles in conferring regional identity and regulating cell specificationalong the anterior – posterior (AP) axis during development of all bilateriananimals Hox gene expression has been genetically manipulatedHox genes and accumulation of secondary effects in gain or lossof function genetic models present serious limitations. The elucidation of theprecise roles that Hox genes play in cell fate specification aswell as the identification of target genes and processes are key goals todeciphering the regulatory network underlying morphogenesis of the body plan.Furthermore, this may allow harnessing their patterning potential in the directeddifferentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS)cells to specific cell types.Two main general approaches have been used, a candidate target gene approach Hox gene expression and specific dorsoventral (DV) patterningcues define specific subclasses of neuronal progenitors in the developing hindbrainand spinal cord Hoxgenes act as integrators of AP and DV patterning mechanisms to generate specificclasses of neuronal progenitors and neurons for the appropriate AP levels of thehindbrain and the spinal cord. For example, Hoxb1 is specificallyexpressed in rhombomere 4 of the developing hindbrain. The specification of thisterritory and subsequent generation of r4 specific neuronal progenitors and neuronsdepend largely on Hoxb1 function. Disruption of the Hoxb1 gene inmice leads to transformation of the r4 territory into an r2-like state Hoxb1 in r2 causes homeotictransformation of r2 to a r4-like identity in chick Hoxb1 expression is responsible for the generation of facialbranchiomotor neurons and the suppression of serotonergic fate specification Hox genesdirect the generation of distinct motor neuron (MN) subtypes at hindbrain, brachial,thoracic and lumbar regions During development of vertebrate neural tube the combinatorial use ofHox gene function in neuraldevelopment we modeled the role of Hox genes in neural cell fate specification usinga mouse ES cell based system that affords the possibility of inducible Hoxb1expression. Using a differentiation protocol that generates a highly homogeneouspopulation of neural stem (NS) cells and inducible expression ofHoxb1 we showed that timely long term induction (8 days) of theHoxb1 transgene in ES cell derived NS cells resulted in thespecification of NS cells toward a hindbrain specific identity through theactivation of a rhombomere 4-specific genetic program and the repression of anteriorneural identity Hoxb1 mediates neural crest cell fate induction. This wassubsequently verified in vivoHoxb1 target genes,Hoxb2, Hoxa2, EphA2 andPhox2bHoxb1 targetgenes.To bypass limitations in delineating in vivo function we used loss and gainof function approaches using the chick and mouse developing embryos as model systemsand investigated the in vivo response of two up  and two down  regulated genes in ESderived NS cells. Hoxb1 is itself regulated by retinoic acid CRABPI and II. On the otherhand, Lhx5 and 9 mediate neuronal differentiationin vivo repression would correlate well with the finding thatHoxb1 blocks ES derived NS cell differentiation after mitogenwithdrawal Hoxb1 downstreamtarget genes in other approaches Hox target genes and processes, define binding sites andelucidate the interactions of Hox genes and extracellular signalsin dictating neural cell fate.Here we use this approach and microarray gene expression profiling to identifypotential novel Hoxb1 target genes and processes. To compare the long and short termeffects of Hoxb1 function and limit the number of potential target genes we used ashort term and a long term induction protocol. To validate the approach andelucidate aspects of Hoxb1 Animal studies were conducted in accordance with international guidelines andafter ethical approval of the competent Veterinary Service of Athens. The Hoxb1mouse mutants were described and genotyped as reported Tet-On/Hoxb1cells were as described previously +) and uninduced (Hoxb1−) cells asdescribed earlier The generation and neural differentiation of the mouse ESTotal RNA was isolated from ES derived NS cells using the RNeasy kit (Qiagen)according to the manufacturer's instructions and digested by RQ1 DNase(Promega) to remove genomic DNA. First strand cDNA synthesis was performed withSuperscript II reverse transcriptase (Invitrogen) using random primers. Realtime PCR analysis was carried out in a Chromo4 DNA engine (Biorad), running thefollowing program: 95°C for 10 min, then 40 cycles of 95°C for 15 s,60°C for 40 s, followed by plate read. PCR reactions included 1x SYBRgreener PCR master mix (Invitrogen), 200 nM primer and 2 ul of template in a 25ul reaction volume. Primers were as follows (5′ to 3′):CRABPI F:GGAGATCAACTTCAAGGTCGGAG,CRABPI R: ATACTCCTCAGGGGAACTCGCATC,CRABPII F: ACATCAAAACCTCCACCACTGTGCGAAC,CRABPII R: CGTCATCTGCTGTCATTGTCAGGATCAGC,Lhx5 F: GACAAGGAAACCGCTAACAACG,Lhx5 R:GTGGACCCCAACATCTCAGACTCG,Lhx9 F: TACTTCAATGGCACTGGCACCG,Lhx9 R: TCCTTGGCATCTGGGTTATGG.in situ hybridization embryos were fixed overnight at4°C in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer saline(PBS). In situ hybridization was performed in whole embryos using probes formouse CRABPI and CRABPIILhx9Lhx9Lhx5in situhybridization, embryos were fixed again overnight at 4°C and then processedfor immunofluorescence. For immunofluorescence embryos were fixed in 4%PFA in PBS for 1–2 h at 4°. Embryos were cryoprotected with 30%sucrose in PBS and cryosectioned. Blocking was carried out in 10% normalgoat serum (NGS) with 0.1% triton for 1 h at RT. The cryosections wereincubated overnight at 4°C with the primary antibody diluted in 1%NGS, 0.1% triton in PBS. Primary antibodies used were as follows: rabbitanti-Hoxb1, 1∶400 (Covance), mouse Lhx5, 1∶100. Secondary antibodieswere anti-mouse and anti-rabbit Alexa 488 or Alexa 568 (Molecular Probes) usedat 1∶500. Images were acquired using a Leica TCS SP5 confocalmicroscope.For Hoxb1 cDNA was inserted into thepCAGGS-IRES-NLS-GFP expression vector in situhybridization or immunofluorescence.Chick embryos were staged according to Hamburger and Hamilton (HH)  and electroporated at HH stage 10–11. Chick embryos wereelectroporated with plasmid DNA at a concentration of 1.5 µg/µl. Thecoding regions of mouse Hoxb1 target genes and processes we usedthe stable line ESTet-On/Hoxb1 that allows for tight dox mediatedinducible expression of the Hoxb1 transgene at both the ES celland NS cell stages. However, inducible expression of the transgene couldmobilize the endogenous Hoxb1 autoregulatory loop only at theNS cell stage demonstrating the importance of cellular context forHoxb1 function and its analysis. Hoxb1induction using an 8-day long dox exposure resulted in the generation of r4specific neuronal progenitors Hoxb1 downstream effectors andcompare the short term and long term effects of Hoxb1expression we performed microarray gene expression analysis after a two day longexposure to dox  (HHstage 20) by whole mount in situ hybridization with the chickLhx5 in situ hybridization probe cLxh5 at HH is expressed in twodorsomedial stripes in r2 and r3  analysis showed that up regulatedand down regulated genes related to strictly distinct processes. The Hoxb1repressing activity was directed primarily towards differentiation related processeswhereas its activating functions were directed primarily towards early development,wnt and cell surface receptor linked signal transduction and cell-to-cellcommunication  upon cell cycle exit. To in vivo validate some of ourfindings we corroborated the effects of Hoxb1 on the expression patterns ofCRABPI, CRABPII, Lhx5 andLhx9 using in vivo loss and gain of functionmodels. CRABPI, CRABPII and Lhx5had a Hoxb1 dependent r4 specific expression pattern. It is worth noting that noneof them was identified as such in the aforementioned screens underlining thesensitivity of the approach presented here.Three other screens have been conducted to identify CRABP expression was initially associated with structures thatwere more sensitive to excess of RA CRABPI and II was activated byHoxb1 in ES derived NS and these findings were validated in themouse embryo since expression of both was down regulated in the r4 ofHoxb1−/− reverting to expression patterns identical tothose of r2. Intriguingly, CRABPI is up regulated whereasCRABPII is down regulated in the resident territory of r4 motorneurons suggesting that maturation and/or specification of this subpopulation needsparticular shielding from RA exposure. Ectopic Hoxb1 expression inr2 through timely supply of extraneous RA converts the r2 trigeminal motor neuronsinto r4 facial motor neurons CRABPIand II expression. However, further studies are needed to provethis hypothesis and establish whether CRABPI/II are direct Hoxb1target genes. The localized expression of RARa in r4 and thelocalized expression of Cyp1B1, an atypical RA generatingcytochrome, in the ventral r4 RARa as a Hoxb1 downstream target in r4. The ES derivedNS cells are a mixture of different DV characters and this limits the detectioncapacity for markers that are exclusively expressed in distinct and narrow DVlevels. This can be bypassed by dorsalising or ventralising these cells withappropriate DV morphogenetic signals Cyp1b1 is induced in shh treated ES derivedHoxb1+ NS cells as well.Within the developing neural tube the diverse cellular distribution patterns ofretinoid receptors and retinoid binding proteins indicates that it is necessary tofine-tune levels of RA signaling for the specification of diverse of neuralsubpopulations. CRABPI and II are located in the cytoplasm and bind RA, a key playerin CNS pattern formation, neural specification and differentiation.Lhx genes) was regulated byHoxb1 in ES derived NS cells. Click here for additional data file.Table S2List of genes regulated in both short (s) and long (l) induction schemes.Classification is according to primary GO process assignment. If not anassignment has been made genes are labelled as non-classified.(XLS)Click here for additional data file.Table S3List of of common genes induced in Hoxb1-/- r4 and also regulated by Hoxb1 inES derived neural progenitors after long induction. At the top part of thelist the observed regulation is in the same direction and after the space itis in the opposite direction.(XLS)Click here for additional data file.Table S4List of common genes repressed in Hoxb1-/- r4 and also regulated in ESderived neural progenitors after long induction. At the top part of the listthe observed regulation is at the same direction and after the space it isin the opposite direction.(XLS)Click here for additional data file.


Sox1, Sox2 and Sox3 are key regulators of NP differentiation and that their roles in CNS development are largely redundant. Nevertheless, mutation of each SoxB1 individually results in a different array of CNS defects, raising the possibility that SoxB1 proteins have subtly different functions in NP cells. To explore the mechanism of SOXB1 functional redundancy, and to identify genes that are most sensitive to loss of the Sox3 gene, we performed genome wide expression profiling of Sox3 null NP cells. Nineteen genes with abnormal expression were identified, including the homeobox gene Dbx1. Analysis of Sox3 null embryos revealed that Dbx1 was significantly reduced in the neural tube and developing brain and that SOX3 bound directly to conserved elements associated with this gene in cultured NP cells and in vivo. These data define Dbx1 as a direct SOX3 target gene whose expression, intriguingly, is not fully rescued by other SOXB1 transcription factors, suggesting that there are inherent differences in SOXB1 protein activity.SoxB1 sub-family of transcriptional regulators are expressed in progenitor (NP) cells throughout the neuroaxis and are generally downregulated during neuronal differentiation. Gain- and loss-of-function studies indicate that  Sry-related HMG box) family of transcription factors, of which 20 members have been identified in mammals. SOX genes generally have developmentally-regulated expression and play important roles in cell specification, self-renewal and differentiation in a broad range of embryonic contexts Sox3, and the closely related genes Sox1 and Sox2 (which together make up the SoxB1 subgroup), are expressed in neuroprogenitor cells throughout the neuroaxis and are down regulated upon differentiation Sox3 null mice exhibit specific CNS defects within the hippocampus, corpus callosum and hypothalamus despite the widespread Sox3 expression in NP cells throughout the developing brain SOX3 have a relatively mild phenotype that includes infundibular hypoplasia, hypothamalic-pituitary axis dysfunction and incompletely penetrance of intellectual disability Sox2 or Sox1 in mice also results in regionally-restricted defects as opposed to a general NP phenotype SOX3 is a member of the SOX  subpopulation of NP cells. This phenomenon can be explained by at least three possibilities. Firstly, given that subtle differences in Sox1, Sox2 and Sox3 gene expression have been identified in the developing CNS Sox3 null embryos despite expression of Sox2 in this structure While these studies provide strong support for SOXB1 functional redundancy, the existence of congenital CNS defects in Sox3 gene, we performed genome wide expression profiling of Sox3 null NP cells. Nineteen genes with abnormal/delayed expression were identified that included the homeobox gene Dbx1. In vivo analysis of Sox3 null embryos revealed that Dbx1 was significantly reduced in the neural tube and developing brain and that SOX3 bound directly to conserved elements associated with this gene in cultured NP cells and in vivo. These data define Dbx1 as a direct SOX3 target gene whose expression, intriguingly, is not fully rescued by other SOXB1 transcription factors, suggesting that there are inherent differences in SOXB1 protein activity.To explore the mechanism of SOXB1 functional redundancy, and to identify genes that are most sensitive to loss of the Sox3 null embryonic stem cells were previously targeted as described in . Sox3 null mice have been published previously R1 ES cells were maintained on irradiated MEFs in standard conditions and neurodifferentiated as monolayers using N2B27 as previously described Sox3 null Day 4 samples using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays (full dataset available at Gene Expression Omnibus: GSE53760). 2 way-ANOVA, using batch as a factor, was used to identify the significantly regulated genes. qRT-PCR validation was performed previously described RNA from differentiated cells was extracted using a RNeasy mini kit (Qiagen) and cDNA generated using a High Capacity RNA-to-cDNA kit (ABI). Expression profiling was performed on four wild type and four IHC was performed as described previously sample (non-enriched region) - Ctsample (peak region)] was >2 following normalisation to CtIgG and CtInput of the corresponding qPCR run.In vitro ChIP was performed as per Animal experiments were approved by the University of Adelaide Animal Ethics Committee (project number: S-2012-242). All studies were conducted in accordance with the principles of animal replacement and reduction and experimental refinement.Sox3 expression during N2B27 neuroinduction, we performed qRT-PCR . Sox3 expression was upregulated by Day 2, and increased further at Day 4 and Day 6. Consistent with these data, weak expression of SOX3 protein was evident in Day 2 cultures. By Day 4, robust expression of SOX3 was detected in most cells. At Day 6, robust expression of SOX3 could still be detected in most cells, although some cells (particularly those at the periphery of colonies) had begun to downregulate SOX3.To begin to identify SOX3 target genes in NP cells, we utilised the N2B27 culture system, which has previously been shown to generate NP cells from ESC with high efficiency  qRT-PCR  and immu qRT-PCR . At the Sox3 deletion on NP differentiation, we performed N2B27 neuroinduction using our previously-generated Sox3 null ES cells that contain a GFP reporter knock-in allele Sox3 has a major impact on induction of neural progenitors, expression of neural progenitor markers (including other SoxB1 genes) was analysed by qRT-PCR. No significant difference in Sox1, Sox2 or Pax6 expression was detected in samples utilized for microarray analysis, at Day 4  and Fezf2 (p = 0.0228) showed significant down regulation in mutant cells (Efnb3 (p = 0.0229), Cspg5 (p = 0.0251), Fam174b (p = 0.0065), Ddr2 (p = 0.0386) and Slit1 (p = 0.0456) was also confirmed in the independent samples. These data identifies a number of potential SOX3 targets in cultured NP cells.To identify putative SOX3 target genes, we compared the global gene expression profile of Day 4 NP cell cultures derived from nt cells . SignifiSox3 null NPs potentially represent genes that are direct SOX3 targets. To investigate this possibility, we examined the differentially expressed genes for SOX3 binding sites using a previously published SOX3 ChIP-seq data set generated using a comparable NP differentiation system Sox3 null NP cells. Over 83% of the differentially expressed genes  as embryos at this stage of development have an abundance of NP cells and a paucity of differentiating neurons. In addition, like Day 4 NP cell cultures, Sox1 and Sox2 expression in not significantly altered in Sox3 null embryos at this stage  using chromatin extracted from 10.5 dpc embryonic heads. Significant enrichment (p≤0.001) of this SOX3 binding this site was observed in comparison to the IgG negative control  can function redundantly with SOX3 to “rescue” the SOX3 deficiency. However, the lack of a complete phenotypic rescue suggests that, at least in some NP cells, the level of SOXB1 protein may be important for neurodevelopment. Alternatively, each of the SOXB1 factors could bind preferentially to a unique subset of SOXB1 targets. As a result, target gene expression resulting from the loss of a single SOXB1 gene would only be partially rescued by the other two SOXB1 factors. To explore this issue in more detail, we attempted to identify genes that were sensitive to the loss of SOX3 in NP cells. To do this we used a previously targeted Sox3 null ES cell line in which the single SOX3 exon was replaced with GFP Sox3 null NP cells resulted in rapid upregulation of Sox3/GFP at the mRNA and protein level. The NP markers Sox1 and Pax6 were also rapidly induced and showed comparable kinetics in the control and mutant cultures. In addition, expression of Sox2 was comparable between the two populations. Notably, no overt defects in the morphology or global gene expression (see below) were identified in Sox3 null NP cells. This is likely due to the expression of Sox1 and Sox2 in N2B27-induced NP cells and is consistent with the mild CNS phenotype of Sox3 null embryos.Despite the widespread expression of Sox3 were expressed in the majority of NP cells. Of over 24,000 genes assayed by microarray, only 19 had significantly different expression levels >1.4-fold at Day 4. Expression differences (when tested by qRT-PCR) were validated on independent samples for a majority of the targets, thereby confirming the authenticity of the microarray data. However, not all differentially expressed genes validated on independent samples . The relatively small number of differentially expressed genes is interesting to consider in the context of a recently-published ChIP-seq analysis of a similar NP cell type, which showed that SOX3 binds at thousands of genomic sites, many of which are likely to have regulatory functions To maximise the likelihood of identifying genes that are directly regulated by SOX3, we performed gene expression profiling at Day 4, the earliest time point at which high levels of Cspg5 has been associated with intellectual disability disorders Slit1 expression has been published in mice with abnormal corpus callosum development SOX3 mutations Sox3 null mice have abnormal corpus callosum development Interestingly, many of the genes identified by the microarray analysis have established roles in neurodevelopment. For example, Fezf2. Fezf2 has been shown to be important for the early development of the posterior hypothalamus/thalamus in zebrafish and mouse Fezf2 expression has been previously published to be downstream of the SOXC group genes Sox4 and Sox11, double deletion of which leads to a loss of Fezf2 expression. Regulation of Fezf2 by SOX4/11 gene is mediated by a cis acting enhancer binding site which, interestingly, was also identified as a SOX3 binding site in NP cells by ChIP-seq and independently within our lab  had significantly different expression that matched the genome-wide expression profiling analysis. The lack of in vivo validation for other targets may reflect a phenotypic or stage-specific difference in the NP cells induced by N2B27 differentiation compared with their 9.5 dpc embryonic counterparts. Consistent with this idea, expression analysis of Sox3 targets in Day 6 NP cells revealed that many genes no longer had significantly different expression levels or had greatly reduced expression fold changes  could be used to generate NP cells that have an equivalent SOXB1 dosage but which lack SOX3. In addition to the spinal cord, we identified a reduction in Dbx1 expression in the head of 10.5 dpc Sox3 null embryos. However, we were unable to determine whether this reflected a lack of Dbx1 expression in a specific region or general reduction in the Dbx1 expression level (data not shown).Dbx1 is a direct target of Sox3 Dbx1 expression in Sox3 null NPs. The data also suggest that the regulation of Dbx1 by SOX3 is complex and subtle differences between early or later expression or spinal cord/brain expression might be regulated any number of these sites. Further studies are needed to address this issue. Given the increasing ease with which the genome can be manipulated Dbx1 locus and assess their impact on Dbx1 expression in vitro and in vivo.A recent report by Oosterveen et al also indicated that Figure S1Expression of putative Sox3 targets at day 6 of neural progenitor differentiation. A: Putative targets Dbx1 and Slit1 relative gene expression levels by qRT-PCR at day 6 of N2B27 differentiation, . B: Putative targets Cp, Fezf2 and Efnb3 showing reduced and/or insignificant fold change at day 6 of N2B27 differentiation . RQ: relative quantification normalised to β-actin.(TIF)Click here for additional data file.


We report that the high level of 5hmC present in neurons is enriched in active transcription units, and that surprisingly strong depletion of 5mC over gene bodies is observed for these genes. However, the relative contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndrome causing mutation of residue R133C in the MeCP2 methyl-CpG binding domain (MBD) preferentially inhibit 5hmC binding. Loss of MeCP2 does not alter the genomic distribution of 5hmC. These findings demonstrate that 5hmC and MeCP2 constitute a novel, cell specific epigenetic mechanism for regulation of chromatin structure and gene expression in the mammalian nervous system, and they provide new mechanistic insights into the pathophysiology of Rett Syndrome (RTT).The very high levels of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes and its accumulation within gene bodies suggests that the epigenetic mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem (ES) cells. Here we present the first quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC) and gene expression in identified, terminally differentiated CNS cell types


Avian influenza H5N1 virus is highly pathogenic partially because its H5 hemagglutinin contains a polybasic cleavage site that can be processed by proteases in multiple organs.Monoclonal antibodies (mAb) specific to the synthetic peptide of hemagglutinin polybasic cleavage site of H5N1 virus were raised and tested for their neutralizing potential.Purified mAb showed suppression of H5N1 pseudovirus infection on Madin-Darby Canine Kidney (MDCK) cells but the efficacy was less than 50%. Since those mAb are specific to the intact uncut polybasic cleavage site of hemagglutinin, their efficacy depends on the extent of hemagglutinin cleavage on the viral surface.Proteolytic analysis suggests the low efficacy associated with those mAb may be due to proteolytic cleavage already present on the majority of hemagglutinin prior to the infection of virus. Avian H5N1 highly pathogenic influenza virus was first isolated from sick geese in China during 1996 and later transmitted to human in Hon Kong during 1997 . The rapThere are many antiinfluenza measures available. For example, vaccination is a good defense against highly pathogenic influenza like the avian H5N1 virus ,16, but An antibody targeting the conserved epitopes on viral surface may be able to circumvent the antigenic drift and thus avoid the hit-and-miss situation associated with influenza vaccines. For example, the ectodomain of M2 channel protein is highly conserved among most strains of influenza A viruses and has been targeted as a broad spectrum epitope, but the antibody will only work on influenza A viruses and as the mutations accumulate at the ectodomain of M2 protein, they count against the efficacy of such antibody . The HA2The polybasic cleavage site on hemagglutinin is highly conserved among those highly pathogenic H5N1 viruses and its polybasic residue constituent should make this peptide fairly antigenic but discernable from other hydrophobic peptides and, therefore, this polybasic peptide is an interesting candidate as a broad spectrum epitope. Because the proteolytic cleavage of HA is a necessary step for an influenza virus to become infectious, we hypothesize that monoclonal antibody (mAb) specific to the polybasic cleavage site on hemagglutinin may be able to suppress virus infection by preventing HA cleavage by host proteases.The polybasic cleavage site on hemagglutinin is highly conserved among H5N1 avian influenza viruses ,30,31, bThose 7 hybridomas were cultured to raise ascitic fluid in nude mice and the mAb was purified by protein A resin . Briefly, monoclonal antibodies in the ascitic fluid was precipitated by 50% saturation of ammonium sulfate and redissolved in a minimal volume of phosphate buffered saline (PBS) before an overnight dialysis. Dialysed ascitic fluid was loaded onto a protein A column that had been equilibrated with 20 mM sodium phosphate pH7 buffer. The monoclonal antibody was eluted with 100 mM acetic acid pH2.8 and neutralized by 1 M Tris/HCl pH9. The immunoglobulin in elution was concentrated with Centricon  and the contents were determined by Bradford protein assay .One mg of pepsin (Sigma P6887) was predissolved in 200 μL of 0.1 M glycine acetate (pH 2.6). Twenty μL of mAb was added with an equal volume of pepsin and digested at 37°C for 40 min before 1 μL of 1 M Tris (pH 9) was added to alleviate the protein precipitation due to acidity. The mixture was further incubated for 30 min before 4 μL of 1 M Tris (pH9) was added to stop the reaction. An aliquot of 225 μL D-MEM was added to the antibody/pepsin mixture and 50 μL of the mixture was used for pseudovirus neutralization assay.nd antibody was used to probe the membrane at 1:5000 dilution for 1 hr. The immunoreactive spots were developed with a chemiluminescent substrate  and scanned with a typhoon scanner (Amersham).Three synthetic peptides were used to evaluate the epitope binding property of those 7 purified monoclonal antibodies. The full length 18 meric peptide, RERRRKKRGLFGAIAGFI, the N-half 8 meric peptide, RERRRKKR, and the C-half 10 meric peptide, GLFGAIAGFI, were synthesized at the Genomic Research Center of Academia Sinica. All 3 peptides were dissolved in DMSO at 2 mg/mL concentration. An aliquot of 5 μL (10 μg) was spotted on a 1 cm square of PVDF membrane . Four ten-fold serial dilutions were also spotted on the membrane similarly. The PVDF membranes were allowed for air dry and subject to immunoblotting after the membrane being rewetted by methanol/PBS. Blocking of the PVDF membrane was carried out with 2% non-fat milk dissolved in PBS. The purified monoclonal antibodies were diluted 500 fold in 2% non-fat milk and allowed for 1 hr incubation with the membrane. Horseradish peroxidase (HRP) conjugated anti-mouse 2GERRRKKRGLFGAIAGFIEGG, mutation underlined; 3) Anhui 2005 H5N1 pseudovirus, RERRR_KRGLFGAIAGFIEGG, which has a deletion of K344 or K345 at the polybasic site. The peptide sequence of antigens and viral cleavage sites were listed in Table To avoid using those highly pathogenic H5N1 viruses, pseudoviruses comprised of HA5 and NA1 antigens, Pol and Gag of HIV and a firefly luciferase reporter gene  were kinPseudovirus infection assay was performed on Madin-Darby Canine Kidney (MDCK) cells which were cultured in D-MEM supplemented with 10% FBS. Overnight cultured MDCK cells were incubated with pseudovirus in the presence or absence of mAb for 1 or 4 hours at 37°C before the pseudovirus mixture was removed by aspiration. Pseudovirus treated MDCK cells were cultured 48 hours further before the infectivity was determined. The infectivity of pseudovirus was determined by assaying the transfected and expressed luciferase activity in those MDCK cells. The MDCK cells were rinsed with PBS before being solublized with 60 μL Glo Lysis Buffer of Promega  for 2 hours. Fifty μL of cell lysate was mixed with an equal volume of Bright-Glo Luciferase Substrate in an opaque well/plate. The chemiluminescence was detected with a TopCount NXT of Perkin Elmer .The immunostain blot was carried out with a 4-20% gradient SDS-polyacrylamide gel and transferred to a PVDF membrane in a semidry blot transfer apparatus (BioRad). Both the primary and the secondary antibodies were diluted 1:5000 fold in PBS containing 2% non-fat milk. The stains were developed with chemiluminescent substrates of Invitrogen  and the band intensities were measured by a Typhoon scanner and analyzed by ImageQuant TL of GE Healthcare .All 7 mAb were purified to near homogeneousity by protein A resin Figure , but the2 can result in a better suppression during the pseudovirus infection assay. After pepsin treatment (Figure 2 remained low and none of the clones provided more than 50% suppression on pseudovirus infection (Figure Since H5N1 avian influenza virus is highly pathogenic and is not available for general laboratory use, we opted for a pseudovirus that expresses H5 and N1 surface proteins and contains a firefly luciferase reporter gene so that its infectivity can be assessed by luciferase activity. A preliminary neutralization scan was summarized in Figure t Figure , the effAll 7 monoclonal antibodies were subjected to epitope peptide analysis by dot blot. The result showed that clone A, C, 3C4 and 6B8 recognize the full length 18 meric peptide only and are reactive to as little as 0.1 μg of full length peptide (Figure To verify if these purified mAb can bind to hemagglutinin, mAb C, 3C4, and 6B8 were subjected to immunoblotting against recombinant hemagglutinin or virus lysate, as shown in Figure Because our mAb cannot recognize those cleaved HA1 or HA2 Figures  and 6, tThe authors declare that they have no competing interests.HJT is responsible for the overall research; LAC carried out the epitope peptide analysis and 293 cell expressed HA immunostain study; ALY supervised the overall progress. All authors have read and approved the final manuscript.


Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed  in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from  Systems biology involves the study of molecules in context as part of a larger system or network rather than in isolation and the development of mathematical models of the particular system being studied. Both the wet (laboratory-based) and dry (computer-based) experiments are used to inform each other and together generate a greater understanding of the biological system ,2. Over Arabidopsis thaliana seedlings. Depending on the available equipment, the method can be performed manually using multichannel pipettes or automated with a liquid handling robot. The developed protocols enable the study of gene expression by qPCR  in large numbers of samples .Historically, RNA extraction has been performed using organic solvents and phenol-chloroform . Similarlater solution .• RNA• 1.5 ml eppendorf tubes.http://www.mbpinc.com/, Cat.# 3453). NOTE: If using alternative tubes, ensure that they are sufficiently strong to withstand bead beating in a mixer mill.• 2 ml eppendorf tubes .• 5 mm stainless steel cone balls  which in addition to the binding, wash and elution plates, contains buffers RA1, RA2, RA3 & RA4 and DNaseI. The kit requires the addition of 1% β-mercaptoethanol to buffer RA1 and ethanol to buffers RA3 and RA4 prior to use.• illustra RNAspin 96 RNA isolation kit .http://www.starlab.co.uk/, Cat.# E2896-0110).• 1.1 ml 96 deepwell plates . To process 1× 96 well plate requires 8× 200 μl tips  and 144× 1000 μl tips .• Disposable tips for robot .http://www.binder-world.com).• Oven for preparing beads .• Bead-mill .• Plate centrifuge at room temperature capable of speed of 3,220× g (e.g. Eppendorf 5810R centrifuge fitted with an A-4-81 swing-bucket rotor).http://www.nanodrop.com).• NanoDrop spectrophotometer Agilent 2100 Bioanalyser  with 8-channel liquid handling arm fitted with disposable tips (200 μl and 1 ml as detailed above) and a TeVacS vacuum unit. Although we used the vacuum unit with a custom built spacer to ensure the wash plate  was positioned at the correct height directly below the binding plate, a standard size spacer (number 6) can be used. Details of the robot deck layout are depicted in Figure Although our method was developed and performed on a high specification liquid handling robot, it can be easily transferred to other platforms.• Freedom Evo-2 150 liquid handling robot  seedlings carrying the ELF3:LUC transgene (EARLY FLOWERING 3) [CIRCADIAN CLOCK ASSOCIATED 1) seedlings [CAB2:LUC transgene (CHLOROPHYLL A/B-BINDING PROTEIN 2) [-2·s-1) at 22°C for 9 days.Wildtype ERING 3)  or CCA1-eedlings  carryingOTEIN 2)  were groThe concentration of purified RNA was measured using a NanoDrop spectrophotometer (ND1000). Typically 2 μl of each sample was used.http://www.agilent.com/) according to the manufacturer's instructions. The ladder used was RNA 6000 ladder  and 150-200 ng of each sample was loaded. All samples and the ladder were denatured at 70°C for 2 mins prior to analysis. The results were analysed using Agilent 2100 Expert software.The integrity and quality of purified RNA were analysed using an Agilent 2100 Bioanalyzer and a RNA 6000 Nano Assay kit  according to the manufacturer's instructions. cDNA was diluted 1/10 and 1 μl used for subsequent qPCR. qPCR analysis for isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) in a final volume of 10 μl was then performed using LightCycler 480 SYBR Green I Master mix on a LightCycler480 . The following primers each at 300 nM were used: IPP2 forward primer GTATGAGTTGCTTCTCCAGCAAAG and IPP2 reverse primer GAGGATGGCTGCAACAAGTGT [Purified total RNA (1 μg) was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis kit with oligo dT primers . Only half of the lysed sample was processed further to complete the RNA extraction procedure while the other half was stored in the freezer (-20°C) after lysis as backup in the case of some experimental catastrophe.later.1) Using clean tweezers, harvest 45-50 mg Arabidopsis seedlings per sample directly into 1.5 ml eppendorf tubes containing 500 μl RNAlater solution.2) Store samples overnight at 4°C to allow full penetration of the RNA: At this point, samples can safely be stored at -20°C for up to 10 months with no detrimental effect - see comments section below.3) Store samples at -20°C until sample lysis and completion of the RNA extraction procedure. NOTEIf the balls are not rinsed properly the acid not only causes rust and corrosion damage to the balls but would also degrade the RNA sample.4) Before use, 5 mm stainless steel cone balls (one per sample) are prepared by washing in 0.4 M HCl for 1 h, thoroughly rinsed using RNase-free water and then baked at 240°C for at least 4 h. NOTE: 5) Add prepared cone balls to 350 μl RA1 (containing 1% β-mercaptoethanol) in a 2 ml eppendorf tube (one per sample per tube).later solution and dab dry on some tissue paper.6) Carefully remove seedlings from RNA7) Transfer seedlings to prepared eppendorf tube containing stainless steel cone ball and RA1 lysis buffer (containing 1% β-mercaptoethanol).-1 for 2 × 2 mins. The tube positions are rotated between shaking steps as described in the manufacturer's operating manual. NOTE: Although the TissueLyser equipment we used is capable of processing 192 samples (2 × 96) in 1.5 ml tubes simultaneously, the size of the bead  required for efficient sample disruption and homogenisation necessitated the use of 2 ml eppendorf tubes. Consequently, samples have to be processed in two batches of 48. Although it is possible to use smaller stainless steel balls (e.g. 3-4 mm) and thus process samples in larger batches of 96 × 1.5 ml eppendorf tubes, we have found that this only works well with wild type samples or similar. In our hands, smaller bead sizes do not work well with mutant plant samples which often grow poorly and the resulting yield is very low. As our experiments usually include a variety of genetic manipulations, we routinely use 5 mm cone balls for efficient disruption and homogenisation of all samples.8) Homogenise the samples in two batches of 2× 24 tubes using a TissueLyser bead mill at a frequency of 25 s9) Centrifuge lysed samples at 11,000 × g for 5 mins.10) Transfer supernatant to a filter plate.: Missing out this step subsequently results in clogged wells in the binding plate and drastically reduced yields.11) Centrifuge filter plate (over a 1.1 ml deepwell plate) at 3,200 × g for 10 mins to remove debris. NOTEAt this point, lysed samples can be safely stored at -20°C for up to 6 months with no detrimental effects on purified RNA - see comments section below.12) 150 μl of the cleared lysed solution is transferred to another 1.1 ml deepwell plate to be processed for RNA extraction while the remainder is stored at -20°C as backup in case of experimental disaster. NOTE: either a centrifuge or vacuum manifold to pass the solutions through the plate. NOTE: We have modified the kit manufacturer's protocol to be used on plate centrifuges commonly found in labs  and all centrifugation steps were carried out in an Eppendorf 5810R centrifuge with an A-4-81 swing-bucket rotor. The plate sandwich formed by the binding plate combined with the plate included in the kit was too deep to be used in our centrifuge set up. Therefore the plate sandwich was composed of a binding plate over a 1.1 ml 96 deepwell plate not the plate provided with the kit. All vacuum steps were performed at -200 mbar for 2 × 60 secs. In our hands, greater pressure resulted in splashing of the sample and possible cross well contamination.The manual RNA extraction procedure is performed using multichannel pipettes and 13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare cleared lysed samples  for binding to the silica membrane by adding an equal volume of RA4 buffer (150 μl) to each well of 1.1 ml deepwell plate.15) Mix thoroughly by pipetting up and down 12 times.16) Transfer samples (300 μl) to the wells of the binding plate.17) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.18) Add 460 μl buffer RA3 to each well of the binding plates .19) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.20) Add 29 μl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.21) Add 460 μl RA2 buffer to each well of the binding plate.22) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.23) Add 800 μl RA3 buffer to each well of the binding plate.24) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.25) Add 460 μl RA4 buffer to each well of the binding plate.26) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.27) Proceed to elution (step 30 below).The automated RNA extraction procedure is performed using a liquid handling robot with an integrated vacuum unit on the robot deck to pass the solutions through the binding plate.13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare the robotic workstation and check that the actual deck layout matches the virtual one shown on the robot script with all labware  containing appropriate solutions  in the correct locations on the robot deck - see Figure 15) Place 1.1 ml deepwell plate containing 96 lysed samples  onto the appropriate position of the robot deck.16) Select appropriate robot script and run which will perform steps 17-28 as detailed below.17) Prepare lysed samples for binding to the silica membrane by the addition of an equal volume of RA4 buffer (150 μl) and mix thoroughly by pipetting (150 μl) up and down 12 times.18) Transfer samples (300 μl) to the wells of the binding plate.19) Apply vacuum  to pull the solution through the binding plate.20) Add 460 μl buffer RA3 to each well of the binding plates .21) Apply vacuum  to pull the solution through the binding plate.22) Add 29 μl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.23) Add 460 μl RA2 buffer to each well of the binding plate.24) Apply vacuum  to pull the solution through the binding plate.25) Add 800 μl RA3 buffer to each well of the binding plate.26) Apply vacuum  to pull the solution through the binding plate.27) Add 460 μl RA4 buffer to each well of the binding plate.28) Apply vacuum  to pull the solution through the binding plate.29) Remove binding plate from robot and proceed to elution (step 30 below).Regardless of whether the manual or automated method is used, the same elution procedure is followed.30) Centrifuge the plate at 3,220 × g for 10 mins to ensure all ethanol is removed prior to elution.31) Add 50 μl RNase-free water to each well of the binding plate and incubate for 3 mins.32) Place binding plate over the 96 well elution plate (contained in the illustra kit) and elute purified RNA by centrifugation at 3,220 × g for 3 mins.CAB2:LUC transgene which are elongated [ELF3:LUC transgene.The yields of extracted RNA purified using the manual methods (using a centrifuge or vacuum) and the automated (using vacuum) procedure were similar Figure . Althouglongated . As the longated , these 9260/280 ratio of between 1.8 and 2.2 (average 2.09) indicative of highly pure RNA [260/230 ratios, which are a measure of polysaccharide contamination, were much more variable. This often occurs with plant samples which typically have relatively high polysaccharide contents [260/230 ratio seemed to have no detrimental effect on subsequent use of the purified RNA for reverse transcription and real time-qPCR (see comments below). This may not be the case for other applications which would have to be individually assessed.The quality of the HTP purified RNA was assessed by UV spectrometry using a Nanodrop spectrophotometer Figure . All purpure RNA . The A26contents -18 howevThe integrity and quality of purified RNA can be analysed using denaturing gel electrophoresis and visualising the ribosomal RNA bands. However, this technique requires a relatively large amount of RNA to detect clearly visible bands and the RNA is not recoverable for subsequent use. Recently, this analysis has been largely replaced by the use of Bioanalyzers which use microfluidics and capilliary electrophoresis on chips. Bioanalyzers require much smaller amounts of RNA and are ideal when limited amounts of starting sample are available. We therefore assessed the purified RNA using Bioanalyzer microfluidic chips which demonstrated its consistently high quality producing distinct, sharp ribosomal peaks Figure .isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) gene expression was performed on eight scattered samples  from one of the 96 well plates  is sufficient for subsequent cDNA synthesis and analysis of expression by qPCR of over 500 genes in triplicate (based on single-plex qPCR) using our usual procedures [[Plant tissue contains large amounts of polysaccharides and a number of endogenous PCR inhibitors such as polyphenolic compounds which can co-purify with the RNA and inhibit downstream applications such as qPCR -18. The bidopsis . All tescedures [ and unpuTraditional methods of RNA extraction such as phenol-chloroform are cheaper to perform and produce a greater yield than kit based silica methods . However, modern health and safety practice encourages the use of safer alternatives (where available) to replace harmful or dangerous chemicals so, even on low throughput, many labs now routinely use silica-based methods (such as spin columns). In addition to simplifying sample handling procedures, the use of a 96 well plate format is cost effective as the cost per sample in the 96 well format is less than half that incurred when using individual mini spin columns. This is before other considerations such as staff time are factored in and represents a considerable saving for larger scale studies involving hundreds of samples. The 96 well plate format also lends itself to automation. Liquid handling robots used to be the preserve of industry however they are becoming more commonplace in academia particularly with the recent move to a more systems-based approach to studying biology. Liquid handling robots are also decreasing in price and becoming more affordable both for core facilities and individual laboratories. Small systems cost around the same as a plate format qPCR machine and can be used for multiple applications as many individual labs will not have sufficient sample throughput for a dedicated robot specifically for RNA extraction. Our optimised protocol can easily be implemented on other robot platforms providing flexibility to the available system.The automated RNA purification procedure is based on the adsorption of nucleic acids to silica membranes and can be performed on the open bench in contrast to a recently published HTP method using phenol chloroform  which relater used in our protocol) can be used. These have recently become commercially available and increasingly popular  or 10 months . Storage at 4°C also affects the quality of the RNA.Many of the issues encountered with plant RNA extraction protocols are thought to originate from the initial sampling procedure . In orde example ). These C Figure . AlthougWe also investigated the effects of freezing the lysed samples in RA1, the lysis buffer supplied with the illustra kit. Samples were processed immediately or after storage at -20°C for 2 days or 6 months prior to completion of the extraction procedure Figure . No signlater) and the ability to freeze lysed samples with no detrimental affects on the subsequently isolated RNA, provide a flexible sampling and storage scheme. When this is used in conjunction with our automated HTP RNA extraction procedure, it enables complex time series experiments critical for systems-based plant research.We describe a HTP silica membrane-based method for total RNA extraction from Arabidopsis seedlings in a 96 plate format. This procedure can be performed manually by using appropriate multichannel pipettes or automated with the use of a specialised liquid handling robot. The described protocols isolate RNA in sufficient quantity and of high quality suitable for sensitive downstream processes such as reverse transcription and qPCR. The procedure is time efficient and cost effective for the processing of large numbers of samples. We have used our automated method to investigate and verify sample storage options during the procedure. The combined use of an aqueous RNA stabilising solution (RNAThe authors declare that they have no competing interests.ESC and SK performed the experimental work and performed the data analysis. LEK conceived and coordinated the project and wrote the manuscript. All authors have read and approved the final manuscript.


To the Editor: As someone with an interest in case reports (1-3), I read your editorial regarding the comeback of the case report with great interest (4). The three case reports that you published (5-7) are a great read. The case report by Nosan et al (6) mentions an “antimongoloid palpebral slant,” which is a term with pejorative connotations . A more informative and appropriate term would be downward-slanting palpebral fissures (10). One can only hope that with time inappropriate terms will disappear from medical discourse save for historical mention and medical journals should play a very important part in discouraging their use (11).


Diffusion-weighted magnetic resonance imaging (DW-MRI) is a quantitative MRI technique that provides physiological information by measuring the degree of water molecule diffusion within the extracellular space. It gives a quantitative measurement known as the apparent diffusion coefficient (ADC) value. The aim of the study is to show the influence of the menstrual cycle on breast ADC values and the relationship of the ADC to transverse relaxation (T2) value.b values . The T2 relaxation time was measured using T2w turbo spin echo (TSE) with four echo times . ADC and T2 maps were generated automatically by standard Philips software.Female volunteers had one MRI scan per week over 4 weeks using a 3 T MRI scanner. The ADC of the fibroglandular tissue was measured using a single-shot SE-EPI with four The study was performed on 11 healthy volunteers (23 to 41 years old) with a regular menstrual cycle. There is no significant difference between ADC and T2 values for the 4 weeks. Pearson's correlation coefficient indicated a negative correlation between ADC and T2 values. See Table The study shows that ADC values are not affected by the normal hormonal fluctuations during the menstrual cycle.