Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented.Both host and pathogen factors contribute to disease outcome in P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome.A custom made oligonucleotide array with probes based on the in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins.The data demonstrated that P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity.Whole genome transcription analysis of  Plasmodium falciparum infection remains a major health problem worldwide. Its complex life cycle has hampered standard methods for the study of pathogenesis. New approaches to elucidate parasite biology using whole genomic methods have provided insight into gene function, transcriptional regulation and stage specific biology [in vivo biology of this pathogen, through adaptation of a whole genome approach, would provide insight into the host-parasite relationship, parasite virulence factors and inform new strategies for intervention. Genomic scale transcriptional profiling of P. falciparum during a natural infection is presented. Small amounts of parasite RNA, isolated from a few milliliters of a blood sample are found to be sufficient for whole genome transcriptional analysis. This data show that several genes are differentially expressed in vivo, indicating differences between the transcriptional program of 3D7 laboratory strain parasites growing in culture and naturally occurring infections in the human host. biology -4. Charain vivo conditions [Pasteurella multocida in vivo as compared to in vitro conditions [P. falciparum gene expression patterns, particularly the subset of genes that are specifically expressed in the in vivo state may identify unique parasite biology when it resides in the host environment. Processes involving parasite metabolism, immune evasion and transmission may be altered in the highly specialized environment of the human host as compared to in vitro conditions. In addition, approximately 12% of P. falciparum's predicted genes have not been found to be expressed in any of the life cycle stages previously studied [in vivo may reveal the unique expression of such genes in vivo, providing additional targets for intervention.Whole genome expression has been used in studies of bacterial pathogenesis to identify genes that are specifically transcribed under nditions -7. For enditions . Similar studied . Whole gP. falciparum chloroquine resistance study in Senegal [P. falciparum malaria gave consent for the study and were enrolled at an outpatient health clinic. Patients underwent venipuncture using K3 EDTA coated Vacutainers (Beckton Dickinson) and from this sample, 1.6–2.5 ml of whole blood was collected and passed through a white cell depletion filter using a 20 ml syringe. The filtered sample was centrifuged for 5 minutes at 3,200 rpm in a clinical centrifuge and placed in Tri-Reagent BD (Molecular Research Center). The samples were vortexed and stored at minus 70°C. Samples were thawed in a room temperature bath one month later and isolation of RNA was completed per Molecular Research Centers protocol. Three samples obtained in Senegal that had the highest parasitemia and largest blood volume are presented. A 14 ml blood sample from a P. falciparum infected traveler from Nigeria was similarly processed in Boston.This study was conducted as part of an ongoing  Senegal . PatientLabeling and hybridization of total RNA was performed as described . Expressin vivo sample and 3D7 ring stage total RNA using Super Script 1st Strand synthesis system (Invitrogen). Duplicate reactions using real time PCR were performed with 1 μl cDNA with gene specific primers in 50 μl reaction volume using fluorescent dye SYBR Green . The reactions were carried out on an ABI PRISM model 7700-sequence detector and all PCR reactions amplified a single product as determined by dissociation curve analysis .To confirm array data, a subset of genes  that vary from high to low level abundance by array were quantified using real time PCR from cDNA. PFB0120: forward primer 5'-CAG CCC TCT TAG CTC TCA ACT TC-3', reverse primer 5'-AGC AAC AGC AGA GGC TAT AGA ACT-3', PFB0100: forward primer 5'-CAC CAA ATG GCT ATG CTT ATG GA-3', reverse primer 5'-TTC CAG GAG CAC CAT TAA ATC CT-3', PFB0270: forward primer 5'-ACA CTT ACT GGT ATT TCG GAA TTT-3', reverse primer 5'-TAA TTG TCC ATA TTC TTC AAT ATA T-3', PFB0355: forward primer 5'-ATT GTA AGA AAT AGT TGG GGT-3', reverse primer 5'-TAT ATC ATG CTC CTT CTT ATC A-3', PFB0065: forward primer 5'-CGT TGG TAG TGC GTT CCT TAC AA-3', reverse primer 5'-GTT CCT GCT ATA TCA GGA GCA CCA-3'. Sequence analysis confirmed the identity of the amplification products. 3D7 strain parasites were cultivated under standard conditions and synchronized with 5% sorbitol to obtain ring stage parasites for extraction of total RNA . cDNA waPlasmodium genes between the 10th and 90th percentile with at least six probes. Analysis based on rank was a second method used; in each experiment the probe intensity was ranked and this resulted in equivalent quantile distribution for all probes between two experiments. This method is more conservative and will define relative rank changes between experiments and is independent of potential normalization artifacts.Variation between samples was assessed using Kruskal-Wallis method (non-parametric ANOVA) to test the null hypothesis. To normalize samples, the mean gene expression level was calculated for all Patient blood samples were collected after informed consent was obtained. The study was approved by the institutional review boards at Harvard School of Public Health, Brigham and Women's Hospital and Cheikh Anta Diop University.in vivo isolated samples a denaturing RNA gel was carried out . The most abundant transcript of human origin in the in vivo was haemoglobin RNA . Further analysis was performed on the Nigerian sample. Only one parasite line was detected in this sample through DNA genotyping of the K1, MAD20, RO33 alleles of msp1 and FC27 and IC1 alleles of msp2 using primers and methods previously reported [Plasmodium genes on the array [P. falciparum chromosome 2 expression levels utilizing 15 μg to 30 μg or 15 μg to 60 μg cRNA were 0.95 and 0.92, respectively. However, as cRNA concentration was increased to 120 μg, the background to noise ratio increased significantly, resulting in a decreased correlation coefficient (R = 0.72)  Figure  and 2c.in vivo sample are listed in Table in vivo sample compared to 3D7 ring stage previously reported using the Kruskal-Wallis method [in vivo.The most abundant transcripts detected from the Nigerian s method . Notablyin vivo overexpression of a SERA antigen (PFB0355), the relative expression of five genes that had varying transcript abundance by array was carried out using real time PCR of cDNA generated from total RNA isolated from the Nigerian in vivo sample and a 3D7 in vitro ring stage sample. There is good correlation between the array results and those obtained by real time PCR (Figure in vivo and in vitro samples, the data is normalized to cDNA of PFB0120 to account for differences in starting parasite cDNA, secondary to human cDNA. The in vivo sample contained 0.15 ng cDNA of PFB0355c and 3D7 ring stage cDNA had 0.09 ng by real time PCR. When PFB0355c is normalized to PFB0120c, it was found to be ten fold overexpressed in vivo as compared to in vitro, consistent with the array results.To confirm the accuracy of the results from the oligonucleotide array and to confirm the igen PFB055, the rin vivo whole genomic expression can be performed despite the potential technical challenges of scarce RNA contained in a small blood volume sample and presence of abundant human RNA. Tri-Reagent BD was used to stabilize RNA and the samples were stored at -80°C before transport to the US. The denaturing gel suggests that the RNA remains intact using this reagent. Notably Kyes et al. reported that minus 80°C rather than 4°C or minus 20°C is the optimal temperature to store field sample RNA for detection of long transcripts such as var [in vitro sample [in vivo samples as compared to the 3D7 in vitro sample. Although human red cells are used for culturing in the in vitro system, reticulocytes which contribute to the haemoglobin detected are not abundant in in vitro samples. This is most likely due to the observation that reticulocyte levels display a 75% loss at 48 hours, when placed at 37°C, which is the condition of in vitro culture [in vivo samples and may be secondary to white cell contamination. Cross hybridization of human RNA to parasite probes may occur. However, Zhou et al have shown that human genomic DNA does not highly cross hybridize to the parasite probes on this custom array [in vivo parasite transcripts when using these probes.This data confirms that h as var . Surpris culture . In addiom array . The higom array ,19. Due in vivo samples it was not possible to determine how much parasite RNA is required for whole genome analysis. The samples from Senegal were of low parasitemia and small volumes, whereas the 14 ml blood sample with 0.4% parasitemia was sufficient to detect a greater number of chromosome 2 transcripts. Four to five mls of packed blood in a patient with a greater than 2% parasitemia should provide sufficient material for whole genome analysis using these methods. The data demonstrates the reproducibility of the method by independent hybridizations and that maximal senstivity can be achieved with up to 60 μg of cRNA, using this array.The amount of blood volume necessary for comprehensive detection of transcription depends on level of parasitemia and method of microarray analysis. This analysis utilized the Affymetrix system which requires very little starting RNA as compared to other methods . Due to in vivo transcript in all samples encoded on chromosome 2. This gene is a member of a recently described gene family, etramps, expressed at early ring stage encoding a protein thought to be involved in erythrocyte remodeling; this was also the most highly expressed transcript in in vitro ring stage cultures [in vivo expressed genes from the Nigerian sample was compared to that of the in vitro 3D7 ring stage chromosome 2 transcriptome [in vivo ring stage transcriptome and the 3D7 ring stage transcriptome. Most of the genes that are expressed in vivo are also expressed in vitro particularly those involved in cellular function and genes encoding hypothetical proteins (Table in vivo samples. Previously this gene has been demonstrated to be expressed and transcribed only in the sexual transmission stage [in vivo samples may have more parasites that are undergoing or are committed to sexual development than is detectable by microscopy. Several genes encoding membrane proteins, including SERA and rifin genes, were also found to be differentially expressed. These genes are members of multigene family that encode surface proteins which are thought to be involved in immune evasion [in vivo could be due either to genetic or transcriptional modulation of the parasite's defense repertoire or geographic variation. Differences between the in vivo and in vitro expression of SERA antigens may be due to differences in the in vivo stage of development as this gene family is transcribed at later stages in the in vitro life cycle [in vitro samples due to higher parasite counts and a subset of genes were found to be overexpressed in vitro after normalization. This analysis focussed on genes overexpressed in vivo as these results would not be influenced by normalization algorithms. Overall, this data demonstrates that P. falciparum transcripts can be detected from in vivo samples, and that there are potentially important differences between trancription of in vivo samples and that of the 3D7 in vitro trancription profile.PFB0120w is the most abundant cultures ,22. The on stage . Since n evasion ,24. The fe cycle . There wP. falciparum can be studied in vivo from a small blood sample of an infected patient. The in vivo sample however contains human RNA, whose quantity may vary from sample to sample and therefore differenes in parasite transcript level between samples must be reported relative to a reference transcript. Despite the abundance of human RNA the genomes are sufficiently different with resultant probes specificity. Predictably, there was a high correlation of in vivo expression with the in vitro ring stage 3D7 transcriptome [in vivo and in vitro expression levels in genes typically found in transmissable forms and encoding variant surface proteins. Evaluation of trancription of genes specific for gametocyte development in specific patient populations may uncover the in vivo conditions that favor development of transmissable forms. Similarily, a whole genome analysis can comprehensively characterize expression of multigene families that encode variant surface proteins under in vivo conditions. Further exploration of the in vivo biology of P. falciparum using specific probes to all annotated genes will be undertaken to confirm and explore other important biological differences. This new approach will further the understanding of the host-pathogen interaction and may result in the development of new strategies to combat this disease.In summary, this study provides evidence that whole genome gene expression in criptome . Importa


Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells.Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS.Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions Multiple large-scale analytical techniques to assess gene expression in defined cell populations have been developed. These include microarray analysis, EST enumeration, SAGE and MPSS. Each of these techniques offers unique advantages and disadvantages. Technique selection largely depends on the expertise of the investigator, the cost, the availability of the techniques, the amount of RNA/DNA that is available, and the existence of the genome databases. The human genome dataset is the best annotated one available ,2- makinHuman ES cells have been isolated relatively recently and ES cell genes are underrepresented in current databases. More importantly, recent evidence has suggested that mouse ES and human ES cells differ significantly in their fundamental biology ,4 and onWe selected MPSS for this analysis as it offers some unique advantages over other methods including SAGE ,16. MPSSWe have utilized MPSS using a pooled sample of three human ES cell lines grown in feeder-free culture conditions over multiple passages ,18 to asA pooled sample of undifferentiated human ES cell lines H1, H7, and H9 grown in feeder-cell free conditions  was usedPooled mRNA of the three hESC lines was subjected to MPSS analysis at Lynx Therapeutics , generating 22,136 distinct and significant signature sequences from a total of 2,786,765 sequences  that were elevated in ES cells when compared to neural stem cells examined in a similar manner was used. This list is provided in supplementary materials . A list Recently we and others have begun examining hESC with EST scan  and micrComparison with an EST scan analysis of 37,081 EST sequenced from a similar pooled sample of hESC ,10 also Richards et al  have useOur results provide a global overview of the gene expression pattern of undifferentiated human ES cells and allow comparisons with other data sets. These results suggest the hESC are an actively dividing population of cells that exhibit high metabolic activity. Our analysis detected expression of approximately 10,600 unique transcripts, a figure that about a third of the total number of mapped genes. Unlike other cell types, however, a much larger fraction of unknown or novel genes was present. This high ratio likely represents the paucity of information available in existing libraries on this relatively newly characterized cell population rather than the possibility that ES cells use radically different pathways for self-renewal, survival, proliferation and differentiation.Our results confirm the reported differences between rodent and human ES cells. We confirm the absence of expression of ERAS, Ehox and the orthologs PEPP1 and 2. The apparent lack of LIF requirement of hESC is reflected by the absence or low tpm levels for genes of the LIF pathway and high tpm for suppressors of LIF mediated signalling (see supporting information). The high level of expression of genes in the FGF pathway likely reflects the requirement of hESC for bFGF. The high level of FGFR1 expression suggests that FGFR1 is an important signal transducer and that FGF's other than FGF4 are important in hESC self-renewal. The high tpm of the fibronectin receptor also suggest that fibronectin or vitronectin are likely useful substitutes for matrigel and that activation of ras mediated signalling is likely critical, as has been described in the rodent ES cell analysis .Comparing data from the MPSS analysis with microarray, SAGE and EST scan analyses suggest that MPSS is a powerful alternative to these techniques. MPSS identified virtually all of the genes highlighted as genes common between six different human ES cell lines surveyed by microarray. We noted that most genes detected by microarray were expressed at high tpm indicating that MPSS is more sensitive than microarray analysis. MPSS however appeared to be able to identify genes detected by microarray. Analysing an additional 400 markers detected by MPSS using focused microarray or RT-PCR confirmed their expression ,  we predict that even a 5% contamination can confound results or detailed comparisons across different laboratories.We note also that gene transcription from both the X and Y chromosome is observed indicating that at least subtle differences will exist between male and female lines even in the undifferentiated state. Sex-based gene expression, along with MHC gene expression and ratio of expression of imprinted genes could serve to distinguish between different ES cell populations. The present results further suggest that analysing embryoid bodies that differentiate stochastically or analysing tissue samples (with variable proportions of cells) by MPSS will prove more difficult and that results will be variable. We suggest that variability can be reduced by pooling samples, normalizing by careful testing for known markers of differentiation, by semi quantitative PCR, or by focused microarray analysis.While MPSS is cost-effective and sensitive, it is by no means perfect. MPSS is limited by the requirement that DpnII sites (GATC) be present in a gene and be present in a unique locus such that the signature obtained is unique. For example, SNRF expression could not be assessed directly, as no GATC site is present. The signatures for ZFP42 are ambiguous and map to multiple transcripts. Although MPSS can distinguish between alternate transcript termination sites, MPSS cannot distinguish between alternative splicing events and possible incomplete digestion during the sample preparation process. Signature lengths are relatively short and it is possible to have to select between multiple genome hits  that had been maintained in feeder free culture conditions and evaluated for the presence of ES cell markers and absence of markers of differentiation. The mRNA was converted to cDNA and digested with DpnII. The last DpnII site and the downstream 16 bases were cloned into Megaclone vectors and their sequences determined according to the MPSS protocol ,16,25. Avirtual signatures") from the human genome sequence, the human Unigene sequences, and human mitochondrion. Each virtual signature was ranked, as outlined in the table To generate a complete, annotated human signature database, we extracted all the possible signatures . By thiTotal RNA was isolated from cell pellets using RNAeasy Qiagen mini protocol and kit. cDNA was synthesized using 100 ng of total RNA in a 20-μl reaction. Superscript II (Gibco-BRL), a modified Maloney murine leukemia virus RT, and Oligo (dT)12–18 primers were used according to the manufacturer's instructions (Gibco-BRL). The list of primers used for RT-PCR and annealing conditions are described previously ).RB, IK and MR were primarily responsible for the data analysis and writing and editing the manuscript. ST generated the ES cell samples and verified their quality, TM performed the RT-PCR and JC performed the immunocytochemistry. RP generated the microarray data and TV and JL provided support and supervision for RB, IK, ST.The document describing details of MPSS analysis of the HuES cells performed at Lynx, and algorithm for initial MPSS signature annotation and classification.Click here for fileThe file contains MPSS data for 22,136 significant and reliable signatures, annotated with genome version hg15  and the human Unigene build Hs.161.Click here for fileThe file contains MPSS data for 22,136 significant and reliable signatures, annotated with genome version hg16  and the human Unigene build Hs.169.Click here for filetable containing data for 8679 unigene clusters, 11 mitochondrial genes, and including 1991 signatures that did not map to unigene but uniquely matched genomic sequences .Click here for fileTop 200 rows from the table HuES17_onetpmHs169hg16.xls.Click here for fileList of tpm of genes identified by Sperger et al [11] present in the MPSS dataset.Click here for fileList of tpm of genes identified by Richards et al [8] present in the MPSS dataset.Click here for fileList of tpm of genes identified by MPSS in the BG02 dataset.Click here for fileThis file lists the genes located on the X and Y chromosome for which a MPSS signature sequence was found in the HuES cells. The X and Y chromosome genes are listed in separate worksheets. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Seq_id: Repeat if hit genome more than 100 locations; UniGene cluster ID if hit one or more UniGene clusters; Chromosome number if hit one genome location; MultiGenome if hit genome multiple times; Description: description of annotation (see Appendix B for more details).Click here for fileList of all UniGene clusters (release 161) located on the X and Y chromosome. The X and Y chromosome genes are listed in separate worksheets. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Locusid: Locus Link identifier UniGene cluster: UniGene cluster ID Description: description derived from UniGeneClick here for fileGraphical representation of hotspots and coldspots for all human chromosomes. Cytobands with significantly higher number of genes in HuES cells are marked with '+' and cytobands with lower number of genes in HuES cells are marked with '-'. The number of '+' or '-' signs is proportional to the difference, with one '+' or '-' representing the fold difference. For example '++' represents a 2-fold higher number of genes expressed in HuES cells compared to the expected number based on the number of genes known to be located in the same cytoband.Click here for fileList of all chromosal regions that appeared statistically distinct.Click here for fileList of genes expressed in HuES cells and located in the hotspots and coldspots. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Seq_id: Repeat if hit genome more than 100 locations; UniGene cluster ID if hit one or more UniGene clusters; Chromosome number if hit one genome location; MultiGenome if hit genome multiple times; Description: description of annotation.Click here for fileList of all UniGene clusters located in the hotspot and coldspot regions. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband UniGene ID: UniGene cluster ID Locusid: Locus Link identifier Description: description derived from UniGene;Click here for fileThis file contains the mapping information of MPSS signatures to the EST scan genes for major signaling pathways. Each pathway is presented in a separate worksheet Columns: Locusid: Locus Link identifier Pathway: Pathway the gene has been assigned to. Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Stdev: standard deviation of the mean abundance from multiple MPSS runs Hit genome: HitGenome – Numbers of genomic locations a signature maps to HitUniGene: Numbers of UniGene Clusters a signature maps to; Seq_id: UniGene cluster ID ('Hs.' Has been omitted). NULL if there is no MPPS signature but an EST from the EST scan. Class: signature class (see Appendix B for more details); Title: description, derived from UniGeneClick here for fileThis file contains additional lists of potential homeodomain proteins, genes of unknown function, zinc finger proteins in separate worksheets. Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Stdev: standard deviation of the mean abundance from multiple MPSS runs Hit genome: HitGenome – Numbers of genomic locations a signature maps to HitUniGene: Numbers of UniGene Clusters a signature maps to; Seq_id: UniGene cluster ID. Class: signature class (see Appendix B for more details); Title: description, derived from UniGene.Click here for fileList of tpm of potential 400 ES enriched genes in other cell lines examined.Click here for fileList of all tpm of potential 13 ES novel genes in all tissues and cell lines examined.Click here for file


Retrovirology provide possible molecular mechanisms for the pro-apoptotic effect of Tat, which involve the cell's microtubular network and the mitochondrial pathway of apoptosis.The Tat protein of HIV-1 is a powerful activator of viral gene expression. Besides this essential function at the HIV-1 promoter, the protein also exerts a remarkable number of other biological activities, among which the induction of cellular apoptosis. Two papers now published in  Although more than 20 years have passed since the identification of HIV as the cause of AIDS, several essential questions about its pathogenicity remain as yet unanswered. In particular, a central, still unresolved issue is the mechanism underlying the progressive development of immunodeficiency. It is now well established that HIV infection determines a rapid turnover of infected CD4 cells ,2; consiIn this context, the finding that the virus-encoded Tat protein is released by the infected cells and can be taken up by neighboring, uninfected cells via an endocytic mechanism ,7 has loA link between microtubule polymerization and the pro-apoptotic effect of Tat has first been suggested a few years ago in the observation that Tat directly interacts with the αβ-tubulin dimers and polymerized microtubules in the cytoplasm of the cell . The funRetrovirology extend the link between the microtubule network, the mitochondrial pathway of apoptosis, and Tat. De Mareuil and coworkers show that Tat enhances tubulin polymerization into microtubules, an effect similar to that exerted by the MTPAs, and physically associates with the polymerized microtubuli [Two papers now published in rotubuli . As oppoThe accompanying manuscript by Epie and coworkers describes the identification of a microtubule-associated protein, LIS1, which specifically binds Tat . In the What might be the relevance of these findings in the context of HIV-1 infection? They clearly provide a mechanism for CD4 T-cell apoptosis and for the extension of the apoptotic effect to bystander, uninfected cells in the lymph node. Moreover, the interaction of Tat with the microtubular network might explain the occurrence of neuropathogenesis accompanying the progression of HIV disease, since many human neurodegenerative conditions are elicited by a reorganization of the neuronal cytoskeleton . Thus, tAs commonly happens in biology, the findings reported in these manuscripts raise more questions than answers. First, the Tat domains involved in the described interactions are different, a surprising finding given the very small size of Tat. This observation might possibly suggest that Tat is part of a large multi-molecular complex associated with the tubular network, making multiple contacts with different proteins. This issue can be experimentally addressed biochemically, or even within the cell, by taking advantage of the biophysical techniques available to investigate protein-protein interactions in vivo . SecondlMTPAs: microtubule-targeted, tubulin-polymerizing agentsCTD: carboxy-terminal domainThe author(s) declare that they have no competing interests.


GGPP synthase gene in Coleus forskohlii Briq.Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.The open reading frame for full-length This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. Coleus forskohlii Briq. (Lamiaceae)   in order to produce a lacZ fusion protein. pGGPPS was then transformed into E. coli DH10B carrying the plasmid pACCAR25ΔcrtE in which the crtE encoding GGPP synthase had been deleted. The yellow color of carotenoid was observed in the transformant, indicating that pGGPPS carried the gene substituting the function of the crtE gene   were subcloned into the HindIII-EcoRI site of the pBI121 vector and then transformed into A. tumefaciens LBA4404. The transformants were cultured at 28°C for two days in YEB liquid medium containing 25 μg/ml of kanamycin and 25 μg/ml of rifampicin. The transformants were washed twice and re-suspended in YEB medium. Agrobacterium transformants (108 cells) were applied to four ml of five-day-old BY-2 suspension culture. The culture was incubated at 28°C for two days under dark conditions. GFP and GFP fusion protein were analysed by fluorescence microscopy using Nikon Eclipse TE2000-U model. Cells were observed at a 400 × magnification.A 240 bp fragment of the N-terminal region of argeting ,30. GFP,C. forskohlii GGPP synthase was amplified by PCR using specific primers M and O. A PCR product was digested; purified and cloned into the KpnI-SalI site of pBluescript II KS- vector, namely pGGPPS. This plasmid was transformed into E. coli XL1-Blue MRF' for over-expression. The transformants were cultured in LB liquid medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol. The culture was induced with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) and incubated for six hours at 37°C. The cells were harvested and washed with 50 mM Tris-HCl pH 8.0 by centrifugation. The pellet was re-suspended, lysozyme was added and the mixture was incubated for 30 minutes. The mixture was then sonicated for four cycles of 15 seconds at one minute intervals. The soluble fraction was obtained after centrifugation at 10,000 × g for 10 minutes. SDS-PAGE was conducted in order to detect the proteins [The coding region of a cDNA of proteins .crtE plasmid contains the gene cluster crtB, crtI, crtX, crtY and crtZ encoding carotenoid biosynthetic enzymes with the exception of crtE (encoding GGPP synthase). The plasmid pBAA containing mouse GGPP synthase (positive control plasmid) and E. coli DH10B carrying the plasmid pACCAR25ΔcrtE was provided by Dr. M. Kawamukai, Shimane University, Japan [- vector, pBS, was used as negative control. pGGPPS, pBAA and pBS were transformed into E. coli DH10B carrying the plasmid pACCAR25ΔcrtE. All transformants were plated on LB agar medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol and then incubated for two to three days at 25°C.The pACCAR25Δy, Japan . pBluescC. forskohlii was analysed in twelve separate parts; leaf (L1–L4), stem (S1–S5) and root (R1–R3). The numbering is based on the maturation of organs. Total RNA was extracted from each part of plant. One microgram of total RNA was used as the template for the synthesis of the first strand cDNA . Primers M and O, the first strand cDNA and KOD-polymerase were used for the amplification of C. forskohlii GGPP synthase with the condition of denaturation, 98°C, 15 seconds; annealing, 60°C, 2 seconds and extension, 74°C, 5 seconds. The 18S rRNA fragment used as an internal control was amplified using primers R and S under the same conditions of C. forskohlii GGPP synthase amplification. The amplified PCR products were analysed by 1.0% agarose gel electrophoresis.An eight-month-old C. forskohlii plantlets were treated with various concentrations of fosmidomycin and then investigated for forskolin content using the HPLC method as previously described [escribed . Forskolcrt, carotenogenic gene; FARM, first aspartate-rich motif; GFP, green fluorescent protein; GGPP, geranylgeranyl diphosphate; MEP, 2C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; SARM, second aspatate-rich motifSE carried out the molecular genetic studies, participated in the sequence alignment, forskolin analysis and drafted the manuscript. TF participated in the design of the study and coordination. MK participated in genetic complementation and coordination. YS conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development.Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C  peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C.The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed.Systemic variation of cellular endopolyploidy in different tissues during growth and development of  Arabidopsis [In the classical cell cycle, the nuclear DNA contents vary only within the range of 2C and 4C, where C is the haploid DNA content per nucleus. When mitotic DNA replication in somatic cells is not followed by cell division , variation of cellular ploidy levels (designated as somatic polyploidy or endopolyploidy) can result . Endopolbidopsis  and brasbidopsis ,9. Presebidopsis ,11.Vanilla [Vanda seedlings [Spathoglottis plicata, a common tropical terrestrial orchid species, from field-grown plants and in vitro cultures was investigated.In orchids, endoreduplication has been described in the raphid crystal idioblasts of Vanilla  and in peedlings ,14. The eedlings . Many ofeedlings . The expeedlings ,17. Howeeedlings . In thisFlow cytometry analysis of nuclear preparations from entire young leaves of 1–3 cm in length revealed that there were three peaks of fluorescence corresponding to 2C, 4C and 8C DNA content of somatic cells Fig. . About 5More detailed analysis was done on mature leaves of 48 cm in length. Tissues taken from different regions of the mature leaves showed that the pattern of ploidy levels was different at different regions. For tissues taken from the basal (petiolar end) of the leaves, there were three fluorescence peaks corresponding to 2C, 4C and 8C nuclear DNA content Fig. . HoweverIn the young root tips, 2C, 4C and 8C peaks were observed Fig.  and the Preparations from floral pedicels Fig. , columnsS. plicata were found to have 2C, 4C and 8C nuclei  and root tips from greenhouse-grown plants. Similarly, endopolyploid cells were found in protocorms, young leaves and root tips from S. plicata seedlings in tissue culture. The common feature for protocorms, young leaves of 1–3 cm and root tips are that they are young and active in cell division and growth. In other orchids such as Dendrobium, endopolyploidy was found in root tips and newly expanded young leaves [S. plicata, endopolyploidy was neither found in tissue-cultured plants nor it was detected in greenhouse-grown plants. These results imply that the presence of the endopolyploidy during S. plicata development is an intrinsic programme, and it is not much affected by the growth condition.The patterns of endopolyploidy may be affected by plant growth conditions in some plants. For example, leaves of eenhouse ,23. Howeg leaves . In the Dendrobium species and cultivars [S. plicata. Furthermore, when the tip, middle and basal parts of the mature leaf were examined, different patterns of ploidy levels were obtained. Endopolyploidy was found only in leaf base part of mature leaves. A mature leaf represents a continuous developmental system, with the young, less green meristem cells at the basal petiolar end and the older, photosynthetically active cells at the tip [S. plicata are fully developed. Endoreduplication in these tissues is unlikely since it would lead to further cell expansion. The pattern of DNA ploidy variation within the mature leaf is closely associated with the developmental status. The mechanism that resulted in endopolyploidy, however, remains unclear.Besides root tips and newly developing young leaves, endopolyploidy was observed in mature leaves in a few ultivars . Endopol the tip . Previou the tip ,27. The S. plicata, endopolyploidy was present in some floral tissues such as columns, growing flower stems and pedicels of both un-open and freshly open flowers. However, other floral tissues like sepals, petals and ovary tissues were found to have only 2C nuclei. In the growing un-open flower pedicels, the highest ploidy level even reached 16C. In cabbage, endopolyploidy was reported in cabbage flowers [Arabidopsis, endopolyploidy was found in hypocotyls, cotyledonary leaves, rosette leaves, stems of bolting plants and floral leaves, but was not found in inflorescences [Arabidopsis floral buds, and the small population of endopolyploid nuclei found in columns and pedicels of S. plicata in this study, the minute population of the endopolyploid nuclei could easily be neglected when the whole floral buds were used for sampling. In S. plicata, it was found that the average size of nuclei was larger in columns and pedicels that have a measurable amount of endopolyploid cells than in other flora tissues without endopolyploidy (unpublished results). Similarly, a correlation was found between cell size and ploidy levels during cabbage petal development [Dendrobium, the post-pollination physiological changes were found to be different between floral tissues such as columns, ovary tissues, sepals and petals [In  flowers , and det flowers . In cabb flowers . In the  flowers . This st flowers . In Arabescences . Given telopment . In Dendd petals . Edgar ad petals  suggesteArabidopsis hypocotyls [Drosophila melanogaster and some small genome plants like Arabidopsis, it is thought that endoreduplication is developmentally regulated [Further evidence was found in legumes where cell differentiation to a specialized function as pod wall tissues was accompanied by endoreduplication, and higher ploidy levels coincided with maximum pod growth . During pocotyls . In cabbegulated ,10,32.S. plicata raises the question of its possible implications. In tobacco, it was reported that the morphogenetic response of the tissues culture was related to the nuclear DNA content variation within stem explants of different ages [Oncidium Gower Ramsey, a hybrid orchid, only root tips, cut surfaces of stem segments and young leaves were able to form callus in tissue culture. Other explants such as old leaves and the roots without meristem tips could not form any callus [The systemic endopolyploidy revealed within different tissues of ent ages . In Onciy callus . Moleculy callus . In variy callus ,9,32. ThThe role of endoreduplication in plant development is still not well understood. The presence of endopolyploidy was proposed to be associated with several factors, such as taxonomic position of a species, life cycle, genome size, and organ type . Recent S. plicata as revealed in this study suggests that endopolyploidy and DNA content variation in explants might be a cause for somaclonal variation in tissue culture derived orchid plantlets. Thus, the pre-knowledge about the ploidy variation in different explant tissues is valuable for clonal propagation or for deliberate induction of variants in culture. Further systemic investigation of the relationship between somaclonal variation and type and endopolyploid level of source explants will provide indepth knowledge for micropropagation of orchids.This research may also have an impact on the orchid industry. Orchidaceae is the largest family of the flowering plants, and many of its members are commercially hybridized . Clonal Spathoglottis plicata from field-grown plants and in vitro cultures was developmentally regulated. Pattern of endopolyploidy is a character of the developmental and physiological properties of the tissue. This finding provides useful information for understanding of the plant development and for industrial propagation of orchids.Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata L. is a common tropical terrestrial orchid. The plants were grown in pots and placed in the greenhouse at 28 ± 4°C without artificial lighting. The following materials were taken for analysis: a) young leaves (1–3 cm in length), b) mature leaf (48 cm in length), c) root tips (2 cm including the tip), d) root segments from region with root hairs (2 cm away from the root tip), e) newly opened flowers, f) growing flower stems and g) developing floral pedicels of un-open flower (4–6 days before flowering).-1m-2 s-1). The following materials from in vitro cultures were examined: a) protocorms (6 weeks after germination), b) young leaves (1–2 cm in length) from seedlings, c) root tips (1 cm segments from tips), d) root segments with root hairs (at least 1 cm away from the root tips).Seedpods were surface-sterilized for 20 min in 20% (v/v) Clorox™ solution and subsequently rinsed 3 times with autoclaved water. Seeds were germinated aseptically in 9 cm diameter petri dishes containing 25 ml Knudson C orchid medium  with 2% (w/v) sucrose and 0.8% (w/v) agar. All cultures were incubated at 25 ± 2°C under a 16 h photoperiod  and extracted for 45 minutes. After filtering through a 45 μm Falcon cell strainer, 100 μl of propidium iodide (1 mg/ml) and 2.5 μl of 500 μg/ml DNase-free RNase  were added to each sample followed by 30 min incubation at 37°C.Extraction of nuclei and staining of DNA were performed according to the method of Arumuganathan and Earle ,45 with ® Elite ESP with 15 mW 488 nm Cyonics Argon air-cooled laser flow cytometer was used to measure the relative fluorescence of nuclei. For each sample, at least 10,000 nuclei were analyzed. Data were analyzed with WinMDI27B software (Joseph Trotter™).A Coulter EPICSC, DNA content of the unreplicated haploid chromosome complementPI, propidium iodidePMT4, photo multiplier tubeMY carried out experiments in the project. CSL and MY prepared the manuscript.


Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines , have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated.B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β.B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant  However, a variety of practical problems limits the use of biotechnological protein drugs. Stability of the active proteins, need for parenteral administration, and high costs of the final purified materials are among the most significant drawbacks. A way of circumventing these issues is represented by the direct administration of recombinant bacteria, acting simultaneously as cell factory and delivery system for pharmacologically active proteins. This approach has been already extensively experimented for the mucosal delivery of vaccine antigens ,2. In reversus-host disease, and multiple sclerosis  has received vast attention. IL-1 is a family of cytokines highly active in the modulation of immune amplification and inflammation. The IL-1 family includes two agonist proteins, IL-1α and IL-1β, and one antagonist protein, IL-1Ra. IL-1β is a very potent immunostimulatory and inflammatory cytokine, responsible for initiating and amplifying the host response to invasion. If not properly controlled, IL-1 can cause fever, acute inflammation, tissue destruction, organ failure, and eventually shock and death (reviewed in ). IL-1Raiewed in ,12,16). iewed in ,12,16,18iewed in ,12,19-21iewed in ,23. The in vivo cell factory, here we describe a novel system of local delivery of IL-1Ra, able to achieve systemic effects. The system exploits the ability of certain bacteria (such as Bacillus subtilis) to undergo autolysis in non-permissive conditions  thereby releasing the cytoplasmic proteins. Intra-colon instillation of B. subtilis expressing recombinant human mature IL-1Ra induces significant serum levels of the recombinant protein in rats and rabbits, and prevents the inflammatory effects of systemic IL-1. Intra-peritoneal administration of recombinant B. subtilis in the mouse could inhibit LPS-induced shock and death. Further experimental evidence with a B. subtilis strain producing human IL-1β demonstrates that this delivery system can be generalised to other recombinant proteins.Based on previous experience of using recombinant bacteria as B. subtilis to generate spores by autolysis of the cell wall, thereby releasing cytoplasmic proteins, was exploited as system for delivery of proteins in vivo. Following a sporulation signal  bacteria undergo an autolytic process with release of most of their cellular components and formation of a highly resistant spore containing DNA and few essential proteins  caused the release of large amounts of intact and active recombinant protein within a few hours after the sporulation signal  and Spo- bacteria, i.e. genetically modified cells which, in response to the sporulation signal, start the autolysis process but are unable to form a complete spore thus undergoing complete cell destruction /mg vs. 0.9 × 106 IU/mg of reference standard IL-1Ra TdR  and their proliferation was measured as radiolabel incorporation with a β-counter.The classical assay of co-stimulation of murine thymocyte proliferation was used to evaluate the bioactivity of IL-1 and IL-1Ra. Briefly, thymocytes from 5–8 week-old C3H/HeJ mice  were cultured at 6 × 10h 5% CO2  and a suThe biological activity of IL-1Ra was evaluated as inhibition of IL-1β-dependent thymocyte proliferation. To this end, cells were stimulated to proliferate (with IL-1β and PHA) in the presence of increasing concentrations of human recombinant IL-1Ra  or seriai.e. the dilution previously found to inhibit 50% of the activity of 10 ng/ml IL-1Ra in the thymocyte assay (not shown).To assess that the effect of IL-1Ra-containing serum was indeed due to IL-1Ra, a polyclonal rabbit antibody against human IL-1Ra  was addeB. subtilis suspensions (2 × 109 live cells/rabbit) were instilled in the distal colon 1 h before i.v. administration of 50–100 ng/ml highly purified LPS-free human recombinant IL-1β in pyrogen-free saline through the marginal ear vein. Temperature was recorded every 20 min for 3 h starting from IL-1β administration. In experiments with IL-1β-producing strain pSM261, rabbits received an intra-colonic administration of 1 × 109 pSM214 (control) or pSM261 (IL-1β) bacteria, or 250 μg purified human IL-1β. Temperature was recorded up to 22 h after treatment.Rabbits were lightly restrained in conventional stocks throughout the experiment, and accustomed to the stocks over a period of 2 h, to minimise variations in body temperature. Body temperature was measured by means of a cutaneous thermistor probe  placed between the left posterior paw and the abdomen and allowed to stabilise for 2 min. B. subtilis strains pSM214 and pSM539 were instilled in the distal colon (1 × 109 cells/kg), 2 h before administration of IL-1β. Blood samples were drawn 2, 4, 6, 8 and 24 h after intra-peritoneal inoculum of 0.1 μg/kg human recombinant IL-1β. The number of circulating neutrophils was assessed by flow cytometry. The plasma iron concentration was determined colorimetrically with a commercially available kit . Hypoferremia (60–75% decrease of plasma iron level) was evident from 4 to 24 h after IL-1β inoculum. The blood glucose concentration was measured in serum samples by the glucose/glucose oxidase/peroxidase method with commercially available kits  or by biosensor detection with devices for diagnostic monitoring . Overlapping results were obtained in rats and rabbits.Live cells of 6 bacteria/mouse), 24 h before i.p. administration of 15–20 mg/kg of LPS . LPS inoculum was delayed to 24 h after bacteria administration to avoid interference of pre-inoculum. In fact, preliminary experiments showed that intra-peritoneal inoculum of PBS decreased significantly LPS toxicity when administered at shorter times before LPS (data not shown). Mice were observed for 7 days after LPS administration and deaths recorded.LPS-sensitive C3H/HeOuJ mice received an intra-peritoneal inoculum of 0.5 ml PBS alone or containing bacterial suspensions .Results are presented as mean ± SEM. Statistical significance was assesed by two-tailed Student's IL, interleukin; IL-1, interleukin-1; IL-1Ra, interleukin-1 receptor antagonist; IBD, inflammatory bowel disease; LPS, bacterial lipopolysaccharide, AUC, area under the curve; PMN, polymorphonuclear leukocytes; GRAS, generally regarded as safe; GMO, genetically modified organisms.in vivo and pharmacokinetics studies in rats and rabbits, and performed the statistical analysis.SP carried out the PB designed and performed the bioactivity studies.PR designed and performed the microbiological and biochemical work.DB coordinated the bioactivity studies, organised the data, and wrote the manuscript.AT designed and coordinated the entire study.All authors read and approved the final manuscript.


We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4+ T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4+ and higher levels of activated T-cells have greatly reduced levels of FoxP3+CD4+CD25hi T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.A T-cell subset, defined as CD4 Human regulatory T-cells cells (Treg) -- or conventional T cells that have been genetically modified to generate Tregs -- express the HIV-coreceptor CCR5 and are susceptible to HIV infection There is now compelling evidence that a subset of T-cells with regulatory activity suppresses T-cell activation in both mice and humans . Regulat+ T-cells that also express the α subunit of the IL-2 receptor (CD25), which is currently the best marker for identifying these cells   stimulation and by their ability to suppress activation of conventional T-cells , 2002. Tse cells . Howeveress CD25 . In adulNFRSF18) . Upon acNFRSF18) . InteresNFRSF18) .FOXP3 gene carried by the scurfy mouse strain or a knockout of this gene causes a CD4+ T-cell-mediated lymphoproliferative disease characterized by cachexia and multiorgan lymphocytic infiltrates  express CD25 (+ T-cells within the memory subset (CD45RO+) express high levels of CD25 (CD25hi) (+CD25hi subset (+CD25hi (referred to as Treg), CD45RO+CD25low/neg (memory T) and CD45RO−CD25neg (naïve T) cells. Treg cells expressed higher levels of GITR and HLA-DR  A. Previoi subset . We firsd HLA-DR B, consisd HLA-DR . Treg ced HLA-DR B.low/neg memory CD4+ T-cells were labeled with CFSE and stimulated under suboptimal T-cell activation conditions in the presence of unlabeled purified autologous Treg, naïve, or memory T-cells. Coculture with Treg cells significantly slowed the proliferation of TCR-stimulated resting naïve and memory CD4+ T-cells as compared to the coculture with either unlabeled naïve or memory T-cells , or replication-defective viruses pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G.HIV) that encode green fluorescent protein (GFP) as a marker of infection . Treg anFoxP3 cDNA into a HIV-derived vector (HDV) that encodes murine CD24 (mCD24) as a marker  and adult blood (AB), activated through the TCR and transduced with FoxP3-expressing HDV (HDV.FoxP3) or control HDV as described previously .Treg cells constitute less than 1%–2% of total human T-cells see . Althouga marker . CD4+ T-eviously . Express+ cells as described previously  and 24 HIV-infected individuals . Total RNA was then isolated from each subset and FoxP3 mRNA expression was quantified using real-time PCR analysis. In order to normalize for experimental variability, FoxP3 expression of the CD4+CD25hi cells was normalized to GAPDH levels from the same samples and compared to the naïve T-cell subset isolated from the same donor. We found that within HIV-negative subjects there was on average a 49-fold higher level of expression of FoxP3 in CD4+CD25hi cells as compared to naïve T-cells (FoxP3 expression as compared to memory T-cells (data not shown). In HIV-positive subjects FoxP3 expression was only increased a mean of 25-fold in CD4+CD25hi cells. In contrast to healthy donors, we also observed that in about half of the HIV-positive subjects, CD4+CD25hi cells expressed very low to undetectable levels of FoxP3 . Because a portion of HIV-positive individuals contain high levels of activated T-cells, simple cell surface analysis would not be sufficiently reliable to quantify Treg cells in these donors. Therefore, we utilized FoxP3 expression as the most reliable marker that defines Treg cells. To accomplish this, we sorted CD4 T-cells A. The lo T-cells A. There of FoxP3 A. FoxP3 subjects A.+ T-cell percentages and increased levels of activated T-cells. We hypothesized that this hyperactivation may be due to a loss of Treg cells. Therefore, to further evaluate relationships between FoxP3 expression and these parameters in HIV-infected individuals, samples were divided into low FoxP3 expressors  versus high FoxP3 expressors . HIV-positive subjects with a FoxP3-low profile had significantly lower CD4+ T-cell percentages, while FoxP3-high HIV-positive subjects had CD4+ T-cell percentages comparable to HIV-seronegative subjects (+T-cells (CD4+HLA-DR+) than either HIV-positive subjects with FoxP3-high profiles or HIV-seronegative subjects . These findings demonstrate that a decrease in Treg cells is associated with HIV disease progression and suggest that loss of Treg cells may contribute to increased T-cell hyperactivation.Progressive HIV disease is associated with decreased CD4subjects B. Similasubjects C. Interepressors D. These pressors C. Among +CD25hi T-cells are greatly decreased in a portion of HIV-infected individuals with low CD4 and high activated T-cells, suggesting a loss of Treg cells. We therefore propose that infection and disruption of Treg cells during HIV infection could have a major influence on T-cell homeostasis and immune regulation.In this study we demonstrated that human Treg cells are highly susceptible to HIV infection and that ectopic expression of FoxP3 genetically reprograms conventional naïve T-cells, phenotypically and functionally, into Treg cells. Remarkably, overexpression of FoxP3 also greatly enhances the susceptibility of activated T-cells to HIV infection. Although Treg cells constitute a small subset of the total T-cells in humans (less than 1%–2%) and thus may not be a significant target population for HIV, they appear to have very potent suppressive activity against activation of T-cells . Here wehi subset of CD4+ T-cells. However, it is not clear if Treg cells are the only population represented in the CD25hi subset since they share this phenotype with recently activated T-cells. Indeed, a portion of purified CD25hi cells proliferated and their suppressive function was less efficient as compared to FoxP3-expressing cells (see + memory T-cells), it is conceivable that there is sizable contamination of non-Treg cells in these sorted preparations. In addition, the differences seen in the infection susceptibility of Tregs as compared to FoxP3-expressing cells may also be partly due to presence of non-Treg activated T-cells within the purified cells. Identification of the Treg subset in disease conditions with chronic T-cell activation, such as HIV, is even more problematic because a large proportion of CD4+CD25hi cells possibly represent recently activated T-cells. Availability of large numbers of genetically reprogrammed Treg cells should facilitate the identification of novel markers that can reliably detect human Treg cells.Similar to previous reports, our findings demonstrate that human Treg cells appear to be enriched within the CD25ells see . BecauseOur results clearly demonstrate that, similar to the mouse system, ectopic expression of FoxP3 is sufficient to recapitulate all of the characteristics of Treg cells, including lower cytokine secretion, higher expression of CD25 and GITR, and their suppressive functions. This system has allowed us to generate large numbers of Treg cells, which will be invaluable in characterizing their suppressive function as well as mechanisms of enhanced HIV susceptibility. The ability to genetically manipulate primary T-cells to reprogram them into the Treg phenotype also could have profound implications for preventing graft-versus-host disease, a serious clinical condition that can be manifested following hematopoietic cell transplantation .It is interesting to note that naïve T-cells are more prone to reprogramming into a Treg phenotype than memory T-cells are. This loss in flexibility of reprogramming with a master transcription factor is reminiscent of Th1- and Th2-type T-cell reprogramming with ectopic expression of lineage-specific transcription factors T-bet and GATA-3, respectively . The los+ cells and the reason for high T-cell activation or turnover during HIV infection remains controversial . Indeed, preactivated T-cells require reactivation to render them susceptible to infection after about 1–2 weeks in culture (unpublished results). We do not yet know the mechanisms by which FoxP3 renders activated T-cells more susceptible to infection; however, two possibilities can be considered: (1) FoxP3 may be overcoming innate resistance factor(s) that accumulate in activated T-cells that block HIV infection, or (2) FoxP3 expression may be inducing critical host factors that are required for efficient completion of the HIV life cycle in primary T-cells. It is important to note that since FoxP3 expression enhances VSV-G.HIV single-round infections, it likely affects an early, postentry step in viral replication. Determining the mechanisms by which FoxP3 enhances HIV infection could reveal host factors involved in this process.+CD25+ T-cells are greatly reduced in HIV patients with low CD4+ T-cell percentages and increased T-cell activation suggests that loss of Treg cells may contribute to HIV disease progression. Further prospective studies will be required to unravel the role of this important subset in HIV infection. The ability to genetically reprogram conventional human T-cells to generate Treg cells will also lead the way to identifying unique markers expressed on this population in order to further investigate their status in HIV-infected individuals. Moreover, understanding how FoxP3 enhances HIV infection and programs T-cells into the Treg subset could help in the identification of novel host factors that mediate HIV infection in primary T-cells and decoding the molecular mechanisms by which Treg cells mediate their suppressive function.In summary, our results indicate that both naturally occurring and genetically reprogrammed Treg cells are susceptible to HIV infection and that ectopic FoxP3 expression greatly increases the susceptibility of T-cells to HIV infection. Our finding that FoxP3-expressing CD4n = 11) were adults who were HIV-negative and with no history of chronic viral infections such as Hepatitis B or C. Blood samples from adults with HIV infection (n = 24) were obtained during routine primary care visits at the Comprehensive Care Center, Vanderbilt University Medical School, Nashville, Tennessee, United States. There were no selection criteria based on race or sex. All subjects provided written consent, and the study was approved by the Vanderbilt Institutional Review Board. Continuous variables were compared by a Mann–Whitney U test, and categorical variables by an χ2 test. All significance levels were based on two-tailed tests. Statistical analyses were performed using SPSS, version 12.0 .Healthy subjects , and CD4+CD45RO+CD25hi and CD4+CD45RO−CD25low/neg cells were sorted using flow cytometry . For some experiments, adult CD4+ T-cells were sorted into CD45RO+ (memory) and CD45RO− (naïve) T-cells with anti-CD45RO conjugated magnetic beads  using AutoMACS (Miltenyi Biotec). Purified resting T-cells were activated by cross-linking with plate-bound anti-CD3 antibody  and soluble anti-CD28 antibody . The plates were first coated with goat antimouse IgG  followed by either 3 μg/ml anti-CD3 for optimal TCR stimulation or 100 ng/ml anti-CD3 for suboptimal stimulation. The culture medium used in all experiments was RPMI  and was prepared as previously described  were separated from buffy coats of healthy and HIV-positive donors through Ficoll–Hypaque separation . Resting CD4escribed . This puescribed . All cytescribed . Superanescribed .nef gene as previously described (6 ifu/ml for replication-competent viruses and 10–30 × 106 for VSV-G.HIV. T-cells were infected at varying multiplicities of infection (MOI), and infection was quantified by GFP expression using flow cytometry. In some experiments, cells inoculated with virus were centrifuged for 1 h at 2,000 rpm to enhance infectivity, as described by VSV-G.HIV particles were generated as previously described . R5-HIV escribed . TypicalCell division was measured by labeling the T-cells with CFSE . Purified cells were first washed and resuspended in PBS. While vortexing the cells, CFSE was added at a final concentration of 5 μM. The mixture was vortexed for an additional 15 s and incubated at 37 °C for 3 min. Labeling was quenched by addition of 50% fetal calf serum in PBS. Cells were washed one more time with 50% serum PBS, followed by two washes with RPMI-supplemented medium. All CFSE labeling and culturing were performed under dark conditions.T-cells were stained with the relevant antibody on ice for 30 min in PBS buffer containing 2% fetal calf serum and 0.1% sodium azide. Cells were then washed twice, fixed with 1% paraformaldehyde, and analyzed with a FACSCalibur four-color cytometer. Live cells were gated based on forward- and side-scatter properties, and analysis was performed using FlowJo software . The following antihuman antibodies were used for staining: CD3, CD4, CD45RO, CD45RA, CD25, GITR, HLA-DR, CCR5, CCR4, CCR7, CXCR4, CXCR3, and antimouse CD24, all obtained from PharMingen . Cytokines  in the supernatants were assayed using a commercially available cytometric bead array (CBA) (BD Biosciences Pharmingen)  and analFoxP3 was PCR amplified from T-cell cDNA with the following primers: FoxP3 forward, 5′-AGATATCCCAGCCATGCCCAACCCCAGGCCTGGCAAG-3′; FoxP3 reverse, 5′-TCAGGGGCCAGGTGTAGGGTTGGAACACCT-3′. The forward primer included an EcoRV restriction site to facilitate cloning. The FoxP3 PCR product was subcloned into a TOPO shuttle vector . Following an EcoRV digest, FoxP3 was ligated into an HDV-encoding mCD24 down stream of an internal ribosome entry site . To synthesize cDNA 100 ng RNA was used . try site . The FoxGAPDH primer mix assay ID Hs99999905_m1; FoxP3 primer mix assay ID Hs00203958_m1.RNA was extracted, as described above, from cells transduced with HDV or HDV.FoxP3 or sorted Treg, naïve, and memory T-cells from HIV-positive and HIV-negative subjects. RNA (100 ng) was used to synthesize cDNA (as described above). Taqman Assays-on-Demand Gene Expression Primers  were used in real-time PCR analyses: FoxP3 or GAPDH primer mix. The reactions were amplified as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min and 65 °C for 1 min. Expression of FoxP3 was normalized to GAPDH expression in each sample.Real-time PCR was performed using the ABI 7700 apparatus . The reaction mixtures  contained 2 μl of serially diluted cDNA, 10 μl of Taqman Universal PCR Master Mix (PE Applied Biosystems), and 1 μl of either http://www.ncbi.nlm.nih.gov/) accession number for the gene FoxP3 discussed in this paper is AF277993.The GenBank (


A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle.We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules.We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle. Microtubules undergo striking reorganization during anaphase and telophase. During anaphase, antiparallel, interdigitating microtubules and many associated proteins become organized into discrete bundles in the spindle midzone , the regRecent progress suggested that, in addition to cytokinesis, the spindle midzone might be involved in additional functions. For example, midzone and midbody microtubules are associated with many regulatory proteins apparently unrelated to cortical contraction, such as the DNA replication initiator Orc6 , the inhTo address this possibility, we severed cells at anaphase or telophase by microsurgery, to bypass the normal mechanism of cytokinesis. The functional role of the spindle midzone or midbody in the following cell cycle was then tested by manipulating the position of the microsurgery or by applying pharmacological agents. Using extended time-lapse microscopy, we found that anaphase midzone microtubules play an important role in the progression of the subsequent interphase. However, there was no evidence of the involvement of telophase midbody in cell cycle progression.To investigate if anaphase spindle midzone affects interphase progression in daughter cells, we cut NRK52E cells at early anaphase, as soon as sister chromosomes have separated completely, with a fine glass fiber to form daughter cells with or without spindle midzone structures. Staining of severed daughter cells with antibodies against aurora B Figure , which iA total of eight manipulated cells were followed by extended time-lapse imaging through the following interphase. All the daughter cell with spindle midzone showed cytokinesis-like cortical contraction activities, while the daughter cell without spindle midzone showed no cortical contraction Figure . The dauThe inhibition of interphase progression was affected by the position of severing. When cells were severed near the equator to divide midzone structures between the two daughter cells, both cells showed cytokinesis-like cortical contractions and no effect on the progression of interphase. This observation was repeated with 4 cells. To test the possibility that the duration of interphase was affected by the size of the daughter cell, we took pairs of normally divided daughter cells and cut away a lateral region near the pole from one of the daughter cells. In all 5 cases, both daughter cells entered the subsequent mitosis with a timing similar to that of neighboring unmanipulated cells Figure . TogetheTo test if cortical contraction is the primary determining factor of the rate of interphase progression, we treated cells with cytochalasin D at early anaphase for 10 min then severed them at various positions in the spindle midzone, at a time when midbody started to form and ingression started to appear in control cells. Cytochalasin D was removed 20 min later to ensure that there was no lingering cytokinesis activity upon removal of the drug  see Meth, but remWe then asked if midzone microtubules, or proteins associated with the anaphase spindle midzone, are involved in interphase progression. Treatment of cells with nocodazole for 6–13 min at early anaphase before severing, with or without additional incubation with the drug, caused a significant delay  in the progression of interphase for both daughter cells, as compared to neighboring cells  during anaphase . DegradaOur results suggest that anaphase midzone not only play a role in the stimulation of cytokinesis in cultured cells, but also provide a scaffold for the activation/deactivation of factors essential for the progression of subsequent cell cycle.Normal Rat Kidney epithelial cells  were cultured in Kaighn's modified F12 (F12K) medium supplemented with 10% FBS , 50 U/ml penicillin, and 50 μg/ml streptomycin, on glass chamber dishes as previously described . The celGlass needles for microsurgery were prepared with a David-Kopf Model 700 vertical puller. The tip of the needle was melted and elongated into a fine fiber with a Narishige microforge (Model MF900). Microsurgery of the cells was achieved by carefully lowering a fiber onto the target cell followed by slow dragging with a micromanipulator .Cytochalasin D  were stored at -20°C as 2.5 mM stock in DMSO, and diluted into warm medium before application to cells. We found that treatment of early anaphase cells with cytochalasin D for 30 min completely inhibited cytokinesis even upon removal of the drug, similar to what was reported with dihydrocytochalasin B . Thus, eAurora B-GFP was constructed and transfected into NRK cells as described previously . ImmunofMMH carried out all the experimental work and drafted the manuscript. GS and YLW participated in its design and coordination of the research and edited the manuscript. All authors read and approved the final manuscript.Interphase progression of the daughter cells with and without spindle midzone. An NRK cell was severed at early anaphase to form daughter cells with and without spindle midzone. Subsequently, time-lapse imaging was performed to investigate if anaphase spindle midzone was involved in interphase progression. The daughter cell with spindle midzone showed a cytokinsis-like cortical contraction and entered into the subsequent mitosis 11 h 23 min after microsurgery, while the daughter cell without spindle midzone showed no cortical contraction and entered into mitosis 23 h 8 min after microsurgery.Click here for fileInterphase progression of the daughter cells with and without midbody. An NRK cell was severed at telophase to form daughter cells with and without midbody. Subsequently, time-lapse imaging was performed to investigate if midbody was involved in interphase progression. The daughter cell with midbody entered into the subsequent mitosis 7 h 35 min after microsurgery, while the daughter cell without midbody entered into mitosis 9 h 25 min after microsurgery.Click here for file


Many three-dimensional (3D) images are routinely collected in biomedical research and a number of digital atlases with associated anatomical and other information have been published. A number of tools are available for viewing this data ranging from commercial visualization packages to freely available, typically system architecture dependent, solutions. Here we discuss an atlas viewer implemented to run on any workstation using the architecture neutral Java programming language.We report the development of a freely available Java based viewer for 3D image data, descibe the structure and functionality of the viewer and how automated tools can be developed to manage the Java Native Interface code. The viewer allows arbitrary re-sectioning of the data and interactive browsing through the volume. With appropriately formatted data, for example as provided for the Electronic Atlas of the Developing Human Brain, a 3D surface view and anatomical browsing is available. The interface is developed in Java with Java3D providing the 3D rendering. For efficiency the image data is manipulated using the Woolz image-processing library provided as a dynamically linked module for each machine architecture.We conclude that Java provides an appropriate environment for efficient development of these tools and techniques exist to allow computationally efficient image-processing libraries to be integrated relatively easily. Three-dimensional 3D) images are now commonplace in biomedical research. Techniques for direct capture of 3D data are widespread and new techniques are becoming available, ,2 to comD images For the expert viewer, digital re-sectioning is sufficient for data-analysis but for others, panning through the volume at non-standard angles leads to disorientation. In addition if used in conjunction with atlas information in which the histology images are segmented in terms of the recognisable tissues, the building of a 3D view of the tissue/anatomical components is very difficult, particularly when learning the anatomy. This orientation and structural visualisation problem is solved by using 3D visualisation of the underlying tissue coupled with interactive feedback of the section location within the volume.The basic structure of the JAtlasView is therefore a combination of a 3D feedback window with a number of section views. Each section view is independent and feedback is provided by displaying the position of the section within the 3D volume either as a simple polygon indicating the plane of section or as a full grey-level image, displayed appropriately in 3D. In addition each section will display the intersection with all other sections currently being viewed.In this short note we describe the structure of the software and the functionality of the interface. This application is directed to the use of the EADHB and EMAP atlases and for browsing 3D grey-level data. In the first instance the data is formatted as a Woolz image structure , tools aThe software design has been developed to meet a number of code requirements:• portability to all major architectures – Unix/X11, Microsoft Windows and Macintosh,• fast and efficient image processing, compatible with existing formats and interfaces,• freely available code and modular design so display elements and functionality can be easily included in other applications and• the user-interface should be mappable to the "look and feel" of the specific machine window system.The portability and user-interface behaviour requirements are satisfied by using Java as the language and environment for the user interface level. For image processing we have adopted the ANSI standard C image processing library Woolz. This already includes the required functionality for calculating and manipulating section views through 3D voxel images and is open-source software.A potential problem with Java is that it can be very inefficient for heavy numerical work (such as image processing) and the effort required to port existing libraries (for example Woolz is 185 K lines of code) to Java is too high. To solve this we use the tools within Java for accessing "native" code so that the computational work is undertaken in C. The management and coding of the interface is potentially time-consuming and prone to error with any small change in the C code requiring complementary effort to modify the native interface code. We have addressed this problem by implementing an automated method which will build the interface directly from the C-library header files. By adopting a standard convention for function prototyping it is possible to use a parser generator, javacc to buildTwo other key choices have been made in the design of the code structure. The first is that the 3D visualisation and feedback should be developed at a level independent of the underlying hardware within an environment that allows a high level of abstraction of the 3D view. The java 3D extension to the core Java environment provides such a model and we have adopted this as standard. Java 3D is available for all Java 2 platforms.CVS[gmake for compilation. The interfaces are developed using Borland JBuilder[The second key choice is that the software will be delivered using Java Webstart. This isCVS for vers JBuilder or a sta JBuilder.Help is provided in two ways, the first is a simple popup "balloon" help on mouse-over and as a series of help files arranged using JavaHelp which prmodel-view-controller (MVC) design pattern [java-bean, that components should meet to guarantee the MVC behaviour and enable easy re-use in other applications using CASE tools. We have adopted this standard for the JAtlasViewer application so that individual interface elements, e.g. the section panel or even our extended view of the slider, can be used simply and conveniently in other code.Java is now widely used and the first choice for new applications that require portability across machine architectures. It is a strict object-oriented language and interfaces adhere to the  pattern . Java alThe user interface, shown in figure With appropriate data, the JAtlasViewer will import a mapped "anatomy". This is in two parts, a hierarchy of terms and a set of "domains" linked to specific terms in the hierarchy. The domains are 3D binary images which identify the region of space or set of voxels within the grey-level image associated with that term. The anatomy will then be used to provide feedback within the section views. These anatomy options, the controls for the section views and the main window options are discussed below.When the application is invoked a top-level dialog is presented to the user. Before anything can be displayed the user must select a grey-level 3D image. Currently this must be formatted as a Woolz image, but converters for many 3D formats are available from the EMAP web site. Once read in, the bounding box of the 3D voxel image will be displayed in the main window along with a surface representation of the data if available. This 3D view can be manipulated interactively using the cursor to provide views from arbitrary orientations and positions.The menu options of the primary window are:Commands to open image data, save views, save and restore settings, recent file-list and quit.Select a section view through the voxel model. A new window will display one of the pre-set sections which are transverse, frontal and sagittal planes if the image model is appropriately aligned. Each of these can be set to display views at arbitrary orientations and locations within the 3D volume image.If the voxel model data is configured with a set of anatomical regions, these can be selected from the menu and displayed in the section views. For the EADHB and EMAP atlases the menu hierarchy corresponds to the HUMAT and EMAP anatomy ontologies.Options to control the 3D visualisation in the main window, toggle the visibility of 3D surface, bounding box and intersection lines, display the focus section and selected anatomy.Preset 3D orientations to provide standard viewing directions.On-line help menu.The 3D view window displays the bounding box of the opened volume and a transparent view of the embryo surface. This surface is pre-determined and stored in the visualization toolkit (VTK) format.If an anatomy hierarchy and associated data files are provided then an additional window will allow browsing through the ontology and selection of components for display both in the section views and the 3D view window. As for the embryo surface, the surface models are pre-calculated and stored in VTK format. The data layout recognized by the JAtlasViewer is described in detail on the EMAP web-site.pitch, yaw and roll respectively. These viewing controls are hidden by default.Each Section View is displayed in its own Section Viewer, either inside the main window (Microsoft Windows style) or in an independent external window. Section Viewers are Java components that can be easily imported into other applications. The primary viewing control is to move the view plane-parallel through the image volume as a form of "digital microtome" with section thickness determined by the underlying resolution of the 3D image, i.e. moving the microtome by a single step will move to the next voxel in the stack. The assumption is that once the section orientation has been determined the typical use will be to explore the volume in this fashion. The section position is determined by the "distance" parameter which is the voxel distance from the fixed point (by default in the centre of the bounding-box). Section orientation is selected by setting a number of view-angles. These control the view-direction which is perpendicular to the view-plane. We use the standard viewing angles defined by  which arView-mode: options for automatic roll determination in terms of the pitch and yaw values.In addition to the primary view-direction controls there are options to assist navigation. These are Fixed-point: select the fixed point used as the centre of rotation. The effect of setting this is to keep that voxel in view for all view-directions provided the "distance" is zero.Fixed-line: set a second fixed-point and constrain the view so that both fixed points remain in the section. The effect of this is to reduce the degrees of freedom to a single parameter of rotation around the line between the two points.The remaining controls for each section view are to set the feedback options including between section views, between the section view and the 3D view and to allow saving of the view and its settings (view parameters). The within view and between views feedback options are provided by the "Show" menu. This provides toggle controls to enable:• cursor position in the reference image coordinate space and image grey-value to be displayed,• line of intersection with section views. If two views intersect then the line of intersection is displayed in the appropriate colour,• anatomy feedback – shows the domain and name of the anatomical component under the current cursor position,• visible fixed point,• visible fixed line.The 3D view can provide feedback of the viewed sections in terms of the 3D volume. For most users these are important aids to understanding the position and direction of the viewed section. Most publications adhere to a convention for displaying section images, with this interface it is possible to view section data at any orientation and direction, i.e. depending on the view-direction the section may appear "reversed", so positional and directional feedback is critical. The positional feedback is provided in a number of forms but all indicate the intersection of the viewed plane with the bounding-box of the reference image. The most informative choice is to use texture mapping to render the grey-level image of the section into the 3D view. This is computationally expensive and so two other options are provided. These use the intersection polygon between the section plane and bounding box, either as-is, or filled with solid colour. The directional feedback is optional and provided by an arrow displayed in the 3D view.atlases. These comprise a grey-level  reference image and a set of domains or regions which are associated with terms in a text hierarchy. For a geographic atlas these would correspond to the physical geography and the areas associated with individual countries. The hierarchy would then list the country names, perhaps under continents and split into counties. For EADHB and EMAP the reference image is the voxel reconstruction of the embryo and the domains are delineated anatomical components. The hierarchy of terms are the corresponding anatomy ontologies [Anatomy Manager  and the final part of the name is capitalised. An asterisk following a name indicates that this is an atomic component, referred to in the anatomy menu as a (domain). Anatomical components are selected from the anatomy menu using a left mouse click. Higher level components or structures may also be selected from the anatomy menu using a right mouse click or a combination of the shift key and (left) mouse click. The anatomy name may be scrolled by dragging the mouse left or right inside the text box.The delete is a toggle control which removes an anatomy component from the table.The visibility toggles select whether a component is displayed in the section views and in the 3D feedback window. This fine control helps the process of analysis and allows the user to build up a visualization showing all or parts of the anatomy. The 3D images are in widespread use in medical and biological research and there are a large number of options to view this data, but many of these are commercial and expensive, and are architecture and operating system dependent. More recently atlases and spatially mapped databases in biomedicine have been developed and whilst these packages can provide solutions for browsing this data we believe a simple, free-to-use, open-source and architecture-neutral solution provides a useful tool for biological research and teaching. The JAtlasViewer is intended to fill this requirement. The viewer provides the browsing functionality to locate and display arbitrary sections through the data with simultaneous 3D display. The JAtlasVIewer can also read and display a full anatomy atlas.The JAtlasViewer is programmed in Java. The 3D programming technology is Java3D, which is a wrapper to the OpenGL or DirectX libraries. The Java and Java3D runtime environment are freely available from the Sun Microsystems web site and in most systems Java is pre-installed. These techniques minimize the coding work and developing time. The file size of the JAtlasViewer is less than 1.5 MB. Java WebStart manages the deployment, installation, upgrade and launch via a simple click on a html page link or an icon in the WebStart application. It is portable to any operating system to which Java has been ported and is currently available for Windows, Linux, Solaris and Mac OS.The JAtlasViewer design is of reusable and extensible components. Based on the viewer a 3D tie-point collector for capturing 3D to 3D correspondences, and an atlas viewer that can also import gene expression data, have been developed.• Project name: The Mouse Atlas Project• Project home page: • Application download: • Operating system(s): Solaris, Linux, Mac OSX, MS Windows.• Programming language: Java, ANSI C.• Other requirements: Java 1.4, JavaDoc, Java 3D.• License: GNU GPL• Any restrictions to use by non-academics: NoneAuthors GF and NB undertook the main Java development and implementation, BH and RB develop and maintain the Woolz image processing library and BH implemented the automatic generation of the JNI. DD, JK, MS and SL all contributed to the design and testing of the interface and the preparation of the Atlas data for use with the tool.