The Microarray Gene Expression Data Society believe that the time is right for journals to require that microarray data be deposited in public repositories, as a condition for publication While some journals have already made this a condition of acceptance, we feel that submission requirements should be applied consistently and that journals should recognize ArrayExpress , European Bioinformatics Institute (EBI), and National Center for Biotechnology Information (NCBI). Similarly, we, members of the Microarray Gene Expression Data Society  as a means of achieving this goal. (2) Scientific journals should require that all primary microarray data are submitted to one of the public repositories—ArrayExpress, GEO, or CIBEX—in a format that complies with the MIAME guidelines. (3) Public databases should work with authors and scientific journals to establish data submission and release protocols to assure compliance with MIAME guidelines. (4) To assist with the review process, the databases should continue to work in collaboration with publishers to provide qualified referees with secure means of accessing prepublication data. Authors should be strongly encouraged to submit data to the databases during review.To this end, the members of MGED propose the following as a new paradigm for the publication of microarray-based studies. (1) Authors should continue to take primary responsibility for ensuring that all data collected and analyzed in their experiments adhere to the “Minimum Information about a Microarray Experiment” (MIAME) guidelines and should continue to use the MIAME checklist , ArrayExpress (www.ebi.ac.uk/arrayexpress), and CIBEX (cibex.nig.ac.jp), as well as a growing number of commercial and academic software packages capable of writing MAGE-ML documents  These established repositories have a commitment to continued community service and to providing some level of assurance that published gene expression datasets will continue to be available into the future. (2) Having the data available in these public repositories in a standardized format will not only make them more accessible, but it will allow expression data to be integrated with other relevant data, including the available genome sequences, single nucleotide polymorphism and haplotype mapping information, the literature, and other resources that can aid in further interpretation of expression patterns. Although many authors now provide some or all of this information, the established databases are much more likely to assure that these links are maintained and current. (3) Curation of data submitted to public data repositories will assist authors, reviewers, and publishers in assuring that the data comply with the MIAME requirements, further enhancing their utility. (4) The standardization of microarray data formats will enable the development of additional data analysis and integration tools and makes it easier for scientists to access, query, and share data. (5) Finally, submission prior to publication will make it easier for referees to access the data confidentially, facilitating the review and publication process.In the same way that availability of sequence data had a profound impact on a wide range of disciplines, we believe that requiring that microarray data be deposited in public repositories as a necessity for publication will accelerate the rate of scientific discovery.What this proposal requires is a change in the way in which we approach the publication of microarray-based studies. Both authors and journals have a responsibility to assure that the requisite data are available, and because submitting MIAME-compliant data can take considerable time and effort, this process should be factored into review and publication timelines. However, while this process may be time consuming and painful at first, we believe that the benefits of building an open repository of microarray data will far outweigh any initial disadvantages. As always, it is our sincere hope that these suggestions stimulate discussion within the community and that together we can arrive at a consensus that ensures that microarray data are widely and easily accessible. Finally we would like to urge the DDBJ, EBI, and NCBI to work together towards exchanging all MIAME-compliant microarray data.


Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes.In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells.RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors. Many recent studies have described the use of microarrays to identify genes expressed in different types of cancers . Our laIn this study, we define a model system for using poly (A) binding protein (PABP) to recover mRNAs from specific cell-types in mixed cell cultures. Using this approach, we were able to determine how the gene expression profiles of endothelial cells change in response to the presence of breast cancer cells. Among the advantages of this approach are: a) no manipulations or treatments are required prior to the preparation of cell extracts, b) the recovered mRNA population can be identified directly using genomic methods, and c) RNA binding proteins can be engineered for expression in different cell types using various molecular tags in order to discriminate cell-specific mRNA populations. These studies provide a methodological basis for creating mouse models in which different types of cells within a tumor express RNA binding proteins to reveal unique populations of posttranscriptionally regulated mRNAs.The goals of these experiments are to validate procedures for the isolation and characterization of discrete mRNA sub-populations associated with RNA binding proteins expressed in specific cell types within a tumor or organ in order to assess the responses of cells to their surroundings. Earlier studies have shown that mRNA subpopulations in single cell types reflect the functions of the RNA binding proteins with which they associate and can provide key information about post-transcriptional regulatory mechanisms of gene expression ,13,15-18Microarray analysis was used to compare the gene expression profiles obtained using total RNA and PABP-associated RNA of PY 4.1 endothelial cells  with proteins (proteome) in the same cell population ,29. The It is possible that gene expression profiles obtained by immunoprecipitating mRNA-PABP complexes may reflect the functional state of protein production from these mRNAs . For theIn order to compare mRNA profiles from mixed cell populations, we prepared two different cell lines stably expressing different epitope tagged PABPs. Figure A potential complication for this type of analysis is that expression of a tagged-RNA binding protein, in this case PABP, could affect cell growth. While these cell lines appeared unaffected morphologically, the levels of PABP in cell lines expressing tagged-PABP and respective control cell lines were evaluated and compared by Western blotting. No substantial change in overall PABP expression was observed when expressing exogenous tagged PABP Figure . This reNo changes in cell growth or mortality of the cell lines used in this study or in other cells lines expressing tagged-PABP were observed. Moreover, the cell cycle kinetics of T98G cells expressing Flag-PABP and cells that were subsequently stimulated by serum addition were compared to those of T98G cells containing the empty vector, pCMVneo and no substantial differences were observed using fluorescent cell sorting Figure . We concEarly studies of PABP binding to mRNA indicated that a dynamic exchange or hopping of PABP from mRNA to mRNA might be an important aspect of its function ,34. For We have also addressed the potential problem of having a pool of free PABP in an extract that could be available to bind mRNA during incubation. Sucrose gradient analysis indicated that this is very unlikely since the majority of PABP was found in heavy polysomes and associated with mRNA, while only a small percentage was found in the upper portion of the sucrose gradients . In total, these results demonstrate that reassortment of PABP in these cell extracts was not a significant limitation to using PABP RNPs for gene expression profiling of bound mRNAs.As noted above, it has been suggested that yeast PABP uses a "hopping" mechanism in vivo by moving from RNA to RNA ,34. Whilmouse angiogenesis (mAngio) and human tumor suppressor (hTS1) RPA probe sets after they were tested for cross species hybridization. Both probe sets showed good specificity of discrimination with the exception of the L32 and GAPDH control genes as expected . The mouse RNA on human Atlas arrays did not show any detectable cross hybridization signal, while the human RNA on mouse Atlas arrays showed a small percent (2–3%) of cross hybridizing mRNAs  were enriched at least 4 fold in the G10 PABP population when compared to the Flag PABP population. For the human genes, 82.4 % (122 out if 148) were enriched at least 4 fold in the Flag PABP population in relation to the G10 PABP population  often precede downstream alterations in the expression of other important genes. Among the RNA binding proteins identified in our screen were stem-loop binding protein, a highly conserved RNA binding protein that binds a stem loop structure in the 3'UTR of histone mRNAs and is required for both processing and translation of histone messages [Drosophila bruno, a translation repressor which functions at the early steps of embryogenesis [As expected, the great majority of the genes expressed in PY4.1 cells were not altered in their levels of expression while in co-culture. Of interest, a small number of genes were consistently upregulated in four independent experiments. To assess the consistency of the fold change, we plotted the p-value (from modified t-test) against the average fold change Figure . A 'volcmessages , Brul4, ogenesis , and quaogenesis . It shouThe presence of several RNA binding proteins among the top-20 genes affected by co-cultivation may result in downstream effects on gene expression, and we plan to examine the target mRNAs of these RNA binding proteins in endothelial cells. This should help elucidate additional post-transcriptional pathways and networks regulating cell growth mechanisms and tumorigenesis .This study describes changes in the gene expression profile of an endothelial cell when co-cultivated with a tumor cell by isolating ribonucleoprotein complexes and identifying their associated mRNAs using genomic arrays. Moreover, it presents a model system that can be used to elucidate post-transcriptional operons in specific types of cells by using various RNA binding proteins from mixed cell cultures as a novel approach to understanding how cell-cell communication affects gene expression during tumorigenesis and organogenesis.Murine endothelial PY4.1 cells were kindly provided by Dr. Christopher Kontos, Duke University Medical Center. Human gliobastoma T98G and murine breast cancer 4T1 cells were obtained from American Type Culture Collection. All cell lines were maintained in DMEM Medium (Gibco) supplemented with 10% Fetal Bovine serum.The ORF of human PABP I containing the Flag tag (GACTACAAGGACGACGATGACAAG) or the G10 tag (CCACCATGGCT AGCATGACTGGTGGACAGCAAATGGGT) at the 5' end was cloned into the pCMV-Neo retroviral vector . Stable Monoclonal anti-G10 antibodies were obtained as previously described . AntibodProtein extracts were prepared from T98G and PY4.1 cell lines by homogenization in polysomal lysis buffer . 50 μg oAnalysis of the cell cycle of T98G cells was performed as described .Cell lysates and immunoprecipitation of mRNP complexes were essentially performed as described . PolyadeTotal or immunoprecipitated RNAs were assayed by RNase protection by using the PharMingen Riboquant assay according to the manufacturer's recommendations (45014K). mAngio-1 (mouse angiogenesis) and hTS1 (human tumor suppressor) template sets were used . Protected riboprobe fragments were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and analyzed by using Molecular Dynamics IMAGE QUANT software (version 5.0).32P α-dATP (Amersham Biosciences). After hybridization, the array membrane was washed and the results were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and stored as .gel files. Images were analyzed by using ATLASIMAGE 2.01 software (CLONTECH). Global normalization was used when arrays being compared had approximately the same number of positive hits.cDNA array analysis was performed by using Atlas Mouse and Human 1.2 Arrays (CLONTECH). Probing of cDNA arrays was performed as described in the CLONTECH Atlas cDNA Expression Arrays User Manual (PT3140-1). Reverse-transcribed probes were radiolabeled with .Printed oligo arrays using the Operon Mouse Oligo set version 2.0  were produced by the Duke Microarray Core Facility. Protocols used for preparation of slides, labeling, amplification, hybridization and scanning are described in GenePix data were normalized with pin-tip specific lowess normalization . DiffereELAV – embryonic lethal abnormal visionRPA – RNase Protection AssayPABP – Poly A binding proteinLOFP was responsible for the experimental design and performed Western blots, immunoprecipitations, RPAs and microarray experiments. MDB helped perform RPAs and microarray experiments. SML performed statistic analysis of microarray data and prepared the webpage with supplementary data. HS generated the stable cell lines used in this study and performed the cell cycle experiment. JDK conceived the project and assisted in experimental design.


Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis,  The study of hair follicle morphogenesis provides insights into how cells within a sheet can be triggered to proliferate, remodel, and form buds - a recurring theme in development Mammalian development involves the morphogenesis of complex three-dimensional structures from seemingly uniform sheets or masses of cells. A simple bud-like structure initiates the formation of many organs, including lungs, spinal cord, mammary glands, and hair follicles . The mulAt the helm of these molecular communication pathways are Wnts, bone morphogenic proteins (BMPs), transforming growth factor βs (TGF-βs), and fibroblast growth factors (FGFs). Through activation of cell surface transmembrane receptors, these external signaling molecules trigger distinct cascades of intracellular events that culminate in changes in gene expression, growth, and differentiation . How thiThe development of a bud requires a number of coordinated changes in the behavior of the targeted cells within an epithelial sheet. The process must be accompanied by alterations in the proliferation, polarity, shape, and adhesiveness of selected cells, as well as by modifications in their underlying basal lamina. Thus, extracellular epithelial-mesenchymal crosstalk must be intricately orchestrated to couple the determination of distinct cell fates with the contemporaneous remodeling of the physical and structural properties of the cell.Among the few dispensable organs, hair follicles offer an excellent model system to study epithelial bud formation. Mammalian skin epithelium begins as a single sheet of multipotent ectodermal cells. During development, specialized mesenchymal cells populate the skin in a spatially defined pattern to initiate the complex epithelial-mesenchymal crosstalk that will specify the bud . Once coPreviously, we delineated how two respective epithelial and mesenchymal signals, Wnts and the BMP-inhibitory factor noggin, function in concert to induce lymphoid enhancer factor-1/β-catenin (LEF-1/β-catenin)-mediated gene transcription within the follicle placode . The dowE-cadherin promoter activity, we were intrigued by the close proximity of the LEF-1/β-catenin binding site to a site known to bind the Snail/Slug family of zinc finger transcriptional repressor proteins [E-cadherin promoter activity [As we probed more deeply into the underlying mechanisms governing proteins ,13,14,15proteins ,17. Bud Snail gene expression in embryonic skin keratinocytes. In contrast, we provide in vitro, transgenic (Tg), and gene targeting evidence to show that TGF-β2 and small phenotype– and mothers against decapentaplegic–related protein 2 (SMAD2) signaling are upstream inducers of Snail gene expression in skin epithelium. In the absence of TGF-β2 signaling and Snail gene expression, hair placodes can form, but further follicle down-growth is blocked. Our studies point to the view that Snail likely functions downstream of cell fate specification, at a stage where the bud begins to exhibit enhanced proliferation and migration.In the present study, we discovered that Snail is expressed briefly at an early stage of hair bud formation, when E-cadherin down-regulation and activation of proliferation take place. Thereafter, Snail disappears and remains absent during subsequent follicle down-growth and maturation. This exquisite pattern appears to be functionally relevant since altering it in vivo correspondingly affects features associated with hair bud formation, including down-regulation of E-cadherin, increased proliferation, and repressed terminal differentiation. Although the temporal spike of Snail in the hair bud is reflected at the mRNA level and seems to follow Wnt signaling and BMP inhibition, LEF-1/β-catenin activation does not appear to induce E-cadherin expression in keratinocytes [Although Snail family members are most frequently associated with EMTs, they also participate in many malignant processes involving a down-regulation but not a quantitative abrogation of intercellular junctions . The raninocytes , coupledinocytes ,19, we tSnail mRNA expression during a period of skin embryogenesis when waves of hair follicles are forming (unpublished data).To pinpoint specifically where Snail mRNA is expressed in the developing skin, we conducted in situ hybridization using a cRNA probe unique to the Snail 3′ untranslated region (UTR). Embryonic day 17.5 (E17.5) was chosen, since the multiple waves of follicle morphogenesis occurring at this time enabled us to evaluate Snail expression at different stages of the process. As shown in Snail mRNA expression during follicle development was most apparent in hybridized skin sections containing follicles from two different waves of morphogenesis -tagged Snail protein . In this respect, the skin epithelium resembled that of the hair bud, where the down-regulation in junction proteins is permissive for cell-cell remodeling without abrogating intercellular adhesion.Snail Tg epidermis and hair buds extended to the hyperproliferative state, leading us to wonder whether the down-regulation of AJ proteins might contribute to this condition. Given the increase in pMAPK staining in Snail Tg epidermis  family of DNA binding proteins ,24,25. Tcription . This sacription  gave no Snail Tg epidermis. In addition to its documented ability to bind α-catenin [Snail Tg mice but not from the corresponding wild-type (WT) animals  may be regulating the Snail gene. A variety of extracellular signals have an impact on the cell type-specific expression of different Snail family members, and many of them, including Wnts, BMPs, FGFs, and TGF-βs, also affect hair bud development [The temporal spike of elopment ,16,28. SE-cadherin promoter and induced a LEF-1/β-catenin-sensitive reporter gene, TOPFLASH [Snail expression in keratinocytes.Previously, we showed that effective transmission of a Wnt-3a signal in cultured keratinocytes can be achieved through their exposure to the BMP inhibitor noggin, which induces LEF-1 expression . In vitrTOPFLASH . In contTOPFLASH ,29,30, oTGF-β genes, only the TGF-β2 null state blocked follicle development at the hair bud stage [Snail expression in keratinocytes isolated from the basal layer of the epidermis. Though there is no cell culture system available to specifically study placodal cells, these keratinocytes are their progenitors and are the closest approximation available to study the behavior of epithelial cells of the placode.TGF-β1 has been shown to induce Snail family members in hepatocytes and heart , 31. In ud stage . Thus, wSnail expression could not be maintained (Snail expression correlated with phosphorylated SMAD2 (pSMAD2) induction, its decline seemed to rely on secondary downstream events.Interestingly, treatment of cultured keratinocytes with as little as 5 ng/ml of TGF-β2 caused a rapid and transient induction of Snail B. Followintained B. Thus, Snail promoter activity in keratinocytes might be sensitive to TGF-β2 signaling  and then assayed for transgene activity over the same time course in which we had observed Snail protein induction. The results of this experiment are presented in The rapid kinetics of Snail expression were reflected at the mRNA level, suggesting that ignaling C. To tesSnail promoter activity had increased 3-fold, and by 2 h, it peaked to approximately 10-fold over control levels  family [Recent studies on hair bud morphogenesis suggest that Wnt signals likely from the epithelium and BMP inhibitory signals from the underlying mesenchyme converge to produce an active transcription factor complex involving β-catenin and LEF-1, which in turn plays a key role in specifying the hair follicle fate ,30,36,37e absent ,38. Thes) family ,40. FurtTGF-β1-null skin, which exhibits an extended growing phase of postnatal hair follicles, TGF-β2-null skin displays an embryonic block in follicle bud progression [TGF-β2 null buds appear unable to progress to the down-growth phase, a feature that cannot be explained readily on the basis of previously established effects of TGF-βs.TGF-βs are known to promote withdrawal of keratinocytes from the cell cycle . Hence, gression . Althouggression , about 5Our finding that TGF-β2 is upstream from Ki67 expression and MAPK activation lends further support to the notion that hair follicle keratinocytes at this early stage of development react to TGF-β2 signaling in a fashion opposite to that typically expected for TGF-β factors. This said, based upon pSMAD2 immunohistochemistry, the immediate steps of downstream signaling appeared to be intact. Thus, we surmise that the proliferative outcome is likely to be rooted in differences in the repertoire of activated SMAD target genes. In this regard, the positive effects of TGF-β2 on proliferation within the hair bud may be more analogous to what has been seen in progression of squamous cell carcinoma to metastatic carcinoma  rather tSnail gene as a potential target of TGF-β signaling [Snail gene expression that occurs in the developing hair bud. The additional correlation between epithelial hyperproliferation and Snail transgene expression further fostered our interest in a possible link between TGF-β2 and Snail. Our functional studies demonstrate that without TGF-β2, Snail expression is abolished in the mutant hair buds, and conversely, in K14-Smad2 skin, Snail is ectopically activated. Moreover, our in vitro studies indicate that even sustained TGF-β2 exposure may cause only a transient induction of Snail, offering a possible explanation as to why Snail is so briefly expressed during hair follicle morphogenesis. An additional point worth mentioning is that prolonged expression of Tg Snail in postnatal skin resulted in morphological and biochemical signs of epithelial to mesenchymal transitions (unpublished data), underscoring why transient Snail expression may be so important during normal hair follicle morphogenesis [The prior identification of the ignaling  was introgenesis .Snail Tg mice seemed indicative of a defect in follicle formation  cell lines, Snail has been shown to block cell cycle progression and promote motility and shape changes for invasion . While oIrrespective of these differences, our in vivo studies do not stand alone, as there are many situations in which a down-regulation in AJ proteins correlate with enhanced proliferation. In fact, a myriad of diverse mechanisms have been implicated in activating epithelial proliferation upon down-regulation of AJ proteins ,23,24,48Snail fused to GST . Recombinant human TGF-β2 was purchased from R&D . Heat inactivated TGF-β2 was generated by heating the recombinant protein at 100 °C for 10 min.Primary antibodies used were against: E-cadherin ; α-catenin, β-catenin, pMAPK, tubulin , Ajuba ; β4 integrin/CD104 , laminin 5 , K5, K1, loricrin (Fuchs Lab), involucrin, fillagrin , MAPK, pSMAD2 ; Grb-2 ; P-cadherin ; HA , vimentin , Ki67 , keratin 6 , cyclin D , and TGF-β2 . FITC-, Texas Red-, or HRP-conjugated secondary antibodies were from Jackson ImmunoResearch . Biotinylated secondary antibodies were from Vector Labs . Dilutions were according to the manufacturer's recommendation. The Snail antibody was generated in Guinea pigs by inoculating them with the N-terminal sequence of murine K14-Snail Tg mouse was generated by digesting the pcDNA3-mm Snail-HA plasmid  with BamHI and NotI and subcloned into the K14 vector [K14-Smad 2 Tg mouse was reported in Ito et al., 2001. The TGF-β2 knockout (KO) mouse was described in [shh KO mouse [The 4 vector . The linribed in . The shhKO mouse  and TOPGProtein extracts from primary keratinocytes were generated either by lysing cells in lysis buffer  or directly in Laemmli bβuffer and boiled. For skin tissue: Frozen tissue was pulverized in a liquid nitrogen-cooled Gevebesmascher and the powder scraped into a chilled microcentrifuge tube. RIPA buffer  and protease inhibitors or Laemmli buffer was added. The cell suspension was sonicated three times for 15 s and centrifuged at 14,000 rpm at 4 °C. The supernatant was separated from the pellet and used in the experiments. Extracts subjected to immunoprecipitation were precleared with Protein G Sepharose  and incubated with antibody with rocking overnight at 4 °C. Protein G Sepharose was added and samples were incubated for 1 h at 4 °C with rocking. Samples were washed three times for 5 min each in lysis buffer, and the Protein G Sepharose-antibody-antigen pellet was resuspended in Laemmli buffer and boiled for 10 min. Samples were run on SDS-PAGE and transferred to nitrocellulose membrane . Western blot signals were developed using the enhanced chemiluminescence kit from AmershamPrimary keratinocytes were culture in low-calcium medium as previously described . TransieSnail promoter was generated by PCR using a forward primer with an XbaI linker sequence, 5′-TCTAGAATTGTTTGCTGCTGTATGGTCTTC-3′, along with a reverse primer with a BglII linker sequence, 5′-AGATCTGTTGGCCAGAGCGACCTAG-GTAG-3′, and mouse genomic DNA as a template. The PCR product was purified with the Gel Extraction Kit  and ligated into pCRII-TOPO TA vector . The promoter was verified by sequencing and digested with XbaI and BglII and subcloned into the pβ-gal BASIC vector . The point mutations in the SMAD binding element was generated with the Quik-Change Kit  using the forward primer 5′-GGGCGGGCTTAGGTGTTTTCATTTACTCTTGAGGAAAAGCTTGGC-3′ and the reverse primer 5′-GCTTTT-CCTCAAGAGTAAATGAAAACACCTAAGCCCGCCCTGCCC-3′. The probes for the Snail in situ hybridization were generated against the 3′ UTR by PCR using the forward primer 5′-ACCTTCTCCCGCATGTCCTTGCTCC-3′ and the reverse primer 5′-CTGCTGAGGCATGGTTACAGCTGG-3′, and genomic DNA as a template. The PCR product was gel purified and ligated into pCRII-TOPO TA vector. The pre-LIM domain of Ajuba was generated essentially as described [The 2.2-kb murine escribed , but wasSnail was used as a template to generate digoxigenin-labeled sense and antisense riboprobes (Roche). The respective probes were obtained by XhoI and BamH1 digestions. In situ hybridizations were performed on 10-μm thick sections of E17.5 mouse embryos. The sections were fixed with 4% PFA for 10 min at room temperature, prehybridized at room temperature for 4.5 h, hybridized with the probe (2 μg/ml) at 55 °C for 12–14 h, blocked with 10% NGS, and treated with anti-dig Fab-AP antibody (Roche #1093274) at a 1:2,500 dilution for 3 h. The sections were incubated with NBT and BCIP until adequate signal was detected.The pCRII-TOPO TA vector containing a region of the 3′ UTR of Tissue samples for immunofluorescence were frozen in OCT and sectioned 10 μm thick on a cryostat. Sections were fixed in 4% paraformaldehyde for 10 min at room temperature, blocked, and stained with antibodies. Tissue samples for immunohistochemistry were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Samples were sectioned on a microtome (10 μm thick) and rehydrated prior to staining with antibody. Samples stained with Snail, pMAPK, pSmad2, and cyclin D were antigen unmasked with 10 mM sodium citrate (pH 6) in an Antigen Retriever 2100 . The DAB substrate kit (Vector Labs) was used according to manufacturer's instructions to develop the signal.Snail forward: 5′-CAGCTGGCCAGGCTCTCGGT-3′; Snail reverse: 5′-GCGAGGGCCTCCGGAGCA-3′; GAPDH forward 5′-CGTAGACAAAATGGTGAAGGTCGG-3′; and GAPDH reverse: 5′-AAGCAGTTGGTGGTGCAGGATG-3′.RNA was extracted from keratinocytes or skin tissue with Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was generated using oligo-dT primers and the Superscript II kit (Invitrogen). The primers used for PCR were


Cytology slides are often unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide. Cytology slides are often "sent out" to other physicians, laboratories or hospitals, typically so that the pathologist at the institution where the patient will receive treatment can review the slides. Less often, a cytology lab sends out the slides for a second opinion or as part of the discovery process in a lawsuit, where they may or may not be defendants. Rarely, unique and irreplaceable cytology slides are lost. This article presents a hypothetical scenario that is based on reported state appellate court decisions. The article discusses some of the legal issues that will affect the defendant cytologist/cytology lab and the "expert cytologist," and suggests some steps a cytologist/cytology lab can take to minimize the risk of repercussions from a lost unique and irreplaceable cytology slide. A WestLaw search  uncovered only a handful of reported appellate cases that directly applied to the issue of lost cytology slides. There are many cases and statutes dealing with lost and altered evidence (other than cytology slides), but the circumstances surrounding cytology slides are unique. I did not see any previous reviews or commentary specifically addressing the topic. Notwithstanding the relative paucity of on-point legal authority, the issue is one commonly addressed by cytology laboratories and cytologists and many have approached the issue thoughtfully and prudently.This review synthesizes the available legal cases and presents to practicing cytologists a short, relatively concise summary in the format of a hypothetical case. The review seeks to incorporate the legal issues with the practical aspects of running a cytology laboratory. The review answers how some state courts might approach the problem of lost and irreplaceable cytology slides, and offers general ideas to cytologist for minimizing the risk from lost slides.The issues surrounding the decision whether to send out diagnostic patient slides are more important in cytology than in surgical pathology because the cytology slides are typically unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide.Slides are routinely "sent out" for a variety of reasons. Most commonly, slides are sent to another institution because the patient's pathology slides will be reviewed before treatment. Some smaller laboratories with only one or two pathologist may send out slides as part of their quality assurance procedures. More rarely, but increasingly, slide's are requested as part of existing or contemplated litigation. Slides might be lost in any of these circumstances. A laboratory or hospital might loose the slides and not discover their absence until the slides are requested.This article will discuss the approach the US legal system takes in addressing what happens when cytology slides are lost, and what steps a prudent laboratory might take to manage the risk. The article also intends to improve cytopathologists' awareness and understanding of some of the legal issues that arise when evidence is missing and perhaps promote an international comparative discussion of alternative legal approaches.In the US, most of medical malpractice law is made and interpreted by state legislatures and state courts. What follows is a hypothetical story based on several legal decisions made by appellate state courts in the United States and reported in the legal literature. Although based on real cases and available to the public, the names have been changed. Importantly, none of this is intended as legal advice and readers should consult their attorney about specific questions.Rugged Labs (RL) is a small, independent laboratory. Part of Rugged Labs' work involves providing Big Giant Lab (BGL) with overflow services for cytopathology. In December of 1995, BGL bought out RL.In 1994 and 1995, a RL cytotechnician interpreted two Pap smears from 30-year-old Ms. Penny as normal. In 1996 a cervical biopsy from Penny showed adenocarcinoma. Ms. Penny underwent a radical hysterectomy, which confirmed the invasive endocervical adenocarcinoma. Ms. Penny is alive today, but endured extended post-operative hospitalization.At the time of the hysterectomy, Penny Plaintiff's oncologist requested that the 1994 and 1995 PAP smear slides be sent to Dr. Experta, who interpreted both Pap smears as containing "abnormal cell groups consistent with adenocarcinoma." Dr. Experta also reviewed the biopsy and in a note concluded that the cells on the Pap smears were consistent with the adenocarcinoma diagnosed on the cervical biopsy.Approximately 6 months later, Plaintiff Penny decided to sue for medical malpractice based on failure to diagnose her endocervical adenocarcinoma on the Pap smears.At some time before the plaintiff filed her lawsuit Dr. Experta's assistant apparently mailed the slides back to BGL. The Pap smear slides are lost, presumably in the mail, by BGL or Dr. Experta's office.RL, the independent laboratory, asked the trial court to grant it summary judgment on the basis that the evidence was lost and no questions of fact remained. Summary judgment means that there are no outstanding questions of fact and the court needs to decide only questions of law. Summary judgment means there is no trial with a jury or judge hearing and weighing evidence. The question of law RL wanted the court to determine on summary judgment was that the lost slides substantially prejudiced RL and summary judgment was, therefore, appropriate. RL included in its motion for summary judgment an affidavit from an expert stating that she could not give an opinion without having the slides to look at. An affidavit by Dr. Experta's secretary stated that the slides were returned to BGL by US mail. BGL submitted an affidavit attesting they did not lose the slides. The parties to the lawsuit stipulated that the slides were lost. The trial court agreed with RL that there should be summary judgment in RL's favor, reasoning that no questions of fact needed to be answered and that a trial would unduly prejudice RL because of the spoliation of evidence. For a summary of the facts, please see Fig. 1.The Plaintiff appealed and the state appellate court reversed the trial court, concluding that the trial court made a mistake in not allowing the case to go to trial. The appeals court reasoned that the case turned on a question of fact; "The slide either showed the presence of cancer cells or it did not." The appellate court envisioned the trial as follows: the cytotechnologist from RL would "testify to her conclusions" and the plaintiff would have Dr. Experta testify to her conclusions. There should be a trial because there were questions of fact including, in the words of the majority, whether the slide had "cancer cells" or not.The majority also discounted the affidavit from BGL, stating that the conclusory statement that BGL had not lost the slides was not valid; just because BGL couldn't find the slides did not mean they never had them. In a footnote, the majority noted that although they aren't accusing anyone, they couldn't help but notice that missing the slides benefited BGL and RL.One judge dissented in the three-judge panel that decided the case. The dissent focused on two points. First, the dissent emphasized that the defendant RL had nothing to do with loosing the slides. Although the plaintiff, Ms. Penny, had no "direct role" in the loss of evidence, the dissent treated Dr. Experta as an agent of the plaintiff and concluded that Dr Experta should have sent the slides back to RL not GBL. The dissent also reasoned that its approach would "encourage experts to treat more carefully evidence delivered into their hands."Secondly, the dissent emphasized that the defendant RL is disadvantaged by the loss of the slide and the plaintiff has gained a significant advantage. The dissent sees a trial where the cytotechnologist's faces a serious credibility problem because her testimony will come across as blatantly self-serving. The cytotech will be limited to "opining that he made no error." Moreover, the fact-finder may conclude that the cytotechnologist is wrong since many may presume that the expert pathologist must be right. Finally, the defendants cannot obtain their own expert to bolster their case, because no cytology slides remain for review.The dissent concludes that summary judgment for RL was proper because the trial court had good reasons to conclude that the lost evidence was going to unduly prejudice the defendant RL.The most obvious issue this case brings up is the difficulty in deciding what to do when it isn't clear who lost the evidence, or when a third party lost the evidence. In contrast, is the situation where one party is responsible for inadvertently losing the evidence or, even worse, where one party deliberately loses or destroys evidence. This is called spoliation of evidence and includes meaningful alteration of the evidence.The court will determine the severity of the sanction for spoliation by the degree of willfulness or bad faith and the extent of the prejudice suffered by the non-responsible party. For example, in one case, surgeons performed a hepatectomy after a small needle core liver biopsy was interpreted as cancer. The hepatectomy specimen showed only cirrhosis and no cancer was found. The small needle liver biopsy was subsequently lost by the hospital. The patient sued for medical malpractice, claiming that the core biopsy was misdiagnosed and lead to an unnecessary hepatectomy. The trial court instructed the jurors that they could "draw the strongest possible inference against [the hospital] as to what the lost cytology slides would have shown." In other words, losing the slide means your opposition can make the slide show whatever they want. Interestingly, in the hepatectomy case, the defendants prevailed by arguing that the diagnosis of cancer and the decision to undergo resection were reasonable, regardless of the cytology results, because the patient had active hepatitis B and a suspicious liver mass on imaging. They argued that even with a negative cytology result the surgeons would have gone ahead with the hepatectomy.But our case is different. Neither party, according to the majority, is responsible for losing the slides, or put another way, either party might be responsible for losing the slides. Several judicial options exist and none are neutral. One is to impose no sanctions and to proceed as usual, only without the slides, as the majority opinion advocated. This likely favors the plaintiff, particularly when there is a subsequent surgical specimen with cancer. A second option is to try to determine which party the court thinks is more prejudiced by the lost slides and then impose a legal remedy, as the trial court did in our hypothetical case by granting summary judgment. The difficulty is that it will not be clear which side is more prejudiced until the slide is recovered. The critical question of whether the negative diagnosis fell below the standard of care can likely be adequately answered only if the parties and their experts can review the slide. A third option might be to not allow Dr. Experta's testimony if the defendant can show that the expert knew or should have known that there was going to be litigation . ArguablInterestingly, each option leads to a different result based on bias about which party is ultimately more responsible or more likely responsible for loosing the slides and a bias about the standard of care. The majority's opinion included a note that the benefit to BGL from losing the slides can't be ignored, a clear statement that the plaintiff had no responsibility for losing the slides, and a simplistic view about the standard of care reflected by the statement that "the slide either showed the cancer cells or it did not." The majority's subtext is that the plaintiff did nothing wrong and they weren't completely sure about the laboratory. The dissenting opinion, in contrast, treated Dr. Experta as the plaintiff's agent and was dissatisfied that Dr. Experta returned the slide to BGL instead of RL, even though at the time BGL had purchased RL. The dissent also showed a more nuanced understanding of the standard of care, conveying skepticism about Dr. Experta's look back conclusion that the Pap smears were "consistent" with adenocarcinoma. The dissent reasoned that perhaps the cells are consistent with malignancy only in the retroscope and that a defense expert might reasonably conclude that it was "not below the standard of care to determine the biopsy negative" were the slides available for review.Penny vs. GBL, a court does not dismiss the plaintiff's case, the absence of the cytology slide will likely impact the defendant cytologist more adversely because many people will assume that the cancerous cells were on the slide and the cytologist or cytotechnologist missed the cancer, which, after all, was present on the subsequent biopsy. In the hypothetical's facts, this was particularly true since Dr. Experta had already opined that the Paps were "consistent with adenocarcinoma." The defendant cytologist/cytology lab is at a serious disadvantage if the court allows Dr. Experta's opinion as admissible evidence. The majority's comment that GBL benefited from the lost slides and the appellate court's decision to allow the plaintiff to go to trial, suggests the court's bias that they believed Dr Experta's interpretation was the correct one.The facts often surrounding a cytopathology medical malpractice case are that there is a subsequent biopsy or surgical specimen with a discrepant diagnosis. If, as in the hypothetical Admittedly, a cytology lab or cytologist does benefit if slides are lost and the court does not attach any responsibility for losing the slides to the potential laboratory or cytologist defendant, because the plaintiff will not have enough evidence to prevail. Similarly, if the slides are discarded after the legal time periods the likelihood of a successful lawsuit is slim because the plaintiff will not have enough evidence, and the defendant complied with legal requirements regarding slide retention. The hypothetical case of Penny vs GBL differs. Remember that once the court allows the case to go to trial, GBL only benefits from absent slides if the defendant initially misinterpreted the slides. If the slides contained no malignant cells, and Dr. Experta over-interpreted the slides, then GBL is prejudiced by not being able to show the slide.Cytology slides are typically in possession the cytology labs. The slides may be sent out for a variety of reasons, but at least initially the lab has possession of the cytology slides. This is both an advantage and a disadvantage for the lab. The disadvantage is that if slides are lost while in the lab's possession a court will typically see it as the lab's responsibility to safeguard the slides. This allows the plaintiff to have the jury infer whatever is best for the plaintiff's case; that the slide had malignant cells when the cytology diagnosis was benign or that there were only benign cells when the diagnosis was malignant, as in the hepatectomy case.The advantage for the cytology lab is that it is in a position of relative control. The lab can implement a system to help reduce the chances of losing a slide and reduce the risk if a slide is lost. Although the lab may want to employ the help of an attorney experienced in these matters, there are several steps every cytology lab can take. First, the lab should ensure that cytology slides are retained for the time that the current federal CLIA regulations, applicable state regulations and the CAP checklist require. All glass cytology slides must be retained for at least 5 years and fine needle aspiration slides retained for 10 years. (Some state regulations may require longer times.) The CAP checklist also requires policies for "protecting and preserving the integrity and retrieval of original slides in cytopathology" and "to ensure defined handling and documentation of the use, circulation, referral, transfer and receipt of original slides to ensure availability of materials for consultation and legal proceedings." Keeping careful records about when and which slides are released to whom is essential for reducing the risk of losing slides. In the send out cases where slides are sent out for a routine second opinion not sought in contemplation of a law suit or because a patient will be treated elsewhere, the lab might obtain the borrower's explicit written agreement that the borrower has responsibility for the slides with an explicit provision about a duty to indemnify the cytology lab for any losses due to a lost slide or slides. A documented telephone call to request the return of tardy slides may also be worthwhile. In cases where slides are requested in contemplation of a lawsuit, the lab may be able to implement a policy that review of slides is done at the lab, ensuring that the lab retains possession. Alternatively, the lab may pursue or agree to a court order requiring production of the slides that clearly addresses who is responsible for the slides and includes an indemnity clause in the event the slides are lost.It is reasonable to make a distinction between sending out non-reproducible slides to an institution that will treat the patient and sending out non-reproducible slides to the plaintiff's expert witness. It is, therefore, important that the lab understands the purpose for which the slides are requested. A documented telephone conversation may clarify the purpose of the outside review and allow the lab to appropriately "triage" the case.Laboratory administrators should understand that the cytology lab serves a public function in safeguarding cytology slides. Court's have recognized a public policy reason to have the laboratory safeguard slides, in part so that the slides are available in the event of malpractice litigation. At the same time, many states have statutes that give patients the right to examine and copy their medical records . These two propositions are not mutually exclusive. One state case considered the question of whether a patient/plaintiff had a right to "immediate possession of pathology slides" and concluded that the plaintiff did not. The court decided that the patient's rights did not exceed the patient's statutory right in the slides. In other words the court was not going to find a common law, or customary, right in the slides that gave the patient a greater right than the applicable statute. The decision noted that the legislative history of the statute included remarks that pathology slides were part of the medical record. The judges then approached the second question about what to do when the "medical record" cannot be duplicated, as with a Pap smear or other cytology slide. In answering this question, the court noted that hospitals and laboratories have public as well as private duties. One of their public duties is to retain slides so that the slides are available in the event of malpractice litigation. This meant that patients do not have a legal right to possess parts of the medical record that cannot be duplicated. The court, however, did not grant the lab complete authority to never release the slides. Since the public policy reason depended fundamentally on preserving slides to help the legal system run smoothly, the decision reminded the laboratory or hospital that, pursuant to the clear terms of a statute, it must send the original slides to a "licensed institution, laboratory or physician" at the patient's written request.Moore vs Regents of the University of California [The dissent characterized the pivotal issue differently and concluded that the patient had a right to immediate possession because the slides contained the patient's cells and she had the right "to control one's body." The dissent reasoned that recent advancement in genetic science and the accompanying difficult privacy issues raised by genetic information strengthened the patient's right to possession of the cells on the glass slide. The majority addressed this argument and, citing the well known case of lifornia , remindePenny v GBL illustrates, lost slides can result in problems for cytologists and the cytopathology laboratory A good place to start is to ensure that existing CLIA regulations, applicable state regulations and other guidelines, such as the CAP checklist, are in place in the laboratory. Thinking about the issue and implementing appropriate risk management strategies with the help of an attorney are also prudent measures.In summary, lost slides can be a problem for cytology laboratories and cytologists, whether responsible for losing the slides or not. The prudent cytologist will minimize the risk of lost slides, because, as the hypothetical case of


Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific.Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes.We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis transcripts.The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000  Arabidopsis thaliana has been limited when compared to other model organisms, such as human and mouse, mainly due to the lack of high-quality cDNA microarrays offering genome-wide coverage. However, during the recent years both academic and commercial alternatives to these cDNA arrays have emerged. The public initiative by the CATMA consortium has aimed at the production of high-quality probes for each of the 29 787 genes predicted in the Arabidopsis genome  and when bound, washed with 1 × TE-buffer . During incubation the beads were kept in suspension by mixing through pipetting every third minute. Elution was achieved by breaking the streptavidin-biotin bond in a 20-μl volume using deionised HFor all the purifications described below an aliquot of the pooled PCR product corresponding to a 50-μl reaction was used. The binding capacity of the beads was studied using an increasing amount  of fresh beads (first use) while keeping the incubation time constant at 20 minutes. To estimate the variability of the method eight independent purifications were carried out (n = 8). The effect of the length of the incubation time was studied using 150 μg of beads and eight different incubation times  with four replicates of each (n = 4). To study the effects of multiple reuses of the beads, the same beads were reconditioned and used up to nine times. The amount of beads in the first capture was 150 μg and the capture time 20 minutes. The variability was estimated using four independent replications. The multiple reuse of the beads was also analysed using a hybridisation based approach. Twelve clones ranging from 0.3 to 2 kb were amplified and purified multiple times using reconditioned beads. The purified products were subsequently printed in eight replicates onto glass slides and quantified using Syto61 . The well-to-well carry-over of product was analysed by first purifying one of the PCR products, followed by a purification reaction with no PCR product added (water-only control). This pattern was repeated six times for all three products, while the same set of beads was used for all purifications. Eluates from all these purifications were printed on slides and hybridised with a Cy5-labelled oligonucleotide complementary to the common vector sequence present in all products. Hybridisation was carried out using 10 pmole of the labelled oligonucleotide for 1 hour at 35°C in a hybridisation solution containing 50% formamide, 5 × SSC and 0.1% SDS. Slides were washed with 2 × SSC containing 0.1% SDS (5 min at room temperature) and three times with 1 × SSC (1 min at room temperature). Scanning using the G2565BA DNA microarray scanner (Agilent Technologies) was carried out at the highest possible photo multiplier tube setting in order to reveal low-level signals.2, 200 nM dNTPs, 20 pmole of each primer  and 1.5 units Platinum Taq DNA Polymerase in a total volume of 50 μl. Biotinylated products from each amplification reaction were bound to 200 μg of Dynabeads M-270 Streptavidin beads (reused up to six times) during a 15-minute incubation at room temperature using a high ionic-strength buffer, washed with 1 × TE-buffer and, finally, eluted with 12 μl deionised water. After each capture round the beads were reconstituted, pooled plate-wise and randomly assigned to a new plate. An equal volume of 99.9% dimethyl sulfoxide (DMSO)  was added and the purified amplification products were arrayed into 22 by 22 patterns in 48 individual blocks with the QArray arrayer  and SMP2.5 pins . When dried, printed products were immobilised to the reactive surface of the Ultra-GAPS slides  using 250 mJ/cm2 UV-light .Initial amplification from BAC-clones or genomic DNA was carried out by the CATMA consortium at different nodes throughout Europe ,2. One pArabidopsis Col-0 seedlings were grown at 22°C in MS medium  supplemented with 0.5% sucrose and using a 24-h photoperiod with 16 h of light at 75 mE m-2 sec-1 PAR. The samples were treated with 1 μM indole-acetic acid for a period of 0, 30, 120 and 240 minutes, washed once with an excess of MS medium with 0.5% sucrose for 5 minutes, frozen in liquid nitrogen and stored at -70°C. For each time point, frozen seedlings from three independent vials were pooled, grinded and total RNA extracted using the RNeasy kit . The quality of the RNA was determined using the RNA 6000 Nano kit and the Bioanalyzer instrument .10-day-old 20VN, MWG Biotech AG) was annealed to 20 μg total RNA after a denaturating step (10 min at 70°C). The cDNA synthesis reaction was carried out at 42°C for 1 h 45 min in a 30-μl reaction containing 2 mM dNTPs , first-strand buffer , 0.01 mM DTT and 400 units Superscript II (Invitrogen AB). The synthesis reaction was terminated by addition of EDTA, the RNA strand hydrolysed with NaOH (15 minutes at 70°C) and the reaction neutralised with HCl . The cDNA strands were purified using the MinElute spin columns (Qiagen GmbH) with the provided wash and elution buffers replaced by 80% ethanol and 100 mM NaHCO3, pH 9.0, respectively. Monofunctional NHS-ester Cy3 or Cy5 fluorophores (Amersham Biosciences) were coupled to the amino-allyl groups during a 90-minute incubation at room temperature after which unincorporated ester groups were inactivated through a hydroxylamine treatment . The pooled labelling reactions were purified using MinElute spin columns and hybridised to the arrays using a two-step protocol in the GeneTac hybridisation station . The pre-hybridisation at 42°C for 45 min , 0.1% SDS, 40 μg poly(dA) (Sigma-Aldrich) and 20 μg tRNA (Sigma-Aldrich)) was followed by a 16–18 h hybridisation at 42°C with the labelled material and a hybridisation buffer containing 5 × SSC, 25% formamide, 0.1% SDS, 40 μg poly(dA) and 20 μg tRNA. The slides were washed with 2 × SSC and 0.1% SDS at 42°C, followed by 0.1 × SSC + 0.1% SDS at room temperature and finally by three repeated washes with 0.1 × SSC at room temperature. Slides were scanned at 10-μm resolution using the G2565BA DNA microarray scanner for which the photo multiplier tube (pmt) setting was adjusted so that the images for the Cy3 and Cy5 channels were in balance as determined by visual observation. Each time point-to-reference sample comparison was carried out on two arrays, with the dye labels exchanged between the replicated hybridisations in order to avoid sequence-dependent labelling and hybridisation effects. A control self-to-self hybridisation was also carried out for the untreated sample in order to assess the level of noise in the experimental system.Ten μg anchored oligo dT primer  and the data with the R environment for statistical computing , BiocondCATMA a complete Arabidopsis thaliana transcriptome microarrayEST expressed sequence tagGST gene sequence tagIAA indole-3-acetic acidMIAME minimum information about a microarray experimentVW carried out the laboratory work and the data analysis and participated in the design of the study and drafting of the manuscript. AH and ML participated in the solid-phase purification procedure. PN participated in the array production. PH designed and provided the CATMA probes. MU participated in the automation of the solid-phase procedure. RB carried out the auxin treatment and participated in the interpretation of the expression data. JL conceived of the study, participated in the drafting of the manuscript and coordinated the study. All authors read and approved the final manuscript.List of genes identified as up-regulated by the indole-3-acetic acid treatment. Group labels refer to (A) previously identified auxin regulated, (B) transcription related, (C) signal transduction related, (D) transport, (E) cell wall establishment related, (F) metabolic enyzmes, (G) light related, (H) disease repsonse related, (I) other and (J) unknown genes.Click here for fileList of genes identified as down-regulated by the indole-3-acetic acid treatment. Group labels refer to (A) previously identified auxin regulated, (B) transcription related, (C) signal transduction related, (D) transport, (E) cell wall establishment related, (F) metabolic enyzmes, (G) light related, (H) disease response related, (I) other and (J) unknown genes.Click here for file


Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays ( This and the following presentation are part of a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in chemical toxicity testing and mechanistic research. It was decided to compare the results obtained from those transgenic mutagenicity test systems where considerable data was available, with other in vivo genotoxicity tests: in the first article (Part 1) with the mouse bone marrow micronucleus test and in the second (Part II) with the mouse spot test.in vivo mammalian test system for the detection of chromosomal aberrations  for which carcinogenic effects are considered to be of a nongenotoxic mechanism. However, chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane gave negative results in transgenic mice, so the comparison of both test system is not essentially affected and the evaluation „nongenotoxic“ supported. For the other 8 substances out of these 11 with false negative results in the micronucleus test these results are explainable (see above): Ortho-anisidine (mutagenic/carcinogenic effects are restricted to the bladder), IQ (bone marrow presumably not target organ of genotoxicity in mice and more gene mutagenic than clastogenic), asbestos , 2,4-diaminotoluene , N-nitrosodiethylamine , N-nitroso-N-propylamine (presumably more gene mutagenic than clastogenic), beta-propiolactone , trisphosphate (systemic effects not related to the bone marrow).The term negative predictability is also low in the transgenic mouse assay due to false negative results on six carcinogenic substances; for three of them, hydrazine, mitomycin C, and tetrachloroethylene  genotoxic mechanisms are presumed. For hydrazine (no repeated application) and tetrachloroethylene (MTD not reached) limitations on the experimentel design might be the reason for the negative results. Mitomycin C is clearly more clastogenic than gene mutagenic, and the transgenic mouse with lacI and lacZ is possibly an unsuitable test system. For 3 out of the 6 substances the carcinogenic effects in mice were attributed to nongenotoxic mechanisms: chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane , all gave negative results in transgenic mice.Only two substances with negative results in long-term carcinogenicity studies are available in the data pool: bromomethane and 2,6-diaminotoluene. Both gave correct negative results in the transgenic mouse assay  but false positive results in the micronucleus test (see term specificity).Generally, the differences between the two test systems might be due to the fact that 1) unequal genotoxic endpoints are investigated (chromosome aberration in the micronucleus test versus gene mutation in the transgenic mouse assay), 2) organotrophy of genotoxic effects  might play an essential role and 3) transgenic rodent assay conditions in the different systems may not be optimal for mutation detection.A comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated.In comparison to other test systems in genotoxicity testing using endogenous target structures the spontaneous mutant frequency in the transgenic mouse assay is relatively high. This might be related to the fact that bacterial DNA is the target gene (high methylation rate) or the transgene is silent and no transcription related repair occurs like in endogenous genes which are more efficiently repaired . In the Comparing the target organs and cells at risk at the time of exposure, the mouse micronucleus test is restricted to one target organ, the bone marrow, especially to the erythroblasts. This limitation is not given in transgenic mouse assays: target cells are cells in all organs .Both test systems are similar in the number of animals used for a valid test. The minimal number of mice needed in the mouse bone marrow assay is 25 per gender  using a treatment schedule with 2 or more applications at 24 h intervals and sampling 18–24 h following the final treatment. In the limit test (for a test substance of low toxicity) only one dose level of 2000 mg/kg bw is necessary .In transgenic mutation assays ca. 20 animals  are recommended per species and gender ,197. In Due to the simplicity of the mouse bone marrow micronucleus assay and the use of systems for automated analysis, this test is less expensive than the transgenic mouse assay.A comparison both test systems is presented in Table ® mouse and the Muta™mouse were compared with those from the more traditional mouse bone marrow micronucleus test. The transgenic animal mutation assay, which is not yet used in toxicological screening, is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation. However, from the 39 substances, the majority gave the same positive or negative result in both test systems. The substances where differences occurred were discussed in more detail. The advantages and disadvantages of the transgenic Big Blue® mouse and the Muta™Mouse transgenic model compared to the micronucleus test were discussed and both systems were found to have a place in mutagenicity testing and to supplement each other. The transgenic animal assay has, however, some distinct advantages over the micronucleus test in that it is not restricted to one target organ and detects systemic as well as local mutagenic effects.In a comparison of the tests available to genetic toxicologists, the results from studies on substances which had been tested in transgenic mutagenicity assays Big BlueUW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.Table: Results in the transgenic mouse assay versus mouse bone marrow micronucleus testClick here for file


The objective of this paper is to describe the numbers, characteristics, and trends in the migration to the United States of physicians trained in sub-Saharan Africa.We used the American Medical Association 2002 Masterfile to identify and describe physicians who received their medical training in sub-Saharan Africa and are currently practicing in the USA.More than 23% of America's 771 491 physicians received their medical training outside the USA, the majority (64%) in low-income or lower middle-income countries. A total of 5334 physicians from sub-Saharan Africa are in that group, a number that represents more than 6% of the physicians practicing in sub-Saharan Africa now. Nearly 86% of these Africans practicing in the USA originate from only three countries: Nigeria, South Africa and Ghana. Furthermore, 79% were trained at only 10 medical schools.Physician migration from poor countries to rich ones contributes to worldwide health workforce imbalances that may be detrimental to the health systems of source countries. The migration of over 5000 doctors from sub-Saharan Africa to the USA has had a significantly negative effect on the doctor-to-population ratio of Africa. The finding that the bulk of migration occurs from only a few countries and medical schools suggests policy interventions in only a few locations could be effective in stemming the brain drain. Doctors migrate from developing countries to wealthier countries in order to further their careers, or improve their economic or social situation. The World Health Organization (WHO) has long recognized that migration of health personnel from developing to developed countries creates unfortunate imbalances in the global health workforce .America's physician workforce has been significantly infused with foreign-trained international medical graduates (IMGs) since World War II. The purpose of this paper is to describe a sub-population of IMGs in the USA, those who have trained in one of the 47 African subcontinent nations.African governments have been very clear about their objections to the wholesale migration of their physicians to rich countries. In 1996, South Africa's then-Deputy President Thabo Mbeki implored the World Health Assembly to take measures to stop the flow of physicians from poor countries to rich ones. In 1995, South Africa itself banned the recruitment of doctors from other Organization of African Unity countries .Nonetheless, large numbers of African-trained physicians leave home upon completion of their medical school training in search of careers in higher-income countries. They leave behind health systems in sub-Saharan Africa that are severely stressed: life expectancy is only 50 years, 162 children in 1000 die before reaching their fifth birthdays, and only half have access to clean water sources . FurtherAfrican country health systems and workforce data are poor, making it difficult to estimate the effects of physician migration on sending countries. The World Bank has documented this data gap, noting "Quantitative data on the health workforce is notoriously unreliable in most countries...In poor countries, government and professional information systems are weak, when they exist at all, and are rarely comprehensive (often there is no information on the private sector) and up-to-date" . Indeed,The 47 nations of sub-Saharan Africa have a total of 87 medical schools, although 11 countries have no medical school at all and 24 have only one each .One of the most common initial points of entry for IMG physicians into the USA medical workforce is residency training program enrollment, even if physicians have already completed postgraduate training in their home countries. The reliance of many inner-city hospitals on IMGs has thwarted calls by medical policy organizations, such as the Council on Graduate Medical Education, to reduce the number of IMGs admitted to residency programs as a means of narrowing the IMG pipeline to the USAThere is little debate within the USA government or other institutions about the social justice implications of obtaining health professionals from poor countries . TypicalThe ethics of health professional migration from poor countries to rich ones is complicated by the competition of legitimate interests – each country's need for an adequate health workforce as opposed to each individual's human right to travel. When health professionals travel to receive training and then return to apply their skills, there are advantages to the home country. Additionally, emigrants of all social classes from poor countries typically send funds home to relatives, although sub-Saharan African remittances, at less than USD 5 billion, comprise the lowest dollar amounts of any other poor world region . FurtherImmigration theory informs us that "push factors" prompt professionals to leave poor countries in favor of settling in higher income countries . NegativThe "world systems framework theory" stresses the more permeable barriers between and among countries created by the standardized curriculum and English language used in world medical schools, the use of common research methods and shared scientific knowledge, the easy articulation of requirements of practice across countries, and the weakened nationalism that occurs as a result of professional training . Other tGiven the enduring migration from poor countries to rich ones, only likely to increase with the international liberalization of trade in health services , concernTo describe the numbers and types of physicians practicing in the USA who earned medical degrees in Africa, we performed a cross-sectional study using the 2002 American Medical Association Physician Masterfile . This daWe reviewed these data for all physicians in the USA who received their training in sub-Saharan Africa (those 47 countries south of the Saharan desert on the African continent). These data included year of birth, gender, year of medical school graduation, name of medical school, current practice location, specialty of practice, and practice activity . Birth country information is missing for 68% of those who graduated from a sub-Saharan African medical school, so we did not analyze birth country data. To detect changes in migrant waves over time, we analyzed the data by cohorts, categorizing physicians who had graduated from medical school during four periods: before 1970, during the 1970s, during the 1980s, and 1990 and beyond.We linked geographic data about practice locations to a four-category, rural-to-urban status and taxonomy, a condensed version of the Rural-Urban Commuting Area (RUCA) codes, to determine whether these physicians are practicing in rural or urban areas. RUCAs are a census tract-based classification scheme, that have also been adapted for zip codes, combining USA Census population data with work commuting information to characterize the types of rural and urban status .Research colleagues in Canada and the UK provided some data on sub-Saharan African physicians in their countries, as well.A total of 179 978 (23.3%) of the 771 491 active non-federal physicians in the USA in the year 2002 received their medical qualification in another country. The largest portion of these, or 115 835 physicians, originate from low and lower-middle income nations, as defined by the World Bank. Indeed, the most frequent countries of origin of IMGs in the USA include India (36 634), the Philippines (17 755), Mexico (10 404), and Pakistan (8563). Canadian physicians conventionally are not included in the IMG count because the body that accredits USA medical schools ) offers reciprocal accreditation to Canadian medical schools . Canadians are, however, still subject to relevant immigration requirements.Sub-Saharan African medical schools in 22 countries have trained approximately 5334 physicians currently practicing in the USA. Only nine nations, however, have lost more than 40 physicians each , the University of Cape Town , the University of Ibadan , the University of Lagos , the University of Nigeria , the University of Ghana , Addis Ababa University , the University of Benin , the University of Ife , and the University of Pretoria , for a total of 4234 physicians.An analysis of the numbers of sub-Saharan African IMGs coming to the USA in each of the last decades illustrates that it takes some time between graduation and emigration. The number of recent graduates currently in a USA residency program is higher than those in previous decades because of the obvious correlation between age and career stage.Among sub-Saharan physicians in the USA, 78.3% are male. The picture is changing over time, however. Of the cohort who were trained in 1969 or earlier, 90% were male, but now only 66.3% of those who graduated from medical school in 1990 or later are male , and South Africans are the oldest (only 20% are under 40).A higher proportion of sub-Saharan physicians were in residency training programs (21.2%) than were USA physicians (14.1%), because many emigrate specifically for that reason. While 41.9% are in generalist specialty areas, compared to 34.7% of USA-trained physicians, the number has been rising with each new cohort. Table While 31.6% of all sub-Saharan African physicians in the USA are identified as family practitioners or general internists, it may be that this ratio of generalists will increase, as 45.4% of those in residency programs are in those two specialties. The next largest specialty groups are pediatrics (9.7%), psychiatry (5.5%), anesthesiology (5.4%), obstetrics and gynecology (3.3%) and general surgery (3.0%).Urban areas attracted 93% of sub-Saharan African physicians (compared to 90.9% of other IMGs), even after excluding residents, who are typically based in urban teaching hospitals. Graduates of USA medical schools distribute themselves similarly, with 87% of USA-trained physicians in urban areas, even though a smaller 81% of the population lives in urban areas . The staThe 1943 physicians trained in South African medical schools are somewhat different from their fellow African trainees from the subcontinent. They are older, more of them are male, they are typically white (94%) and they are more often in a subspecialty practice. This may reflect a particular wave of physicians seeking subspecialty training and practice opportunities in the USA during the political turmoil South Africa experienced in the 1970s and 1980s. It is unlikely they are seeking training abroad that is unavailable in their home country, as the medical training opportunities in South Africa are quite comprehensive.The 5000-plus physicians trained in sub-Saharan Africa who have migrated to the USA comprise only a small proportion of the total number of IMGs practicing in the USA. However, their relatively small numeric addition to the USA medical workforce contrasts markedly with the impact of their migration on the medical workforce in sub-Saharan Africa. Moreover, in absolute terms, the USA has drawn more of the medical workforce of Africa than either Canada, with 2151 African graduates , or the UK, with 3451 , largely because of the relative size of the health care system in the USA.Including the USA, the UK, and Canada, then, 10 936 physicians trained in sub-Saharan Africa are practicing in the three countries, a number that represents 12% of all African physicians. We expect there are many more African physicians who are in the UK, as well, as our figures from there include only those who arrived post-1992. While some of the physicians in residency training will return home, there are unknown others who could be practicing medicine at home but were not able to get licenses abroad and therefore are engaged in other occupations. Almost all the medical schools sending graduates to the USA provide their instruction in English.Our study provides some measures of sub-Saharan Africa physician migration to the USA. A next step is to collect data from other developed countries and begin to create a physician migration data set from multiple countries. Migration from country to country within the subcontinent is also worthy of further examination, and it would be useful to identify the relationship between country of birth and country of training. There are likely to be some medical schools that draw students from several African countries. It should be noted, as well, that there are physicians practicing in Africa who did not train there, most prominently Cubans.Some sub-Saharan countries lose a larger proportion of their physicians to the USA than others. For example, while Ghana has a reported 1210 practicing physicians in its country, 478 graduates of Ghanaian medical schools are practicing in the USA. Without even considering those who have migrated to other countries, these 478 Ghanaian graduates in the USA represent 30% of Ghana's potential medical workforce .The migration of physicians from sub-Saharan Africa represents a lost investment of significant training costs, since graduates of medical schools in Africa are likely to have contributed financially to only a small portion of the costs of their medical education . MedicalThe United Nations Commission for Trade and Development has estimated that each professional leaving Africa costs the continent USD 184 000, or USD 4 billion a year – one third of official development funds to Africa . The losOstensibly, the USA welcomes IMGs for two purposes. First, as a form of foreign aid, it provides specialty training that physicians can take back to their home countries for the benefit of residents of those nations. Second, IMGs fill positions in specialties and locations that are less attractive to their USA counterparts, and may help to correct physician maldistribution in some rural or underserved areas of the USA. Longitudinal tracking of physicians entering the USA has indicated, however, that few IMGs ever leave the USA after arriving for residency training , and theAttracting physicians to rural practice in most countries is difficult, and is accomplished only through a careful set of policies designed to provide incentives for rural service. In most poor as well as rich countries, physicians are concentrated around urban hospitals that offer tertiary care, even though more rational service delivery systems might focus on a geographically decentralized system of primary and preventive care. Poor countries that offer medical training to produce too many physicians with highly technical skills, some of whom who cannot find satisfying jobs, may further contribute to physician migration . India aOur findings show African physicians are unlikely to select small or remote rural practice opportunities in either their home countries or in the USA, but the preponderance of African physicians in American inner-city underserved areas may to some extent be helpful to USA needs by boosting the number of minority physicians in the urban health workforce. The growing number of African immigrant female physicians follows the trend for increasing numbers of female physicians trained in the USA, and probably has similar implications. Some researchers have found, for instance, that female physicians are less likely to practice in rural areas .While the sub-Saharan Africa region as a whole loses many of its physicians, it is apparent that a small handful of medical schools is the sources of the majority of this migration. Ten medical schools in four countries – South Africa, Nigeria, Ghana and Ethiopia – produce 79.4% of the émigré physicians to the USA, out of a total of 87 medical schools in the region. This suggests policy approaches to reducing the "brain drain" from Africa could be targeted at only these few countries or medical schools, a less daunting task than addressing the problem in 47 different countries.Medical migration results from the complex interaction of myriad social, legal and economic forces. Single country policies are unlikely to alter the flows significantly. Even if the USA acknowledges that that it benefits from luring medical professionals here for whose medical school training we do not pay, solutions that would be compatible with social justice principles are not clear. Furthermore, if all African doctors returned to their home countries today, they would not necessarily find satisfactory employment opportunities in cash-strapped health systems.The 57th World Health Assembly, in 2004, adopted a resolution to urge member countries to develop strategies to mitigate the adverse effects of migration of health workers; to develop policies that could provide incentive for health workers to remain in their countries; and, among other issues, requests WHO to help countries set up information systems to monitor the movement of health resources for health, and to include human resources for health development as a top-priority program at WHO from 2006 to 2015 .In an ideal world, freedom of movement is a universal right for individuals, as there is ostensibly no rational reason why anyone would have a stronger right to be in any place more than anyone else . Today, Importing health professionals from poor countries to provide care in rich countries is not consistent with a rational workforce policy rooted in social justice principles. In the short run, Mullan  and otheThe New York Times editorialized that the "obvious long-term solution to the medical brain drain is for wealthier countries to reimburse Africa's health and educational systems for the cost of poaching their professionals, and to greatly increase the financing and technical help for Africa's health systems" [In response to a recent report by the USA Physicians for Human Rights , The Newsystems" . This unThe New York Times response made a strong recommendation that the International Monetary Fund, World Bank, and other donors refrain from withholding loans or grants from countries that increase their spending on "health, education and other sectors and activities needed to promote human development, including to enhance salaries to health staff or to hire new health personnel."The same Physicians for Human Rights report that prompted One of the major limitations African nations face in addressing their health workforce problems is the lack of reliable data on how many health workers have graduated from their schools, how many are working in the country and in what locations, and how many have emigrated. It is urgent that poor countries put together the information systems required to track these data, as a basis for workforce policy and investment decisions.And, finally, the fact that so few medical schools generate the vast majority of emigrants creates an opportunity to focus attention in a strategic way. These schools might be enticed to redirect their missions towards producing graduates who intend to serve their own countries. This would likely require curriculum changes, admissions policy changes, and a change in faculty culture to ensure that emigration is not promoted as a mark of prestige.The author(s) declare that they have no competing interests.AH conceived the project, designed the research and wrote the paper. MJT and KEJ actively participated in the conceptualization and re-writing of the paper. MF conducted the data management and analysis. LGH provided guidance, advice and editing assistance. All authors read and approved the final manuscript.


Every spring and fall, billions of songbirds fly thousands of miles between their summer breeding grounds in North America and their wintering grounds in the more hospitable climes of southern California, Mexico, and Central and South America. While some birds fly during the day, most, including the white-crowned sparrow, fly under cover of night. Many aspects of this remarkable voyage remain obscure, especially if, and how, nocturnal migrators get any sleep at night.A tracking study of the Swainson's thrush found that the roughly seven-inch birds flew up to seven hours straight on six of seven nights, racking up over 930 miles. While the study didn't track their daytime behavior, the birds' migratory pace—as well as the increased activity required to sustain migrations—suggests little time for sleep. Yet field observations indicate that presumably sleep-deprived fliers appear no worse for wear, foraging, navigating, and avoiding predators with aplomb. Researchers are left trying to reconcile this observation with the vast body of evidence linking sleep deprivation to impaired neurobehavioral and physiological function. How do songbirds cope with so little sleep? Do they take power naps? Have they taken “sleep walking” to new heights? Or have they managed to selectively short-circuit the adverse effects of sleep deprivation during migratory stints?To investigate these questions, Ruth Benca and colleagues studied cognitive and sleep behaviors in captive white-crowned sparrows over the course of a year. The sparrows fly nearly 2,700 miles twice a year between their Alaska and southern California homes. In laboratory cages, the birds' migratory instincts manifest as increased restlessness at night during the migratory season, with lots of hopping around and wing flapping.Niels Rattenborg et al. characterized the birds' activity levels with motion-detection measurements and video recordings, and placed sensors on their brains to monitor their seasonal sleep patterns. The brain recordings showed a marked seasonal difference in both the amount and type of sleep during a 24-hour period: migrating birds spent roughly two-thirds less time sleeping than nonmigratory birds and fell into REM sleep  much sooner. Birds displaying active migratory behavior appeared completely awake during such activity. Cognitive tests—birds performed a task that involved pecking a key in exchange for seed—revealed that birds in the nonmigrating state suffered cognitive deficits when sleep-deprived but displayed an “unprecedented” ability to maintain cognitive function in the face of ongoing sleep loss in the migratory state.These results suggest that wild songbirds drastically reduce sleep time during migration, though Benca and colleagues concede it's impossible to know for sure without recording the birds in action. And it is unclear what molecular mechanisms jumpstart the migratory mindset. Such an ability to temporarily circumvent the need for sleep, however, could prove useful for humans in situations that demand continuous performance. Some studies link migration with increased neuroendocrine activity, which is in turn associated with sleep disruption, accelerated timing of REM cycles, and mood disorders in humans. “Like migrating sparrows,” the authors note, “both depressed and manic patients show reduced latency to REM sleep, loss of slow-wave sleep, and reduced amounts of total sleep.” Given the parallels between migratory behaviors and bipolar illness, it's possible that similar mechanisms may be involved in both.Whatever the mechanism, the unprecedented imperviousness of migrating songbirds to sleep deprivation, the authors conclude, clearly warrants further testing. But it also raises interesting questions about the role of sleep, which recent studies suggest is required to incorporate novel perceptions into the brain's memory banks. If this is true, how do songbirds consolidate memories of migratory events with so little sleep? Understanding the mechanisms that power the sleepless flight of songbirds promises to unravel one of the longstanding mysteries of their improbable journey. It may also shed light on the origins of sleep-related seasonal disorders and the much-debated role of sleep itself.