Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives. Microarray technology is rapidly becoming a standard technology used in research laboratories all across the world. Since its first application in the mid 1990s -7 with oDNA microarrays, microscopic arrays of large sets of DNA sequences immobilized on solid substrates, are valuable tools in areas of research that require the identification or quantitation of many specific DNA sequences in complex nucleic acid samples . They ar1. Determination of transcriptional programs of cells for a given cellular function (e.g., cell function, cell differentiation, etc.) or when they are exposed to certain conditions leading to activation, inhibition or apoptosis.2. Compare and contrast transcriptional programs to aid diagnosis of diseases, predict therapeutic response and provide class discovery and sub-classification of diseases.3. Identification of genome-wide binding sites for transcriptional factors that regulate the transcription of genes.4. Prediction of gene function.5. Identification of new therapeutic targets .6. Development of public databases that will help us understand the functioning of complex biological systems.7. Genetics of gene expression: Although this is a relatively new study field, it is advancing rapidly with major implications in complex clinical traits by the identification of promising candidate genes. Thus, we briefly review the current implementations of this novel approach highlighting its necessity in the research field. Treating mRNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Unlike classical quantitative traits, the genetic linkages associated with transcript abundance permits a more precise look at cellular biochemical processes. Schadt et al.  describeDNA microarrays are used to estimate the levels of mRNA in the cell. The process can be described in three steps:Array construction: Currently, there are two widely used microarray technologies:1) • In situ synthetized oligonucleotide (20–25 mers) microarrays-mainly oligonucleotides synthetized by photolithography or ink-jet technology on a glass surface.• Spotted, in glass or nylon membrane matrices, microarrays-mostly created by robotic printing of pre-prepared cDNAs or oligonucleotides (polymerase chain reaction-PCR-generated products from cDNA libraries or clone collections) ,15.Sample preparation and array hybridization: The next step in the microarray experiment is to prepare the material that will be hybridized with the microarray. Gene expression is measured by the amount of mRNA therefore it must be extracted from the sample cells or tissues. For high-density microarrays one has to convert mRNA into cRNA, whereas for spotted arrays one can use mRNA, cDNA, or cRNA. The RNA needs to be labeled so that the detecting machinery can measure the quantity of RNA present. In oligonucleotide microarrays mRNA is extracted from experimental samples and is labeled with a fluorescent oligonucleotide (biotin). The biotin labeled cRNA and each sample is hybridized to a separate array, the array is scanned and absolute expression levels are obtained for each probe by using a dedicated laser scanner. In contrast, in spotted microarrays, mRNA is extracted from a sample and a control and one is labeled with cy-5 (red fluorescent dye) and the other with cy-3 (green fluorescent dye). Expression values are based on the competitive hybridization of the two samples being directly compared on a single array. Conventionally, in radioactive nylon membrane arrays RNA probes are labeled with P33 or P32 dCTP during a reverse transcription reaction. The great advantage of nylon microarrays is that they require relatively small amounts of PCR products because radioactive targets have a higher intrinsic detectability whereas in glass arrays the quality of RNA is not as critical as it is in nylon arrays. Therefore nylon arrays are mainly used when sample material is scarce or a small number of genes need to be assayed [2)  assayed ,17.3) Image analysis and data acquisition: The laser causes excitation of fluorescently or radioactively labeled cDNA probes. Only the spots representing mRNAs in the sample give emission signals. The emission is measured using a scanning confocal laser microscope for fluorescently labeled arrays or a flat-bed scanner for radioactive nylon arrays and finally data is analyzed by appropriate software. In spotted microarrays using fluorescent probes if particular mRNA from the sample is in abundance, the spot with a complementary probe will be red; if the concentration of the particular mRNA is higher in the control, the spot will be green. If both samples contain the same amount of a given mRNA, the spot will be yellow  is a refractory and lethal interstitial lung disease characterized by fibroblast proliferation, extracellular matrix (ECM) deposition and progressive lung scarring. The incidence of IPF is estimated at 15–40 cases per 100.000 per year, and the mean survival from the time of diagnosis is 3–5 yr regardless of treatment ,21. The -/-), thus protected from pulmonary fibrosis, and wild type mice. Interestingly a simple hierarchical cluster analysis identified most of the known TGF-β1 inducible genes preferentially induced in wild type mice. The latter finding provides support for the hypothesis that β6 knockout mice are protected from pulmonary fibrosis as a consequence of failure to activate TGF-β. The great importance of this study results from the identification and the global availability of several genes that are likely to be directly relevant to the fibrotic process. However the inability of microarray technology to detect genes that are not included in the array, identify critical proteins that participate in biological responses and ascribe changes in gene expression in specific cellular types limit the scientific rigidity of the data derived and highlights the necessity for combined application of novel approaches.In another study Kaminski et al.  used oliFurthermore driven by the same perspective idea to investigate the gene expression pattern in bleomycin-induced pulmonary fibrosis, Katsuma et al.  construcin vivo was established in a separate set of confirmational experiments in rats where it was revealed an overexpression of the ID by myofibroblasts within the fibrotic regions. These novel suggestions have major impact on our understanding of the crucial role of TGF-β1 as a fibroblast differentiation factor in response to fibrogenic stimuli. Nonetheless the present study does not determine the precise role of ID in regulating fibroblast responses to TGF-β1. To do so this study should be coupled with independent methods using ID blocking strategies. Moreover the use of incomplete arrays that detect only the included genes, the variability of cellular composition of tissues studied even from the same organ and the inability of this technology to distinguish changes in cellular composition from transcriptional changes pose major limitations to the global application of these results. Furthermore these observations offer plausible explanations for the lack of similar effects of TGF-β1 in previous studies.One of the most informative studies scrutinizing the global gene expression profile of fibroblasts in response to one of their most potent activators, TGF-β1, has been published by Chambers et al.  Gene expMoreover, bleomycin-induced pulmonary fibrosis rat model was extensively studied by Liu et al.  with theThese preliminary results suggest that this technology could identify unexpected molecular participants in IPF and might help in the development of novel targets for improved treatment. The method may also allow molecular fingerprinting that could improve the ability to identify subclassifications of pulmonary fibrosis that might be more informative than the current classification based primarily on histologic and radiographic patterns . NonetheAsthma is one of the most serious allergic diseases associated with both genetic and environmental factors such as allergens, respiratory tract infections, and atmospheric pollutants. Most asthma is associated with atopy, a predisposition to generate immunoglobulin (Ig)-E against environmental allergens . HoweverMicroarray technology offers a new opportunity to gain insight into global gene expression profiles in asthma, leading to the identification of asthma associated genes. Several experimental models have been used for this purpose although no animal disease model is identical to human disease. Zou et al. were theBrutsche et al.  designedSeveral morphologic changes in the airways of patients with asthma have been attributed to the Th-2 produced cytokines such as IL-13 and IL-5. However the molecular mechanisms underlying the contributions of these cytokines to asthma remain largely unknown.in vivo and in vitro.Towards this direction Lee et al.  applied One of the greatest disadvantages of microarrays and at the same time challenges for most of the investigators is the objective difficulty dealing with the results of the experiments resulting from the large quantities of information. Currently, the hurdle faced is the routine interpretation of this information to identify among thousands of dysregulated genes, those who are informative, causal and specific to the phenotypic change of interest. Thus, Temple et al.  comparedMicroarrays apart from their remarkable effectiveness in identifying novel gene expression patterns can also be used to clarify physiological mechanisms underlying the actions of numerous drugs, such as those applied in the management of patients with asthma. Several studies have utilized microarray technology to assess the gene expression profile of cells and tissues before and after treatment with commonly applied drugs such as corticosteroids. Two of them are reviewed here.Recently, Hakonarson et al.  in additMicroarray analysis performed by Laprise et al.  indicateMast cells represent key cells in the initiation and progression of asthma, releasing several mediators of inflammation, such as certain cytokines and chemokines. The past few years several studies have been focused on the identification of new mast cell products through the gene expression analysis. In one of them published by Sayama et al.  applicatFurthermore, Nakajima et al.  in theirMicroarrays using nylon membrane radioactive cDNAs have already been applied in the research field of asthma and much good work has been done with this technology.Brutsche et al.  applied One of the first gene-profiling studies highlighting the potential role of chemokines and their receptors in the pathogenesis of asthma was conducted by Syed et al.  They useIn another study Banerjee et al.  in theirAccumulated evidence from these analyses revealed that microarray analysis can be a powerful tool for identifying mediators of allergic asthmatic disease through a genomic-based strategy using non-human primates and provide us a novel large scale of differentially expressed genes. Additionally, authors compared different cellular models sharing similar experimental paradigm to focus on the most likely informative genes and filter out the bystanders. The application of this approach further streamlined the pivotal role of microarrays in determining transcriptional responses of genes to several inflammatory cytokines and in identifying gene expression patterns and important mediators associated with the initiation and the progression of asthma. Moreover, microarrays coupled with separate set of experiments have provided the investigators with useful knowledge concerning the efficacy of the already applied drugs in the treatment of asthma and helped them to understand their anti-inflammatory role in terms of physiology and molecular biology. Nevertheless, most of the studies exhibited essential weaknesses generated by the heterogeneity of samples studied and compared and by the disability of microarrays to quantify gene expression and yield information about transcriptional responses and post-translational protein modifications. Therefore more in depth analysis of the microarray results is needed in combination with novel approaches that will help us focus on the specific genes and elucidate their role in the cellular function and the pathogenesis of asthma Tables ,3.Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by progressive airflow obstruction, chronic cough and dyspnea in advanced stages, caused by smoking, environmental, and hereditary factors. It is associated with two clinical entities, chronic bronchitis and emphysema. In nowadays, the invention and application of microarray technology offers scientists the opportunity to gain a better understanding on the pathophysiology of COPD through the identification of novel gene expression patterns, leading to illumination of genes candidates for modern therapeutical approaches .It is already known that chronic bronchitis can be induced by several types of environmental pollutants such as diesel exhaust particles (DEP). Though recently a microarray study has been published by Koike et al.  addressiMicroarray approach is already being applied in respiratory clinical pharmacology with the identification of genes {Yamanaka et al. } that caOne of the major limitations in our attempt to elucidate the exact role of specific cell types in the pathogenesis of COPD is the compact anatomy of the lung which makes unraveling specific cell type gene expression changes difficult, requiring immunoelectron microscopy or laser capture microdissection. The first study to perform quantitative cell type-specific gene expression analysis using the pioneering technology of laser capture microdissection in human tissue samples coupled with RT-PCR and cDNA approach was recently published by Fuke et al.  Authors Several inflammatory cytokines have been implicated in the pathogenesis of emphysema, including TNFa, a molecule with versatile pathogenetic mechanisms. As a means to investigate some of them that culminate to lung-pathology, Vuillemenot et al.  applied Despite the clear link of smoking to the risk for chronic bronchitis, only 15–20% of cigarette smokers develop COPD,  suggestiOne of the most exciting aspects of microarrays is their use as tools for actively introducing serendipity to one's research . In expePresently, very few studies dealing with the role of HOX genes in the adult respiratory system are available in the literature. Golpon et al.  investigCollectively these results suggest that microarray analysis with its ability to highlight gene expression profiles on a large scale and coupled with progressive technologies and independently validated data has led researchers to shed further light into transcriptional programs regulating emphysema and to the identification of common mediators and molecular pathways involved in the pathogenesis of both COPD and pulmonary fibrosis, indicating novel targets for therapeutic interventions and useful genetic markers assessing susceptibility to COPD. Although limitations such as inconsistency between findings derived by microarray approach and independent studies, lack of functional changes assessment and significant data variability can be detectable in these studies, however evidence derived from these analyses is valuable and heavily informative Table .4. Novel interactions between factors (antioxidant genes) previously associated with ALI and factors (surfactant proteins) previously not associated with ALI emerged from the application of functional genomics during nickel-induced ALI. Data derived from this experiment and partially confirmed by Northern blot analysis and nuclease protection assays is valuable and consistent with the ongoing attempts to treat ALI with exogenous surfactant-associated proteins [Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults. This syndrome that responds poorly in therapeutic interventions and has a poor prognosis has been associated with a myriad of mediators including cytokines, reactive oxygen and nitrogen species, growth factors and proteolytic enzymes ,55. Despproteins  in combiAlthough the fibroproliferative response to lung injury occurs in high frequency in patients with ALI, the mechanisms of this response are largely unknown. One of the most meaningful and informative studies addressing this important issue was recently published by Olman et al . AuthorsALI is frequently associated with endotoxemia and is characterized by the accumulation in the lungs of large populations of neutrophils activated to produce proinflammatory mediators. Many studies had demonstrated a critical role of the endotoxemia-induced activation of NF-κB in neutrophils in the development of ALI ,60. One To gain a more comprehensive understanding on the complex interplay between several inflammatory cytokines involved in the pathogenesis of pulmonary edema, several group of investigators {Cher et al. , SabbadiThe study of Perkowski et al.  has beenOne of the first gene-profiling studies to address an important disease process, such as sepsis at a multisystem level, was that of Ward et al.  Using DNIn summary, these studies exhibit the crucial role of a novel molecular technology in discovering, through global analysis of gene expression, genes previously identified only by their DNA sequence. Although the array analysis provides in some studies a comprehensive overview of gene expression in the lung during ALI ,58,61,64Sarcoidosis is a chronic systemic disorder characterized by the presence of non-caseating granulomas and accumulation of T-lymphocytes and macrophages in multiple organs . The mecCollectively these findings not only reveal the importance of the microarray platforms in identifying gene expression patterns that give the scientists the opportunity to elucidate the pathophysiological processes of complex diseases, such as sarcoidosis but also illuminate some of their origin disadvantages.The last five years has seen the emergence of a novel technology applied in almost every aspect of respiratory research, a technology that has also great future perspectives and may provide scientists with numerous avenues of investigation that have clinical implications. Since nowadays, microarray technology has been successfully used for the identification of potential target genes for therapeutic intervention in IPF, mechanisManaging and mining the huge amount of data generated by microarray experiments still remains a major challenge and limitation for most investigators. Whether gene expression changes are considered primary vs. reactive for a given disease is a complicated issue, and one can only begin to judge that if the methods and approaches used to generate the data are of sufficient scientific rigidity. The diversity and scope of the data require the creation of multidisciplinary teams consisting of physicians, biologists and bioinformaticians  . Thus, wBecause of the statistical issues raised by microarray technology, it is necessary for any meaningful interpretation that the data is replicated using independent methodological criteria, preferably with separate samples rather than the tissue or RNA used to derive the original targets. A rapid high through-put method for confirmation of microarray data is quantitative RT-PCR. Alternatively, Northern blots or ribonuclease protection assays provide the benefit of direct quantification. So far some studies have started to adapt these approaches Figure  but therCurrently, the use of microarray technology in respiratory research is limited by the tissue sample, incomplete available arrays and the analysis of data generated from this technology. Clinical use of microarrays technology is still in its infancy and remains exploratory. For these array-based methods to become truly revolutionary, they must become an integral part of the daily activities of the typical molecular biology laboratory and biomedical institute. There is plenty of room for technical improvements, further development, and more widespread acceptance and accessibility. Optimistically thinking we expect that over the next few years the pattern of development and use of microarrays will be on a similar trajectory to that seen for computers and other high-tech electronic devices, which started out as exotic and expensive tools in the hands of the few developers and then moved quickly to become easier to use, more available and less expensive. Alternatively authors believe that so far microarrays have not added much to our understanding and the possibility to live up to the great 'hype' that was generated belongs to the distant future.Our view is that the application of these approaches will improve dramatically the effectiveness and reliability of microarray technology in studies of diseases of complex organs like the lung, and will have a major impact on our understanding of molecular pathogenesis for the foreseeable future. Whether our hopes will be fulfilled or disproved remains to be seen.ADA: Adenosine DeaminaseALI: Acute Lung InjuryASM: Airway Smooth Musclea-SMA: a-Smooth Muscle ActinBAL: Bronchoalveolar lavageCAGE: Composite Atopy Gene ExpressionCOPD: Chronic Obstructive Pulmonary DiseaseCCR7: Chemokine Receptor 7DEP: Diesel Exhaust ParticlesECM: Extracellular MatrixeQTL: expression quantitative trait locusG.I: Gene IntensityIg: ImmunoglobulinID: Inhibitor of DifferentiationI/D: Intensity/Background ratioIPF: Idiopathic Pulmonary FibrosisMBP: Major Basic ProteinMCs: Mast CellsMCP: Monocyte Chemoattractant ProteinMMP: Matrix MetalloproteinaseNF-κB: Nuclear Factor-Kb2: snake venom phospholipase A2nsPLART-PCR: real time-polymerase chain reactionTGF-b: Transforming Growth Factor-bTNF: Tumor Necrosis FactorUIP: Usual Interstitial PneumoniaVEGF: Vascular Endothelial Growth FactorThe authors declare that they have no competing interests.AT, GP and DB were involved with the study conception. AT performed the data acquisition and interpretation. DB and GP were involved in revising the article for important intellectual content. All authors read and approved the final manuscript.


Endosymbiotic bacteria live within a host species. There are many and diverse examples of such relationships, the study of which provides important lessons for ecology and evolution Symbiosis, an interdependent relationship between two species, is an important driver of evolutionary novelty and ecological diversity. Microbial symbionts in particular have been major evolutionary catalysts throughout the 4 billion years of life on earth and have largely shaped the evolution of complex organisms. Endosymbiosis is a specific type of symbiosis in which one—typically microbial—partner lives within its host and represents the most intimate contact between interacting organisms. Mitochondria and chloroplasts, for example, result from endosymbiotic events of lasting significance that extended the range of acceptable habitats for life. The wide distribution of intracellular bacteria across diverse hosts and marine and terrestrial habitats testifies to the continued importance of endosymbiosis in evolution.PLoS Biology, the publication of the full genome sequence of the reproductive parasite Wolbachia allows the first genomic comparisons across this range , and their impressive genetic diversity that spans several major bacterial groups. More recently, studies in genomics of obligate mutualists—and now The distinct lifestyle of endosymbionts has clear effects on rates and patterns of molecular evolution. Compared to free-living relatives, endosymbionts are thought to have reduced effective population sizes due to population bottlenecks upon transmission to host offspring and, in the case of obligate mutualists that coevolve with their hosts, limited opportunities for gene exchange. The nearly neutral theory of evolution  predictsIt is increasingly clear the distinct lifestyle of endosymbionts also shapes the architecture and content of their genomes, which include the smallest, most AT-rich bacterial genomes yet characterized . A commoBuchnera of three aphid host species, Wigglesworthia of tsetse flies, and Blochmannia of ants , and a relatively young mutualist of weevils (Sitophilus oryzae primary endosymbiont [SOPE]) show that genome streamlining in the endosymbionts may preclude extracellular existence, and highlight modifications in metabolic pathways to complement specific host physiology and ecology .Facultative mutualist: A beneficial symbiont that associates with the host, but can also live apart from it. Examples include Rhizobium spp. that associate with legumes, but also have a free-living stage to their life cycle.Obligate mutualist: A beneficial symbiont that lives exclusively with its host and depends on the host for survival. Examples include many nutritional endosymbionts of insects, which cannot survive outside of the insect host cell. These associations are reciprocally obligate when the host cannot survive without the endosymbiont.Parasite: A symbiont that has a negative effect on host fitness, in contrast to a mutualist, which increases host fitness.Reproductive parasite: A symbiont that manipulates host reproduction to its own benefit, but at the expense of host fitness. Reproductive parasites typically bias offspring toward infected females.Symbiosis: An association between two more species.


In a previous study we demonstrated the existence of numerical and functional alterations of γδ T cells in healthy elderly. Recently, we analysed the involvement of γδ T lymphocytes in malignant melanoma, describing a lower frequency of circulating γδ T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients.In this study we investigated the existence of numerical and functional alterations of circulating γδ T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects.+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vδ2 T cells whereas the number of Vδ1 T cells was not affected. A higher percentage of γδ+ T cells producing TNF-α was found in old healthy donors, whereas a reduced number of TNF-α-producing γδ+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-γ production. After a 10-day in vitro culture, both the percentage and the expansion index of γδ T cells, and in particular of Vδ2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects.We demonstrated that the number of circulating γδOur study demonstrates that the numerical and functional impairment of γδ T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of γδ T cells found in old healthy subjects. We suggest that a similar impairment of γδ T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma. In huAlthough little is known about the physiologic significance of γδ T cells, their marked reactivity toward mycobacterial and parasitic antigens as well as tumor cells suggests that γδ T cells play a role in the anti-infectious and anti-tumoral immune surveillance ,4. Few dOn the basis of the pivotal role that γδ T cells may have in the elderly and in the immune response against melanoma we tried to find out a possible correlation in the alteration of γδ T cells between aged people and melanoma patients. In this study we evaluated the peripheral representation, the in vitro expansion, and cytokine production γδ T cells from young/adult and old melanoma patients, comparing the results with those obtained in age-matched healthy controls.Human peripheral blood was obtained from 9 young , 12 old  melanoma patients, and 10 young , and 13 old  healthy donors. Healthy subjects were volunteers in good and stable clinical conditions, and had laboratory parameters in the physiological range. We excluded subjects in poor health with degenerative diseases or in therapy with drugs interfering with the immune system. Melanoma patients have been admitted to the Dermatology Unit of the I.N.R.C.A. Hospital of Ancona. Melanoma patients were in good health other than for the existence of melanoma as checked on the basis of clinical and laboratory parameters. The investigations were performed after approval by a local institutional review board. A written informed consent was obtained from each subject. Diagnosis of melanoma was histologically confirmed. All patients brought cutaneous primary non-metastatic melanoma and were staged according to the new American Joint Committee on Cancer staging system for cutaneous melanoma . A blood2+ and Mg2+- free phosphate buffered saline (PBS, Gibco/Life Technologies, Gaithersburg, MD, USA) and resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml)  at a concentration of 1.5 × 106/ml. Mononuclear cells were cultured in the complete medium supplemented with 100 U/ml of IL-2 . Phosphoantigen-specific stimulation of γδ T cells was performed using the nonpeptidic antigen isopentenylpyrophosphate . The cells were incubated at 37°C in an atmosphere of 95% air, 5% carbon dioxide, at 90% relative humidity in 24 well plates.Fresh peripheral blood mononuclear cells (PBMC) were fractionated on Ficoll-Paque  and separated by density gradient centrifugation (400 g, 30 min). Cells from the interface of the gradients were washed twice with CaPBMCs were analysed for cell phenotype through double staining with the following monoclonal antibodies (mAbs): anti-CD3 (PE) and anti-pan γδ T cells (FITC), or anti-TCR Vδ1 or anti-TCR Vδ2. The phycoerythrin (PE) -conjugated monoclonal antibody anti-CD3 was purchased from EuroClone (Devon, UK). The fluorescein isothiocyanate (FITC) -conjugated anti-pan TCR γδ, anti-TCR Vδ1, and anti-TCR Vδ2 were purchased from Endogen (Boston, MA, USA). IgG1 (Becton Dickinson) was used as isotype control.6 PBMCs were washed in PBS containing 0,1% NaN3 plus 5% FBS and labelled with 5 μl of anti-CD3 or anti-TCR Vδ1 MoAbs or 2.5 μl of anti-pan TCR γδ or anti-TCR Vδ2 for 30 min in ice. At the end of the incubation, cells were washed in PBS containing 0,1% NaN3, resuspended in PBS (Gibco) and immediately analysed with a Coulter XL flow cytometer.0.5 × 106 stimulated cells were stained with the anti-pan TCR γδ mAb for 30 min at 4°C. Fixation-permeabilization of cells was performed in PBS/2% paraformaldehyde for 15 min at 4°C, followed by incubation for 30 min at room temperature in the dark with PE-conjugated anti-human IFNγ mAb or anti-human TNF-α mAb diluted in PBS, 1% BSA, and 0.05% saponin. Cells were finally washed twice in PBS, 1% BSA, and 0.01 % saponin and analysed on a XL flow cytometer (Coulter).Mononuclear cells were stimulated with IPP and IL-2 for 18 h, and GolgiPlug (a protein transport inhibitor containing brefeldin A, PharMingen, Milton Keynos, England) was added during the last 12 h of culture to block intracellular transport processes and allow cytokine accumulation. 0.5 × 10PBMC were cultured for up to 10 days in the complete medium supplemented with 100 U/ml of IL-2 and 30 μg/ml of IPP to determine a phosphoantigen-specific stimulation of γδ T cells. After 1 wk of culture, the volume corresponding to half the culture medium was replaced by fresh medium. On day 10 of culture viable cells were determined by trypan blue exclusion and used for FACS analysis. The viability was always greater than 98% as determined by trypan blue exclusion. The expansion of γδ T cells was followed by cytometric analysis through double staining of stimulated cells with anti-CD3 (PE) and anti-pan γδ, or anti Vδ1, or anti Vδ2 T (FITC) mAbs. The absolute number of γδ T cells in each culture was calculated as follow:  × /100. The γδ T cell expansion index was then calculated by dividing the absolute number of γδ T cells in stimulated cultures by the absolute number of γδ T cells before culture .P < 0.05). The statistical analysis was performed with SigmaStat software version 1.03 (Jandel Scientific, Germany).Data were analysed for statistical significance by using parametric or nonparametric tests according to the distribution of the data. Comparisons of variables among groups were made by one-way analysis of variance (ANOVA) or Kruskal-Wallis one-way ANOVA on ranks. When significant differences were found, the differences among groups were made by the Student-Newman-Keuls method or Dunn's method. Difference between means was considered significant at the 5% level . As shown in Table Peripheral blood lymphocytes from 9 young/adult and 12 old melanoma patients and 10 young and 13 old healthy subjects were analysed for the percentage and the absolute number of γδ T cells through double staining with anti-CD3 and anti-γδ mAbs. As shown in Table Since it has been demonstrated that activated γδ T cells produce TNF-α and IFN-γ, we studied the intracellular production of these cytokines in one-day stimulated γδ T cells from healthy subjects and melanoma patients. As shown in Fig. The expansion of γδ T cells was evaluated after 10 days of culture in the presence of IPP and low dose interleukin-2 (IL-2). Both the proportion of γδ T cells, evidenced by double staining FACS analysis, and their relative increase in comparison with the γδ T cell number found on day 0 (expansion index), were evaluated. As shown in Fig. We and others have demonstrated an impaired potential of γδ T cells in aged people, as evidenced by the reduction of the absolute number of circulating γδ T cells, in particular of the Vδ2 T subset, an altered pattern of cytokine production, an impaired in vitro expansion of these cells, and an increased expression of the early activation marker CD69, in aged people in comparison with young subjects ,17,18. RWe demonstrated that both the number of circulating γδ T cells and their in vitro expansion were decreased in melanoma patients and that the impairment did not correlate with the age of patients. Young/adult and old melanoma patients had a similar derangement of γδ T cells, and this impairment had numerical and functional characteristics like to those found in old healthy subjects. This evidence stresses the relevant role that this lymphocyte population may exert, either directly or through the regulation of T cell-mediated specific responses , both inThe reduction of γδ T cell number well correlated with the decrease of the Vδ2 T cell subset, i.e., the most frequent subset of circulating γδ T cells . The Vδ2Under normal conditions, γδ T cells respond to antigen challenge by secreting large quantities of TNF-α and IFN-γ ,21 whichIn conclusion, we demonstrate that the numerical and functional derangement of γδ T cells which we have found in melanoma patients, is not correlated with age of donors, and that old patients with cutaneous primary melanoma have an impairment of γδ T cells similar to that found in old healthy subjects. This evidence suggests a link between γδ T cell deterioration and the low protection against infections and tumor diseases present in the elderly, as well as the inefficacious immune defense against melanoma, both in young/adult and old ages.


The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon.rbcL, matK and rps2) show significant rate heterogeneity among lineages and among genes. Many of the non-photosynthetic plants have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not always immediate upon loss of photosynthesis. Overall there is a poor correlation of synonymous and nonsynonymous rates. There is, however, a strong correlation of synonymous rates across the 3 genes studied and the lineage-speccific pattern for each gene is strikingly similar. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is a weaker correlation across genes for nonsynonymous rates. Here the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene.Phylogenetic reconstructions of relative rates of sequence evolution for three plastid genes  non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors. Rates of DNA sequence evolution vary among taxa and among genes, and the causes of this variation are many. In some cases, generation time has been shown to be correlated with rates in plants. For example, annual plants can sometimes have higher rates of DNA evolution than perennials . In one rN) and synonymous rates (rS). For example, when rN increases relative to rS, relaxation of purifying selection is a possible explanation. However, when rS increases, but the rN/rS ratio is not greatly affected, then an increase in the mutation rate is a possibility. An example of this is Plantago mitochondrial DNA [e) can increase the fixation rate of neutral and slightly deleterious mutations. Thus, if slightly deleterious mutations are common, both rS and rN are expected to be higher when Ne is low [A useful method for distinguishing among the potential causes of rate variation is to separately examine nonsynonymous rates  and nonsynonymous (dN) substitutions can be measured and are used to compare rates and calculate rate ratios.Rates can also vary if the underlying mutation rate varies ,11 or ifEpifagus virginiana, a nonphotosynthetic plant, has an increased rate of sequence evolution for plastid DNA in general [rps2 gene indicate a significant increase for both dN and dS. This suggests that purifying selection is at least partially relaxed and that there has been an increase in the rate at which mutations are entering the population in this species, due to increased mutation rate or lax DNA repair. MatK, another plastid gene, is characterized by a partial relaxation of purifying selection in the clade containing Epifagus, Orobanche and Boschniakia [E. virginiana and 38 of its relatives for three plastid genes: rps2, matK, and rbcL. Each of these genes is present in photosynthetic relatives of Epifagus, is accelerated (or even lost) in Epifagus or related parasitic plants. Although plastid encoded, the three genes encode proteins that participate in different processes in the plastid. rps2 encodes the ribosomal protein S2 in small subunit ribosome, matK is an intron maturase, and rbcL encodes the large subunit in the CO2-fixing enzyme RUBISCO. We ask several questions: When does the rate increase observed in Epifagus begin, relative to the evolutionary loss of photosynthesis? What are the causes? Relaxation of constraint? More mutations entering the population? Are these patterns consistent across multiple plastid genes? general -17 and rchniakia . In thisSchwalbea relative to the Alectra-Orobanche clade, the Bartsia-Melampyrum clade and the Castilleja-Pedicularis clade. It was also unresolved concerning the relationships among the outgroups Mimulus, Kigelia, Hemimeris, Verbascum, Antirrhinum and Veronica.Phylogenies of the Orobanchaceae and relatives were constructed using maximum parsimony (MP) and maximum likelihood (ML). The MP analysis discovered four most parsimonious trees of 3817 steps, with CI = 0.6275, CI (excluding uninformative characters) = 0.5118, and RC = 0.3888. The strict consensus tree was unresolved as to the position of Cistanche-Epifagus clade. When the MP consensus and the ML consensus were combined into a semistrict consensus tree, a completely resolved tree resulted. This tree is shown in Fig. The ML analysis found two trees, with -ln likelihood values of 24525.88663. The strict consensus of these trees was unresolved, but in a different place, regarding the position of the All three gene trees exhibited statistically significant rate heterogeneity (p < 0.0005), as assessed by the Kishino-Hasegawa (K-H) test . Synonymrps2 and rbcL synonymous and nonsynonymous evolution is poorly correlated, but in matK, the correlation is better  have increases in both synonymous and nonsynonymous rates. Rates are not, however, increased in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides.Some of the non-photosynthetic plants,  usually show an identical pattern of who is faster than whom. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way.Synonymous rates vary markedly among taxa. For example, the branches leading to lar Fig. . For exaMatK is much more rapidly evolving than the other two genes for nearly all taxa, but Epifagus' rbcL pseudogene has a similar rate. When looking at taxa across genes, there is much less consistency than with the synonymous rates. There are some big differences, such as the branch lengths for Striga, Cycnium and Schwalbea, and the Euphrasia species. Overall the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene.There is even more extensive variation in nonsynonymous rates, both among taxa and among genes. This is not surprising because these genes have different functions and some of the taxa are photosynthetic while others are not. The scale bars in Figs. matK rate variation is very similar in the synonymous and nonsynonymous figures. This fits well with the fact that it is less constrained overall, as can be seen by comparing the scale bars in Figs. The pattern of Harveya, Hyobanche and Boschniakia do not have higher synonymous rates.Rates were compared by using two categories at a time and testing for significant differences using likelihood ratio tests. These tests are summarized in Table matK: 2×LR = 10.67, df = 1, p = 0.001; rbcL: 2×LR = 31.3, df = 1, p = 2.2 × 10-8; rps2: 2×LR = 8.56, df = 1, p = 0.00343.Purifying selection is relaxed in the nonphotosynthetic plants for all three genes. The test values are as follows. Epifagus pseudogene has been excluded. Its unconstrained evolution is not typical of "nonsynonymous" change and its position on the plot made it an extreme outlier with an enormous influence on the regression line.Another way to describe the difference in the pattern of synonymous and non-synonymous rates is to say that the former are more correlated across genes. This can be seen in Fig. rps2 and rbcL, the synonymous plots are more highly correlated, whereas for matK, which is relatively unconstrained, they are about the same.For Epifagus [Epifagus, Cistanche, and the Orobanche species, have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not immediate upon loss of photosynthesis, since we do not see increases in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides. This pattern is similar to that found using smaller data sets [The dramatic rate increase observed in Epifagus , with brata sets ,20. Separs (and therefore larger ds) than a species-poor sister group. This was suggested as a cause of rate variation in non-coding DNA in the Lentibulareaceae [Orobanche is not). Thus, accurate numbers of species cannot be assigned to individual branches or clades. However, a few things can be noted. Euphrasia, with ~170 spp., is clearly more speciose than its sister group, with 3 spp. It has a somewhat faster ds. However, the Schwalbea lineage, with a single species, has a fairly high ds. Its position is not certain, but its sister group is probably the Bartsia – Melampyrum clade (>300 spp.), the Castilleja – Pedicularis clade, (>700 spp.), or the union of the two. These groups have do not have dramatically higher ds values; in fact the Castilleja – Pedicularis clade's value is slightly lower.The speciation rate hypothesis predicts that more speciose clades should have a faster areaceae . In thisBartsia, Euphrasia and Melampyrum contains mostly annuals [ds values, as one would expect from a generation time effect. However, both Euphrasia and Melampyrum contain almost exclusively annuals and have very different rates. Likewise, the large clade containing Boschniakia and Epifagus contains mostly annuals and has an overall high ds. The perennials Boschniakia and Cistanche have lower ds than their sister taxa, which also supports the generation time hypothesis, but there is as much variation among categories  as between categories.Differences in generation time may play some role in the observed rate variation. However, as was found previously , the pat annuals  and has The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many  non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors. Clearly, generating additional data from nuclear and mitochondrial genes would help us to more clearly distinguish among these hypotheses. Some of the above-mentioned hypotheses (generation-time, speciation rate) would be expected to affect nuclear and mitochondrial genomes in a similar fashion, whereas factors affecting mutation rate or efficiency of DNA repair would not, as these process involve different, though perhaps overlapping, sets of enzymes in each of the three genomes -24.We sampled 15 photosynthetic and 16 nonphotosynthetic Orobanchaceae, and eight outgroup taxa. The specimens used and their GenBank accession numbers are given in Table rps2 as in [matK as in [rbcL as in [rps2, 7 matK, and 5 rbcL sequences.We amplified and sequenced s2 as in , matK astK as in , and rbccL as in . A totalrps2 alignment was simple, containing only two small indels. For matK and rbcL, a search for the best alignment was conducted using Clustal X and a variety of alignment parameters. Alignments were evaluated according to the following optimality criterion: whichever alignment yields the MP tree(s) with the highest consistency is considered the best alignment[), which uses the simple indel coding method of Simmons and Ochoterena [rps2 had just one small indel and was aligned by eye. For matK, the optimal computer alignment was generated using GOP = 5 and gap extension penalty (GEP) = 1. Transitions were weighed the same as transitions. The RC from the analysis with indel characters included was 0.3693. The RC without indel characters was 0.3759. The alignment was then adjusted by eye. This final alignment yielded RC values of 0.3878 (indel characters included) and 0.3763 (indel characters excluded).The alignment. For alialignment and 0.4499 (indel characters excluded).For rps2, positions homologous to positions 48–660 of the Nicotiana gene were used. For matK, the entire gene was used. For rbcL, positions homologous to Nicotiana gene positions 5–1325 were used. The three genes were then combined into a single data set. PAUP* 4.0b8 [Nicotiana tabacum as the outgroup taxon, TBR branch swapping and 100 random addition replicates. Bootstrap analyses were conducted with the same settings, except with only 40 random addition orders. 500 bootstrap replicates were performed.For P* 4.0b8  was usedML analyses excluded indels. Using the hLRT method of the program ModelTest 3.06 , the ML . At least nine of the rbcL "genes" are probably pseudogenes. These are indicated in Figure Orobanche caryophyllaceae and Cistanche phelypaea "genes" have internal stop codons and thus are probably also pseudogenes. There may also be other pseudogenes with intact ORFs, making their pseudogene status less obvious [Overall rate heterogeneity was assessed using the K-H test as implemented in PAUP, using the same ML analyses, except that the starting tree was a neighbor-joining tree and the analysis was limited to 40 rearrangements. Nonsysnonymous and synonymous changes were reconstructed on branches using the codon-based likelihood model of Muse and Gaut , as impl obvious . Once a dN, dS, and the dN/dS ratio (ω) in likelihood ratio tests [Orobanche, Epifagus and Cistanche branches and (2) Harveya, Hyobanche and Boschniakia branches. Each of these tests used the data set from a single gene and compared two nested hypotheses: H1: the photosynthetic and non-photosynthetic branches share a single value (for one of the parameters dN, dS or ω). H2: the photosynthetic and non-photosynthetic branches have two separate values. If the tree has a significantly higher likelihood under H2, that is taken as evidence that the nonphotosynthetic branches have higher rates. Scatter plots and correlation tests were used to examine the degree of correlation between synonymous and nonsynonymous sites within a gene, and also to see if either class of sites was correlated between genes.Rate increases were compared among categories of taxa (such as photosynthetic and non-photosynthetic), using io tests . These tio tests  indicateNDY and CWD conceived of and designed the study together. NDY did the sequencing, data analyses and drafted the manuscript. CWD provided the genomic DNA samples and provided the conducive laboratory environment, both physical and intellectual, as well as many suggestions for the manuscript.


The mining of information from scientific literature using computational tools has tremendous potential for knowledge discovery, but how close are we to realizing this potential? Biological databases offer access to formalized facts about many aspects of biology—genes and gene products, protein structure, metabolic pathways, diseases, organisms, and so on. These databases are becoming increasingly important to researchers. The information that populates databases is generated by research teams and is usually published in peer-reviewed journals. As part of the publication process, some authors deposit data into a database but, more often, it is extracted from the published literature and deposited into the databases by human curators, a painstaking process.www.ebi.ac.uk/embl/) or ArrayExpress (www.ebi.ac.uk/arrayexpress/), nobody receives credit for the submission of a fact to a database without an associated publication. As long as this practice continues, curation will be necessary to add the (re)formalised facts to biological databases.Research literature and scientific databases fulfil different needs. Literature provides ideas and new hypotheses, but is not constrained to provide facts in formats suitable for use in databases. By contrast, databases efficiently provide large quantities of data and information in a standardised schema representing a predefined interpretation of the data. While the acceptance of a paper can enforce the submission of data to a central data repository, such as EMBL , Unified Medical Language System (www.nlm.nih.gov/research/umls/), and MedDRA (www.meddramsso.com/NewWeb2003/index.htm) [Curators fulfil a second important task: they know how to define standards for data consistency, in particular, the most relevant terminology, which has led to the design of standardised ontologies and controlled vocabularies (see dex.htm) . These tControlled vocabulary: A set of terms, to standardise input to a database.F-measure: A statistic that is used to score the success of NE recognition by text mining tools. The F-measure is an average parameter based on precision (how many of the entities found by the tool are correct identifications of an entity) and recall (how many of the entities existing in the text did the tool find).Machine learning: The technology and study of algorithms through which machines (computers) can “learn”, or automatically improve their systems through data gathered in the past (experience).Ontology: A set of terms with clear semantics (language), clear motivations for distinction between the terms, and strict rules for how the terms relate to each other.The problem with curation of data is that it is time consuming and costly, and therefore has to focus on the most relevant facts. This compromises the completeness of the curated data, and curation teams are doomed to stay behind the latest publications. So, is it possible for curation and text mining to work together for rapid retrieval and analysis of facts with precise postprocessing and standardisation of the extracted information?www.gene.ucl.ac.uk/hugo; 20,000 gene and protein names), GO , Uniprot/Swiss-Prot , and other databases. In addition, terms describing diseases, syndromes, and drugs are available from the Unified Medical Language System. Altogether, about 500,000 terms constitute the basis of domain knowledge in life sciences. To gain some perspective of this figure: an average individual handles 2,000 to 20,000 terms in his or her daily language, and Merriam-Webster's Collegiate Dictionary provides definitions for 225,000 terms (www.merriam-webstercollegiate.com/).There are several software tools that perform well in the identification of standardised terms from the literature. Examples include Textpresso and Whatizit ,6,7,8. EThe identification of all terms by a text mining system still sets challenging demands. All variants of a term have to be taken into account, including syntactical variants and synonyms. In the case of ambiguities, relevant findings have to be distinguished from other findings—a process referred to as disambiguation. Depending on the curation task, it might therefore be advantageous to select only part of the terminological resources and thus restrict the domain of the terminology to the curators' needs .Available text mining solutions are concerned with named entity (NE) recognition (entities are, for example, proteins, species, and cell lines), with identification of relationships between NEs (such as protein interactions), and with the classification of text subphrases according to annotation schemata in general (thyroid receptor is a thyroid hormone receptor) ,13,14,15Not all terms used in the literature (NEs) can actually be found in some kind of database . Text mining methods therefore have to detect new terms and map the term to known terminology . If sevewww.pdg.cnb.uam.es/BioLINK/BioCreative.eval.html, and JNLPBA, www.genisis.ch/~natlang/JNLPBA04/) [Over the past several years text mining research teams have presented various approaches that train a software tool to locate representations of gene or protein names (for example, BioCreative, LPBA04/) ,18. ThesLPBA04/) ), which www.cs.cornell.edu/projects/kddcup/) had to select documents from a given corpus that contained relevant experimental results about Drosophila [Additional information-extraction methods have been proposed, for example, for the documentation of mutations in specific genes and for the extraction of the subcellular location of proteins ,13. An eosophila .For all automated information-extraction methods, it is obvious that access to literature is crucial. Electronic access has, of course, already had a huge impact, but the structure and organisation of manuscripts could also be improved. For example, semantic tags could be integrated into the text. The markup would not appear on web pages or when the document is printed, but it would help software to deal with semantic aspects of the document. Inserting tags, for example, to mark protein names would allow retrieval software to find documents about proteins even if they look like common English words, such as “you” or “and”. Retrieval engines currently often ignore such terms. In addition, explicit tags would enable text mining methods, for example, when looking for protein–protein interactions, to use the correct semantic interpretation.Text mining systems already available today, such as Whatizit, can integrate semantic tags during submission, which have to be verified by the author. Text mining is ready to deliver tools whereby information is passed back to the authors about the proper use of terminology within their documents. If the use of a term raises conflicts or ambiguities or if the use of a term is wrong, the author is asked to provide feedback. The curation effort is resolved at the earliest possible time-point. Author, publisher, reviewer, and reader profit from consistent information representation, which leads to better dissemination of documents and journals and easily offsets the additional cost in the generation of an article. Publishers and authors have to agree on standards though.Text mining solutions have found their way into daily work, wherever fast and precise extraction of details from a large volume of text is needed. We have to keep in mind, however, that any text mining tool, just like other bioinformatics resources, will only be suitable for a limited number of tasks. For example, the same text may serve curators from different communities who extract different types of facts, depending on their domain knowledge. Furthermore, different communities have different expectations for accuracy. For example, curators dealing with a small set of proteins prefer tools with high recall, whereas curators dealing with a large number of proteins prefer tools with high precision.Although text mining cannot dissect English sentences completely, and cannot extract the meaning and put the facts into a database, text mining tools are becoming increasingly used and valued. Text mining is ready to deliver handling of complex terminology and nomenclature as a mature service. It is only a matter of time and effort before we are able to extract facts automatically. The consequences are likely to be profound. Not only will we have a more effective approach for the mining of knowledge from the literature, our approach to the publication process itself might change. If a fact is clear enough for automatic extraction, it could be reported in a fact database instead of a publication. As methods improve, authors will see more and more of their text being analysed and formalised in a database. If appropriate quality control is provided, and if authors receive due credit for their deposition of facts into databases, we might well see a shift towards original papers describing new creative ideas and visions rather than just listing facts.


They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.Vacuolar Hin vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells.To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) +-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H Plant V-ATPase has been identified at the vacuolar and various other endomembranes, and also at the plasma membrane [1-part), and VHA-a, c, c', c", d and e for membrane-associated subunits (V0-part) [A. thaliana, oat and barley [A. thaliana [Mesembryanthemum crystallinum [Arabidopsis and Mesembryanthemum express a similar set of subunits [0-sector since its subunit composition is not resolved yet. VHA-a is the subunit with the highest molecular mass within the V-ATPase and reveals a bipartite structure. As first shown in yeast, VHA-a consists of a 50 kDa hydrophilic N-terminal domain and a hydrophobic, membrane-spanning C-terminus [o-domain [Vacuolar Hmembrane -3. The tmembrane . V-ATPasV0-part) . In planV0-part) ,7. Follod barley ,7. Howevthaliana . The sectallinum , and a tsubunits . An impoterminus -13. Inteterminus  could noterminus . Anotherterminus . VHA-H io-domain .The aim of the study was to improve the understanding of V-ATPase distribution in plant cells with emphasis on the localization of VHA-a and VHA-H in the V-ATPase structure. Three specific questions were answered using different methodology: (i) Is VHA-a part of the V-ATPase structure? (ii) Where are different subunits localized within plant cells? (iii) Is FRET a suitable method to investigate subunit interaction within the complex, for example between VHA-a and VHA-c, and VHA-H and VHA-B.M. crystallinum was identified (AI822404) with similarity to known genes coding for VHA-a in yeast. Using RACE-PCR a full length cDNA was obtained with a size of 2783 bp. Its open reading frame encoded a hypothetical protein of 93.1 kDa. Database search with the program FASTA revealed the highest similarities to the entries At2g21410 (77%), At4g39080 (76 %) and At2g28520 (60 %) from the A. thaliana genomic database and also significant similarity on the amino acid level to the genes Stv1 (34 %) and Vph1 (38 %) from S. cerevisiae. These two latter genes are coding for isoforms of the yeast 100 kDa subunit whereas all other subunits of the yeast vacuolar ATPase are encoded by one gene each. The sequence alignment between the newly cloned amino acid sequence from M. crystallinum, the three isogenes from A. thaliana and the two isogenes from yeast  a cDNA fragment from A. thaliana and M. crystallinum to the distinct yeast isoforms Vph1 and Stv1, in order to define orthologous genes, was not possible on basis of the amino acid sequence alignment. The comparison of the C-termini revealed a higher degree of sequence conservation interrupted through various short insertions or deletions. The sequence was analysed for secondary structures (Predict Protein, EMBL, Heidelberg) and membrane topology (THHM, Denmark). The prediction correlated regions of high sequence conservation with the localisation of putative transmembrane domains , introduced and expressed in E. coli. The 42 kDa and 13 kDa polypeptides were purified to homogeneity by chromatography on Ni-NTA and by preparative SDS-PAGE. The 42 kDa polypeptide was recognized by a purified antibody against yeast Vph1 [N-term (not shown). The presence of VHA-a was investigated in plant endomembranes and soluble fractions rapidly prepared from 5 week old M. crystallinum plants. In the soluble and the membrane-fraction, the serum against McVHA-aN-term identified two polypeptides with apparent molecular masses between 65 and 70 kDa . Without protection from proteolysis, a band at about 50 kDa and a doublet band above 60 kDa were predominant. In the sample with protease inhibitor, the 95 kDa band appeared although as one of five bands of similar intensity if decreasing transfer efficiency of high molecular mass polypeptides from the gel to the membrane is assumed. Apparently, VHA-a is prone to degradation. However, the results also indicate that VHA-a is part of V-ATPase. To further prove that tentative conclusion, immunoprecipitation was performed using anti VHA-aN-termand anti VHA-amemb followed by Western blot analysis with anti VHA-E and anti-VHA-A  and a labelling of the Golgi apparatus significantly stronger than with anti-VHA-a  and excitation spectra (acceptor) and need to be situated in close proximity. Half maximum energy transfer takes place at distance of the Förster radius R0. Cyan and yellow fluorescent proteins constitute such a FRET pair and were fused to the C-termini of various subunits of V-ATPase. Under the assumption of freely rotating fluorophores, R0 is close to 5 nm. The size of the V-ATPase complex is about 15 nm (diameter) × 25 nm (length from lumen side to tip of head). Arabidopsis protoplasts were co-transformed with vector constructs of VHA-a fused to YFP and VHA-c fused to CFP under the control of the 35S promotor. Upon excitation of doubly labelled protoplasts at 458 nm both, CFP and YFP showed strong fluorescence  this corresponds to a distance between VHA-a, and VHA-c of ~5.4 to 7.2 nm (mean 6.3 ± 0.8 nm). Here it has to be pointed out that each ring of the rotor V0 contains 5 VHA-c-subunits and one VHA-c"-subunit. Hence, dependent on the position of the VHA-c/CFP-subunits in the ring different distances between CFP-labelled VHA-c subunits and the YFP-labelled VHA-a subunit will result.FRET allows to investigate protein-protein interaction nce Fig. . Fluoresnce Fig. . This faSimilar experiments were performed with onion epidermis cells Fig.  co-transArabidopsis gene products contain all charged amino acids that have been shown to be essential for proton-translocation at conserved positions in the membrane spanning region of the C-terminus  have previously indicated a distribution of V-ATPase among nearly all endomembranes of the secretory pathway including the plasmalemma -3, 33-3-333-35. 1-structure of V-ATPase. The presence of both subunits on all these membranes shows the presence of fully assembled V-ATPase.In our examination the antisera against VHA-A and -E showed a staining of the tonoplast and to a lesser extent also of the ER and Golgi-Apparatus (data not shown). VHA-A and E are part of the cytoplasmically exposed VN-term on the other hand. Immuno-labelling indicates that VHA-aN-term antiserum exclusively labels the ER with some rare association on Golgi stacks. These results are surprising since the sequence features of McVHA-a suggest an essential involvement of VHA-a in proton transport. A possible explanation might be the presence of three different isoforms in A. thaliana and O. sativa which all share the localisation of the charged amino acids responsible for proton-translocation  is exclusively associated with V-ATPase localised on the ER. The hypothesis of a compartment-specific localisation of VHA-a subunits is supported through several findings in plants and yeast. In yeast all known subunits and chaperones of the V-ATPase are encoded by one gene. Only the 100 kDa VHA-a is encoded by two different isoforms (Vph1 and Stv1) [S. cerevisiae and A. thaliana  . In genend Stv1) ,39. In aficiency . The N-tIn plants, Matsuoka et al.  were ablIn vivo-FRET in protoplasts expressing VHA-B/CFP and VHA-H-YFP confirms the orientation of the C-termini of VHA-B and H in close vicinity. It should be noted that co-expression of other pairs of chimeric donors and acceptors such as VHA-E/CFP and VHA-c/YFP did not elicit FRET after excitation of CFP (not shown). The assumed location of the C-terminus of VHA-c in the lumen of the endomembrane compartments and the C-terminus of VHA-E most likely in the vicinity of the head is in agreement with the negative result, i.e. the absence of FRET between VHA-E/CFP and VHA-c/YFP. The studies exemplify the suitability of FRET to analyse structural features of V-ATPase in vivo. The efficient but variable FRET in cells expressing VHA-c/CFP and VHA-a/YFP allows two conclusions: First, the C-termini of VHA-c and VHA-a are likely to be located on the same, i.e. luminal, side of the endomembrane compartments supporting the topological model with nine transmembrane-domains of VHA-a in plants as previously suggested for yeast [Our results from FRET analysis also allows to make some structural assignments: From crystal structure of F-ATP synthase, the C-termini of subunit β, homologous to VHA-A, and subunit α, homologous to VHA-B, are located in close vicinity and oriented to the membrane . Occurreor yeast ,23 , 4 mM dithiothreitol and a few crystals of phenylmethylsulfonylfluoride [® cocktail (Roche, Mannheim, Germany) was added throughout the procedure. Following differential sedimentation and gradient centrifugation, tonoplast enriched membranes were recovered from a 30%/35% sucrose interphase, sedimented, frozen in liquid nitrogen and stored at -80°C.Leaves (50 g) of fluoride . As indi® system according to the supplier (Roche, Mannheim, Germany).Membrane proteins were separated on 12.5% sodium dodecylsulfate polyacrylamide gels, transferred to nitrocellulose and probed with anti-VHA-E , anti-VHFor immunoprecipitation, membranes were solubilised in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% (v/v) Triton X-100, 5 μl anti VHA-a antiserum was added, and the samples were shaken at room temperature for 45 min. Then 150 μl protein A-sepharose equilibrated in the same buffer was added. After 15 min, the suspension was placed on a cushion of 1 ml of 40% sucrose and spun at 10,000 × g for 1 min. The sediment was washed thrice with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1 % (w/v) SDS, and finally once in 125 mM Tris-Cl, pH 6.8. The sediment was boiled in loading buffer and analysed by Western blot using rabbit antisera raised against VHA-E or A.N-term and anti-VHA-aMemb. For both the corresponding cDNA fragments of Mc-VHA-a were amplified by PCR using primer combinations a-nterm-f (ATG CGA TCG GAG CCG ATG CAA) and a-nterm-r (TTC ACC CAA CTC ATC GGT GG) encoding the 42 N-terminally located fragment, and a-memb-f (CTT CCA AAG CCC TTT ATT ATG) and a-memb-r (TCA CTC ATG TCC ACC ATG TCA ATC) encoding the polypeptide loop of about 13 kDa located between transmembrane domain 3 and 4 according to the topological model of Vph1p of S. cerevisiae [E. coli JM109. The 6x-his-tagged proteins were expressed, purified by chromatography on Ni-nitrilotriacetate columns, separated by preparative SDS-PAGE, excised as protein bands, eluted and used for immunization (Pineda, Berlin). In addition, antisera against subunits VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany), VHA-E, calreticulin  in a site-directed manner after amplification from cDNA  using th2, 4 mM morpholinoethane sulfonic acid, KOH , phl 5.7) and checked for sufficient intactness in the microscope. In short, 1 mm leaf slices of 3- to 5-week-old Arabidopsis plants were vacuum-infiltrated and cell walls were digested in media containing 1.5 % (w/v) cellulase R10 and 0.4 % (w/v) macerozyme R10. Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCl2, 4 mM morpholinoethane sulfonic acid, KOH, pH 5.7) and checked for sufficient intactness in the microscope. 110 μl PEG-medium (4 % (w/v) polyethylene glycol 4000, 0.2 M mannitol, 0.1 M CaCl2) and 20 μl plasmid DNA (3 μg/μl) were added to 100 μl protoplast suspension. The samples were incubated at room temperature for 15 min and then consecutively diluted with 0.5, 1, 2 and 4 ml W5-medium with 15 min incubation steps in between (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM morpholinoethane sulfonic acid, KOH, pH 5.7). Following 24 h incubation at 25°C, sedimented protoplasts were used for analysis.Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCI2, 3.33 μl 0.1 M spermidine. Sedimented gold particles were consecutively washed with 70 % and 100 % ethanol and resuspended in 8 μl 100 % ethanol, loaded on a macro carrier for transformation with the Particle Delivery System using a rupture disc of 1100 psi (PDS-1000/He, Biorad, Hercules, USA). The distance between macrocarrier and tissue was 12 cm. The epidermis tissue was incubated for about 20 h at room temperature in the dark prior to analysis.Cells of onion epidermis were placed on filter paper soaked with one-strength MS basal medium in petri dishes and were transiently transformed with a biolistic approach. Gold particles (1.6 μm, 60 mg/ml) were suspended in 50 % glycerol. 8.33 μl of the suspension were mixed with 8.33 μl plasmid DNA (1 μg/ μl), 8.33 μl 2.5 M CaCl4, 10 mM EGTA, 1 × phosphate buffered saline, pH 6.8), washed, permeabilised in 0.5% Triton X-100 and washed again. Following blocking of non-specific binding sites with 1 % bovine serum albumin, primary antibody was added for over night at 4°C. Washed samples were incubated with secondary antibody labelled with Cy3, Cy5 or FITC for 1 h. Double labelling was performed by combined application of primary antibodies from rabbit and guinea pig. Slides were mounted with Citifluor Mounting Medium. Fluorescence analysis was performed with a confocal laser scanning microscope Leica TCS-SP2 (Leica, Heidelberg, Germany) equipped with three lasers and excitation wavelengths of 458, 476, 488, 514, 568 and 633 nm. The double dichroic mirror DD488/543 was used for fluorescein isothiocyanate (FITC), and for Cy5 the triple dichroic mirror TD488/543/633 was used. Background was controlled and photomultiplier voltage (800 V) selected for maximum sensitivity in the linear range.Immuno-labelling was performed according to . In brie2PO4, 50 mM NaH2PO4, pH 7.0) for 45 min, washed with EM buffer and dehydrated with a series of increasing concentration of acetone. Samples were embedded in epoxyresin (Transmit EM, TAAB laboratories equipment, Berkshire, Great Britain), cut into ultra-thin cross-sections of 60–70 nm and immobilized on 200 mesh gold nets. Immuno-decoration was performed with antibody diluted in Tris-buffered saline  NaN3) for an hour. Samples were washed five times and incubated with secondary antibody conjugated to 15 nm gold particles. The samples were stained with 0.1 % (w/v) uranyl acetate for 5 s and afterwards with 2 % lead citrate. The samples were analysed with an electron microscope (H500, Hitachi, Japan) at 75 kV.Cells were fixed in 2.5% glutaraldehyde in EM buffer (50 mM KHo = 5 nm [CFP/bleached - ICFP/unbleached)/ICFP/unbleached and R = ((Ro6/E)-Ro6)1/6, where E is the transfer efficiency, and ICFP the fluorescence emission intensity in the CFP peak.Transformed protoplasts and onion epidermis cells were examined for the localisation of the CFP/YFP-fused proteins using the same CLSM set-up as mentioned above. Autofluorescence of 10 protoplasts, as well as reference spectra of YFP and CFP-derived fluorescence were recorded in the spectral range of 480 to 700 nm, averaged and used for corrections. Excitation was recorded at 458 nm (CFP and FRET) and 514 nm (YFP), respectively. Scan speed was 800 Hz. Acceptor dye was bleached with 100 % laser intensity. Emission spectra were recorded and averaged from 20 transformed protoplasts. For a first estimate of transfer efficiency, a Foerster radius for green fluorescence protein variants of Ro = 5 nm  was used+-ATPase; Vph1: VHA-a subunit isogene in yeast; YFP: yellow fluorescence proteinCFP: cyan fluorescence protein; CLSM: confocal laser scanning microscope; FRET: (Förster) fluorescence resonance energy transfer; PAGE: polyacrylamide gel electrophoresis; Stv1: VHA-a subunit isogene in yeast; VHA: vacuolar HN-term; TS: co-transfection of protoplasts and epidermis cells, CLSM analysis; SB: immuno-co-localisation and discussion; SS: co-immuno-precipitation; MH: preparation of anti VHA-aMemb; BS-J: immuno-co-localisation and discussion; JR and MS: FRET analysis; DG: transformation and construct design, KJD: project design and supervision.CK: VHA-a sequence analysis, immuno-cytochemistry, transient expression systems, preparation of anti VHA-a


Only controlled blocks are capable of confirming the zygapophysial joints (ZJ) as the pain generator in LBP patients. However, previous workers have found that a cluster of clinical signs ("Revel's criteria"), may be valuable in predicting the results of an initial screening ZJ block. It was suggested that these clinical findings are unsuitable for diagnosis, but may be of value in selecting patients for diagnostic blocks of the lumbar ZJ's. To constitute evidence in favour of a clinical management strategy, these results need confirmation. This study evaluates the utility of 'Revel's criteria' as a screening tool for selection of chronic low back pain patients for controlled ZJ diagnostic blocks.This study utilized a prospective blinded concurrent reference standard related validity design. Consecutive chronic LBP patients completed pain drawings, psychosocial distress and disability questionnaires, received a clinical examination and lumbar zygapophysial blocks. Two reference standards were evaluated simultaneously: 1. 75% reduction of pain on a visual analogue scale (replication of previous work), and 2. abolition of the dominant or primary pain. Using "Revel's criteria" as predictors, logistic regression analyses were used to test the model. Estimates of sensitivity, specificity, predictive values and likelihood ratios for selected variables were calculated for the two proposed clinical strategies.Earlier results were not replicated. Sensitivity of "Revel's criteria" was low sensitivity (<17%), and specificity high (approximately 90%). Absence of pain with cough or sneeze just reached significance (p = 0.05) within one model."Revel's criteria" are unsuitable as a clinical screening test to select chronic LBP patients for initial ZJ blocks. However, the criteria may have use in identifying a small subset (11%) of patients likely to respond to the initial block (specificity 93%). It is estimated that 15 – 40% of chronic low back pain patients have pain arising from the lumbar zygapophysial joints (ZJ). PreviouBecause prognosis for acute low back is good, invasive and expensive and invasive diagnostic testing cannot be justified. However, persistent disabling pain needs more intensive investigation in order to determine appropriate management strategies. Back pain of ZJ origin may be treated using intra-articular steroid injection,10., or The current study objectives were to estimate the predictive value of the two clinical strategies of Revel et al (1998), using similar measurement parameters (75% reduction in pain VAS after ZJ block), and apply alternative analytic methods to explore any potential utility of the variables used.This study utilized a prospective, blinded, concurrent, reference standard-related validity design with intra-articular ZJ or medial branch blocks as the reference standard against which clinical variables were compared. Local Institutional Review Board approval was granted at the beginning of the study. A 75% or more reduction in pain following ZJ block on pain VAS was designated as reference standard A, the same standard used by Revel et al (1998). Based on pain drawings and patient self-report, complete abolition of the patient's primary or dominant pain was designated as reference standard B. Dominant pain location was acquired by pain drawing and direct questioning, and documented prior to clinical examination and diagnostic injection in a prospective manner. Post injection dominant pain location was acquired by reference to pain drawings and direct questioning also.Patients with low back pain with or without lower extremity symptoms, referred to a private radiology practice in New Orleans, USA specializing in the diagnosis of spinal pain, were invited to participate in the study. Patients receiving ZJ blocks were either referred specifically for that procedure or had the procedure included in their radiology examination based on pre-injection clinical evaluation by the injectionist (CA). Between May 2001 and October 2002, physical therapists attended the clinic in blocks that ranged from 4 to 8 weeks (ML) and examined patients. Normal scheduling was not affected by the presence of the visiting therapists, so patients were consecutive during these periods. All patients had undergone imaging studies prior to referral from a variety of medical and paramedical practitioners. Some were self-referred.Patients were excluded from the study if they were unwilling to participate, were too frail to tolerate a physical examination, or were deemed by any member of the clinic team to be unable to comprehend the study procedure. Prior to the formal clinical examination, clinic staff recorded basic demographic and medical data.100 mm visual analog scales (VAS) scales for current, best and worst pain. A current pain VAS was repeated after the clinical examination and following ZJ blocks. The 23-point Roland-Morris Disability Questionnaire  was compHistory taking and a structured physical examination were carried out by a physical therapist with 30 years of clinical experience as a manipulative therapist (ML). Some patients were examined by a physical therapist with 17 years experience (SBY). The clinical examination occupied 30 to 60 minutes and included many tests besides those necessary for the current analysis, as part of a larger project. Inconclusive findings or incomplete examinations were documented. The physical examination included a visual assessment of range of motion, recording anatomical location of dominant pain, nerve tension tests, key muscle strength tests, tendon reflex tests, light touch sensitivity, a McKenzie styled ePrior to ZJ blocks, the radiologist reviewed case notes and imaging studies, and conducted a physical examination that guided the type of diagnostic procedure to be employed and the target structures. Intra-articular ZJ joint injection or MBB using standard technique was carrPhysical therapists conducting the clinical examination were blinded to the results of previous imaging studies and diagnostic injections, the Roland, Zung and MSPQ questionnaires. The injectionist was blinded to the results of the physical therapy examination and diagnostic conclusions.Basic statistical values for demographic variables and regression analyses were calculated using statistics software (Minitab version 13.31 © Minitab Inc 2000). Differences between included and excluded patients were evaluated with the student's t, chi square, and Kruskal-Wallis tests where appropriate. Significance for differences was set at p < 0.05.Calculations of sensitivity, specificity, predictive values and likelihood ratios with 95% confidence intervals were performed using Confidence Interval Analysis software © Bryant T.N. 2000.Initial ZJ blocks were carried out on 151 chronic low back pain patients. Thirty-four patients were excluded from analysis as they received another intervention in the same procedure session and did not return for differentiating and confirmatory blocks. One case was excluded through incomplete data on Revel's criteria. Following ZJ block, 27 of 116 patients satisfied reference standard A. Data required for determination of Reference standard B were missing for five of the 116 cases. Eighteen of these 111 patients satisfied reference standard B. Table In specifically evaluating Revel's criteria against reference standard A, logistic regression failed to achieve significance as a model (n = 108, p = 0.46). Two variables; "absence of pain with coughing and sneezing" and "no exacerbation of pain rising from flexion", showed a trend towards significance as predictors within the model (p = 0.07).Using Revel's criteria within a logistic regression with reference standard B as the response variable, a strong trend towards significance was reached (n = 100, p = 0.06). One component variable (age over 65) individually reached significance within the model: . In the whole sample 12.5% were aged over 65 years whereas of the 19 positive responders six were over 65 years (31.6%).The same patients satisfied both strategies of Revel et al. Estimated sensitivity, specificity, predictive values and positive likelihood ratios for both strategies of Revel et al are presented in Table Of the 27 patients satisfying reference standard A, 13 (48%) returned for confirmatory ZJ blocks. Three of these reported 75% or more reduction in pain following the confirmatory block. None of the three patients with confirmed ZJ pain satisfied Revel's criteria.The current data produces results that are in stark contrast to those of Revel et al (1998) with sensitivity low and specificity high. However, 'no pain with cough and sneeze' and 'no exacerbation of pain rising from flexion' approached statistical significance in relation to a 75% reduction pain after ZJ block (reference standard A). These variables are in line with Revel's results. Age over 65 is associated with reference standard B (abolition of primary pain). Likelihood ratios for the criteria are lower in the current data also. Some of the differences in results may be explained by a number of factors:1. Revel et al's study was a placebo controlled design whereas we did not routinely utilize a similar or equivalent control.2. The patients in Revel et al's sample were older (mean 58 versus 43 years), and had a shorter duration of symptoms (mean 78 versus 160 weeks).3. It is also possible that the high level of standardization when acquiring the criteria data in the current study, and the prospective methodology might have contributed to the differences in results.Following anaesthetic blocks, patients frequently state that the pain prompting consultation is abolished, yet a post-procedure pain VAS still registers more than 0/100. In this study reference standard B was evaluated as an alternative to the usual standard, so that pain in areas above the lumbar spine or pain directly attributable to the needle insertion site do not result in inappropriate post procedure VAS scores. In the interests of developing more precise instruments documenting results of diagnostic blocks, we propose that in future studies, the VAS scale should specifically refer to the primary pain complaint for which the diagnostic block is undertaken.Based on the current data, Revel's criteria are not suitable as a screening test to select patients suitable or unsuitable for an initial screening ZJ block. Such a screening device should have high sensitivity to ensure that most patients likely to respond are included in the initial diagnostic block. Our study suggests that, at best, Revel's criteria, might identify a small subset (11%) of patients likely to respond to a screening block (specificity 93%).Neither strategy utilizing Revel's criteria is suitable as a clinical screening device for selection of chronic LBP patients for initial diagnostic ZJ blocks. In contrast to Revel's findings, the current data demonstrated low sensitivity and high specificity for these clinical criteria. The high specificity of the criteria reported in this study relates to a single uncontrolled screening block. Consequently these criteria can not considered diagnostic of painful lumbar ZJ. Only placebo controlled or double ZJ blocks are able to diagnose this source of low back pain.The author(s) declare that they have no competing interests.ML conceived the design of the study, examined all but 10 patients, carried out data analysis and prepared the manuscriptBÖ assisted in project design and manuscript preparationCNA assisted in project design, provided facilities and conducted fluoroscopically guided injectionsBMcD carried out data analysis and assisted in manuscript preparation.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Male suicide rates continued to increase in Scotland when rates in England and Wales declined. Female rates decreased, but at a slower rate than in England and Wales. Previous work has suggested higher than average rates in some rural areas of Scotland. This paper describes trends in suicide and undetermined death in Scotland by age, gender, geographical area and method for 1981 – 1999.Deaths from suicide and undetermined cause in Scotland from 1981 – 1999 were identified using the records of the General Registrar Office. The deaths of people not resident in Scotland were excluded from the analysis. Death rates were calculated by area of residence, age group, gender, and method. Standardised Mortality Ratios (SMRs) and 95% confidence intervals were calculated for rates by geographical area.Male rates of death by suicide and undetermined death increased by 35% between 1981 – 1985 and 1996 – 1999. The largest increases were in the youngest age groups. All age female rates decreased by 7% in the same period, although there were increases in younger female age groups.The commonest methods of suicide in men were hanging, self-poisoning and car exhaust fumes. Hanging in males increased by 96.8% from 45 per million to 89 per million, compared to a 30.7% increase for self-poisoning deaths. In females, the commonest method of suicide was self-poisoning. Female hanging death rates increased in the time period.Male SMRs for 1981 – 1999 were significantly elevated in Western Isles (SMR 138, 95% CI 112 – 171), Highland (135, CI 125 – 147), and Greater Glasgow (120, CI 115 – 125). The female SMR was significantly high only in Greater Glasgow (120, CI 112 – 128).All age suicide rates increased in men and decreased in women in Scotland in 1981 – 1999. Previous findings of higher than expected male rates in some rural areas were supported. Rates were also high in Greater Glasgow, one of the most deprived areas of Scotland. There were changes in the methods used, with an increase in hanging deaths in men, and a smaller increase in hanging in women. Altered choice of method may have contributed to the increased male deaths. Compared to the adjacent countries of England and Wales, Scotland had a low suicide rate through most of the twentieth century . This diSeveral authors have noted the importance of suicide in Scotland as a public health problem ,10. TherWe used anonymised information on deaths by suicide and undetermined deaths provided by the General Register Office for Scotland (GROS). Deaths registered during 1981 – 1999 were included if the cause of death was recorded as suicide or as undetermined cause (ICD-9 E950-E959 and E980-E989 respectively). Undetermined deaths were included as suicide deaths may be misattributed ,14.Population figures were taken from the GROS annual reports for the mid-year of each period. For analyses by area, if a death was registered away from the person's home address, the death was allocated to their area of residence, rather than the area in which they died. Deaths of people resident outside Scotland were identified using country codes, and were excluded. As far as possible, therefore, results reflect the rates of suicide and undetermined deaths of people resident in each area of Scotland. Standardised Mortality Ratios were calculated for National Health Service administrative areas, with 95% confidence intervals. In time period descriptions, the periods 1981 – 1985, 1986 – 1990, 1991 – 1995, and 1996 – 1999 were used. Data were analysed using Excel and SPSS.There were 14502 deaths recorded as suicide or undetermined cause in the time period. Of these deaths, 28.5% occurred in females (n = 4137) and 71.5% in males (n = 10365).Table Examining changes by age group in males Table , there aIn women, there were increases in the three youngest age groups, with a 76% increase in rates in the under 15 year old group, 150% in the 15 – 24 year group and 37% in 25 – 34 year olds  (Table The epidemiology of suicide in Scotland has changed greatly between 1981 and 1999. Male suicide rates have increased in all age groups up to and including 35 – 44 years. The highest male suicide and undetermined death rates in 1996 – 1999 were in the 25 – 34 year age group. In women, rates dropped in age groups from 35 – 44 years up to and including 75 years and over. Rates increased in younger women.There is limited information on the factors underlying individual deaths from suicide in Scotland. Squires and Gorman  reviewedSome methods of self-harm have higher case fatality rates . FirearmThe difference between areas was also of note. The lower rates of increase in the areas with the highest initial rates may reflect to regression to the mean. Method availability  may be iAvailability of method would not explain the differences between apparently similar rural areas. Previous work has found that deprived areas of Scotland tend to have higher suicide rates ,28. DeprUsing routine information allowed a large number of suicide and undetermined deaths to be included in this series. There are, however, limitations to the use of anonymised routine data. No qualitative information was available, and our exploration of the data was limited to trends with no examination of possible underlying causes. The increase in the rate of deaths recorded as suicide or undetermined cause of death, but where no detail on method was included, could conceal recent trends. The increase as a percentage of relevant registrations was small, however, increasing from 7.5% in the first period to 9.1% in the last period studied. The classification of deaths as suicide is often difficult, but the inclusion of undetermined deaths as well as deaths recorded as suicide should have helped to minimise bias from under identification ,30. SquiA divergence between male rates in England and Wales and in Scotland, and in male and female rates within Scotland, had been identified for the first part of the time period described here. This work found that male rates of suicide and undetermined death continued to increase in Scotland, but also identifies increases in younger female age groups. Examination of changes in method by male and female age group will help to establish whether changes in case fatality because of altered method choice  may be pSome rural areas of Scotland had significantly elevated male suicide rates. We have suggested that access to lethal means of suicide may be one contributing mechanism for this, and have also noted the higher than expected suicide numbers in some rural occupations . Rural aThe authors declare that they have no competing interests.CS had the idea for the study, wrote the grant application, contributed to the design and interpretation and drafted the paper. Diane Gibbs and Tracey Rapson analysed the data. Paddy Hopkins contributed to the design, undertook part of the analysis, and commented on the interpretation of the results. Alan Belbin and Alistair Hay contributed to the design and helped interpret the results. All authors read and approved the final draft of the paper.The pre-publication history for this paper can be accessed here: