Natural disasters, war, and terrorist attacks, have been linked to cardiac mortality. We sought to investigate whether a major financial crisis may impact on the medical management and outcomes of acute coronary syndromes.We analyzed the Argentine cohort of the international multicenter Global Registry of Acute Coronary Events (GRACE). The primary objective was to estimate if there was an association between the financial crisis period (April 1999 to December 2002) and in- hospital cardiovascular mortality, with the post-crisis period (January 2003 to September 2004) as the referent. Each period was defined according to the evolution of the Gross Domestic Product. We investigated the demographic characteristics, diagnostic and therapeutic procedures, morbidity and mortality.We analyzed data from 3220 patients, 2246 (69.8%) patients in the crisis period and 974 (30.2%) in the post-crisis frame. The distribution of demographic and clinical baseline characteristics were not significantly different between both periods. During the crisis period the incidence of in-hospital myocardial infarction was higher , as well as congestive heart failure . Time to intervention with angioplasty was longer during the crisis, especially among public sites (median 190 min Vs 27 min). The incidence proportion of mortality during hospitalization was 6.2% Vs 5.1% after crisis. The crude OR for mortality was 1.2 . The odds for mortality were higher among private institutions {1.9 } than for public centers {1.2 }. We did not observe a significant interaction between type of hospital and crisis.Our findings suggest that the financial crisis may have had a negative impact on cardiovascular mortality during hospitalization, and higher incidence of medical complications. The country paid a high toll for this change, with the Gross Domestic Product experiencing a sustained decline from 1998, and unemployment rates reaching approximately 25 percent. By the end of 2001 a rapid cascade of political and economic events opened the road to deep social turmoil and economic unrest that spiraled until December 2001, when the country experienced a virtual halt of vital areas of the economy. With two more years of his period still to be completed, the president left office, and so did several others over the following weeks. Less than a month after the world learned about such unusual events, the interim president addressed the Congress to announce that the country would default from all its national and international debts. Bank deposits were seized and thousands of citizens and businesses were left bankrupt while the national currency was devaluated by almost 200% compared to the US dollar. Shortly after the crisis erupted, many found their investments and personal savings reduced by two thirds when measured in hard currency. During the year of 2002, the gross national product declined by more than 11%, and the level of unemployment blast off[In comparison with other Latin American nations, Argentina used to enjoy a relatively developed economy and a fair distribution of wealth until the early 1980s. During the last decade of the 20Several elegant and landmark epidemiological studies have established a link between trauma produced by war, terrorism, festivities, and mortality -5. HowevThe Global Registry of Acute Coronary Events started in 1999 and continued throughout the crisis and the following period, thus giving us a unique opportunity to get a real time picture of the unfolding morbidity and mortality events by means of an multicenter cohort of patients whose clinical characteristics were registered with standardized methods, definitions and selection procedures. We sought to determine whether the financial crisis was associated with cardiac mortality and if medical procedures and therapies were affected by type of institution, public or private.Full details on the GRACE rationale and methodology have been published ,7. GRACEFor the purposes of this analysis we restricted our study sample to patients enrolled in centers in Argentina between April 1999 and September of 2004, including individuals who had an admission diagnosis of acute coronary syndrome . The seven sites participating in Argentina, were analyzed all together, and also stratified according to their particular profile: Private hospitals , and public hospitals . The period of time examined was divided into the crisis period, which was delimited from April 1999 to December 2002, and the post crisis period, which encompassed the time from January 2003 to September 2004. To define each period we used indicators published by the Census Bureau. We considered the beginning of the negative slope of the gross domestic product curve as the start of the crisis period, which lasted until the domestic product experienced a sustained increase over a full trimester [The primary endpoint of the study was in-hospital all-cause mortality. The secondary endpoint was non-fatal-myocardial infarction defined by the presence of at least one positive increment of cardiac biochemical marker of necrosis  plus chest pain prolonged more than 10 minutes, or new ST-segment deviation seen after the index or qualifying electrocardiogram.Summary statistics are presented as frequencies and percentages. Comparisons between groups were made using two-tailed Wilcoxon rank-sum test for continuous variables and the chi-square or Fisher's exact test for categorical variables. Odds ratios and accompanying 95% confidence intervals were computed to evaluate the effects of the crisis on hospital mortality and morbidity. The standard error for the calculation of the 95% confidence intervals for the odds ratios was calculated by means of the Wald formula, and the errors were handled independently for the different time points. Similar analyses were conducted for public and private hospitals separately. All tests were double sided and considered statistically significant at p-value < 0.5. Statistical analyses were conducted with the SAS V. 9.1 software .Of the 44,991 acute coronary syndrome patients admitted in the global registry, 3220 patients were enrolled in Argentina. The number of patients younger than 65 years old was 1527 (47%), representing the proportion of the population normally expected to be economically active. The remainder 1693 (53%) were older than 65. A final diagnosis of ST-segment-myocardial infarction was made in 1179, and 2041 qualified as unstable angina / non-ST-segment elevation myocardial infarction. Female gender represented 30% (n = 1012) of patients. Baseline characteristics comparing patients during and after crisis did not differ significantly , and related to this a lower proportion was referred to angioplasty , with a larger fraction undergoing coronary bypass surgery . We also observed some evidence of lower adherence to interventions of proven efficacy during the crisis, as shown by a lower proportion of patients receiving aspirin , angiotensin converting enzyme inhibitors , and low molecular weight heparin 43%, n = 950 Vs.60%, n = 585) , with a crude OR of 1.2  } Figure . In a stOur study provides some evidence that there may be an association between the financial and institutional collapse of Argentina and increased in-hospital cardiovascular morbidity and mortality. The link between extraordinary circumstances and increased cardiac mortality has been previously reported. A significant increase in the number of cardiac deaths was observed on the same day of major earthquakes that affected Los Angeles and Athens ,9. Also,The Argentine case is unique in that a major socio-economic collapse occurred in the absence of any natural disaster or war. GRACE provides a useful tool to assess in a standardized, structured manner, the diagnostic and therapeutic approaches performed in a representative cluster of hospitals throughout the crisis and following it. Our observations are intriguing, and pose questions on the mechanisms underlying the increased odds of mortality during the crisis compared to the post crisis period. We analyzed several mechanisms that may be responsible for the worsened outcomes during the crisis period: Differences in baseline clinical risk, in medical interventions, type of hospital, social and psychological factors, bias and chance.We did not observe any significant differences between the crisis and post-crisis period regarding the main demographic characteristics such as age, gender, prior coronary artery disease, co morbidities and Killip class on admission. The overall clinical profile is similar to other cohorts elsewhere for the same period .We anticipated an association between the crisis and access to medical care because of a direct effect on access to technology and imported medical supplies. Our observations provide some evidence to support the presumption that the crisis may have affected the quality of care. On one hand, patients enrolled in the registry were consistently treated according with the guidelines and in a similar manner compared to other regions . The proAnother factor that could have influenced the outcomes is an increased burden of medical care on the public system. The Argentine economic phenomenon has been called a "middle class crisis", namely of those who would normally gain access to health insurance through employment or, for small business owners and entrepreneurs, as an out of pocket expenditure. Approximately 20 million people out of a total country population of 37 million are no longer covered by neither the private sector nor a union-run mandatory health insurance, which represents a huge overload for the network of public hospitals . Public The association between the crisis period and increased in-hospital cardiac mortality could be explained by alterations in socio-economic factors or social support, both variables that were not directly measured by the registry. Socio-economic status has been used as a surrogate marker of a much complex matrix called social support. Several studies have suggested that a meaningful impairment in the quality and width of social support can be associated with higher mortality rates, both from cardiac and non cardiac causes ,19. It iIt may be argued that our observations may be due solely to the availability of better treatment modalities over time. As shown in figure Our analysis is exposed to a potential source of selection bias by the definition of each time period. In the absence of a major natural disaster, disease outbreak or war, it can be argued on the accuracy on our definitions on when the crisis started and when it ended. For that matter, we considered data published by the Census Bureau regarding the National Gross Domestic Product and industrial indicators and unemployment rates, and selected the nadir of the adjusted Gross Domestic Product curve as the onset of the crisis, and the first trimester that showed a sustained increase in the Gross Domestic Product as the arbitrary end of the financial crisis. This is subject to bias in itself and alternative definitions may have yielded different results. Nevertheless, we feel confident in that our definitions are solid and based on hard economic indicators instead of political signs or personal interpretations that are vulnerable to subjective perceptions. There is a consistent match between the evolution of the gross domestic product and other indicators such as the investment indexes, public works and private investments in real estate and construction .Another limitation to our conclusions is that GRACE was not specifically designed to provide information on socio economic status or social support, which would be alternative exposures of interest in the scenario of a deep financial crisis. We considered the broad term "crisis" as the exposure of interest, so we must acknowledge that the mechanisms responsible for our observations are to some extent speculative. Also, the study was not powered to detect strength of association between exposures and mortality for a specific region or country.Even in the absence of a formal level of statistical significance, the odds ratios appear to consistently point in thee direction of worse outcomes during the crisis. Figure This study provides evidence suggestive of an association between a dramatic socio-economic event and increased cardiac mortality. The spike in mortality rates that we observed was striking and above the expected death rates according to prior projections from the Ministry of Health ,21. We oThe GRACE study is supported by an unrestricted grant from Sanofi-Aventis to the Center for Outcomes Research, University of Massachusetts Medical School.Sanofi-Aventis had no involvement in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the paper for publication. The design, conduct, and interpretation of GRACE are undertaken by an independent steering committee.The authors have no conflicts to declare according to the Thrombosis Journal (TJ) Declaration of Competing Interest form. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license on a worldwide basis to the TJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in TJ editions and any other TJ products to exploit all subsidiary rights, as set out in the TJ license conditions. All authors have read and approved this manuscript.. EPG and GEB conceived and designed the study and wrote the manuscript. OD and FA contributed to study design and performed statistical analysis and reviewed the manuscript and BM supervised the study.We thank the physicians and nurses participating in GRACE. The complete list of GRACE Investigators can be found at Keith A.A. Fox, Joel M. Gore (GRACE Co-Chairs); Kim A. Eagle, Philippe Gabriel Steg, (GRACE Publication Committee Co-Chairs); Giancarlo Agnelli, Frederick A. Anderson, Jr, Álvaro Avezum, David Brieger, Andrzej Budaj, Marcus D. Flather, Robert J. Goldberg, Shaun G. Goodman, Christopher B. Granger, Dietrich C. Gulba, Enrique P. Gurfinkel, Brian M. Kennelly, Werner Klein, José López-Sendón, Gilles Montalescot, Frans Van de Werf.


Blood doping is commonplace in competitive athletes who seek to enhance their aerobic performances through illicit techniques.Cobalt, a naturally-occurring element with properties similar to those of iron and nickel, induces a marked and stable polycythemic response through a more efficient transcription of the erythropoietin gene.Although little information is available so far on cobalt metabolism, reference value ranges or supplementation in athletes, there is emerging evidence that cobalt is used as a supplement and increased serum concentrations are occasionally observed in athletes. Therefore, given the athlete's connatural inclination to experiment with innovative, unfair and potentially unhealthy doping techniques, cobalt administration might soon become the most suited complement or surrogate for erythropoiesis-stimulating substances. Nevertheless, cobalt administration is not free from unsafe consequences, which involve toxic effects on heart, liver, kidney, thyroid and cancer promotion.Cobalt is easily purchasable, inexpensive and not currently comprehended within the World Anti-Doping Agency prohibited list. Moreover, available techniques for measuring whole blood, serum, plasma or urinary cobalt involve analytic approaches which are currently not practical for antidoping laboratories. Thus more research on cobalt metabolism in athletes is compelling, along with implementation of effective strategies to unmask this potentially deleterious doping practice Ergogenic drugs are substances commonly used to enhance the athletic performance and include illicit drugs as well as a variety of compounds that are marketed as nutritional supplements. Although such drugs have been widely used by professional and elite athletes for centuries, research indicates that in recent years competitive athletes are increasingly experiencing with illicit compounds to improve both appearance and athletic abilities. Blood doping consists on techniques administered for non-medical reasons to healthy athletes to improve the blood oxygen carrying capacity, increasing oxygen deliver to the muscles, particularly in conditions of demanding physical exercise . Owing tCobalt belongs to Group VIII of the periodic classification of elements and shares properties with nickel and iron. Cobalt is a naturally-occurring element with properties similar to those of iron and nickel. It has been reported that cobalt chloride promotes an hypoxia-like response, involving enhanced erythropoiesis and angiogenesis. The erythropoietin gene is the paradigm of oxygen-regulated genes controlled by the transcription factor Hypoxia Inducible Factor 1a (HIF-1a). HIF-1a is the key regulator of cellular and systemic oxygen homeostasis, through an increased DNA binding activity to the target erythropoietin gene sequence. Under normoxic conditions, the main mediator HIF-1α is rapidly degraded by the proteasome. During hypoxia or cobalt chloride administration, the degradation of HIF-1a is markedly inhibited. Therefore, HIF-1a binds to HIF-1b, crosses the nuclear membrane and powerfully activates the erythropoietin gene transcription .Exposure to 120 or 150 mg/day of cobalt chloride results in the development of polycythemia with a substantial increase of hematocrit and hemoglobin, up to 20% above pre-treatment levels ,6. On thMany athletes either are unaware of or do not consider the possible health risks caused by several doping techniques. Thus, cobalt misuse or abuse in athletes should be regarded in a critical perspective, along with gene doping targeted at enhancing expression and transcription of the erythropoietin gene ,11. UnneOwing to these severe and unpredictable side effects, doping by cobalt salts may reveal as a serious threat for the scientific community and for public health. Cobalt is easily purchasable, inexpensive and not currently comprehended within the World Anti-Doping Agency (WADA) prohibited list . UnfortuAntidoping laws exist to provide a safe and fair environment for participation in sport. These laws should prevent and protect athletes from subjecting themselves to health risks through the use of unsafe, but performance-enhancing compounds. An area of major controversy is the "sports supplement" industry, which is poorly regulated when compared with prescription drugs, but yet is a potential source of doping violations. In this regard, pharmacokinetic characteristics, easy availability through the chemical industry and low costs would make cobalt administration the ideal surrogate or complement for rHuEpo administration, turning out to be the most suited blood doping technique for athletes seeking to improve aerobic performances with little chance of testing positive. At variance with blood doping, cobalt is not mentioned in the WADA prohibited list . NeverthThe author(s) declare that they have no competing interests.GL conceived of the study, and participated in its design and coordination and helped to draft the manuscript; MF participated in study design and helped to draft the manuscript; GCG coordinated the study design and helped to draft the manuscript. All authors read and approved the final manuscript.


Cultures of human MDMs or T-lymphocytes (as a nonphagocytic control) were exposed to carbon or carbon/iron particulates for various time periods and examined by transmission electron microscopy for ultrastructural changes. T-cells failed to internalize either of the particulates and showed no organelle or nuclear changes. Conversely, MDMs avidly phagocytized the particulates. MDMs treated with C particulates exhibited morphologic evidence of macrophage activation but no evidence of lysis of organelles. In contrast, MDMs treated with C/Fe particulates exhibited coalescence of particulate-containing lysosomes. This phenomenon was not observed in the case of C particulates. By 24 hr there was a tendency of the C/Fe particulates to agglomerate into loose or compact clusters. Surrounding the compact C/Fe agglomerates was a uniform zone of nearly total organelle lysis. The lytic changes diminished in proportion to the distance from the agglomerate. In such cells, the nucleus showed loss of chromatin. Although C particles induced no detectable oxidative burst on treated MDMs, C/Fe particles induced a nearly 5-fold increase in the extracellular oxidative burst by treated MDMs compared with untreated controls. Iron bound to C particles catalyzed the decomposition of hydrogen peroxide to generate hydroxyl radicals. Results of these studies suggest that, among particulates of similar size, biologic activity can vary profoundly as a function of particulate physicochemical properties.In this study, we tested the hypothesis that the presence of iron in carbon particulates enhances ultrastructural perturbation in human monocyte-derived macrophages (MDMs) after phagocytosis. We used 1-μm synthetic carbon-based particulates, designed to simulate environmental particulates of mass median aerodynamic diameter ≤ 2.5 μm (PM Respirable particulates containing transition metals such as iron, vanadium, nickel, and copper are known to catalyze the generation of reactive oxygen species (ROSs) such as the highly damaging hydroxyl radical . SequelaThe process of phagocytosis allows uncontrolled entry of iron-containing particulates into cells because these particulates have bypassed control by the protein transferrin  and its Our group has examined two naturally occurring zeolites, including forms whose biologic activity is reported to range from highly pathogenic (erionite) to essentially benign (mordenite). We found that on exposure to the same mass of a specific type of particulate, the oxidative burst increases with decreasing particle size but remains relatively independent of zeolite composition. On the other hand, the Fenton reaction depends on the type of zeolite, suggesting that the surface structure of the iron on the zeolite plays an important role . In anot2.5) that occur as airborne pollution. We made ultrastructural observations and compared human blood monocyte-derived macrophages  with human T-cells (as nonphagocytic controls) on exposure to C or C/Fe particulates. To assess the bioactivity of C and C/Fe particulates, luminescent assays using luminol for oxidative bursts were conducted after exposure of macrophages to the particulates. In addition, the ability of the particulates to produce hydroxyl radicals from hydrogen peroxide (H2O2) was studied (Fenton reaction).In the present study, we used synthetic 1-μm carbon and carbon/iron particulates, designed to simulate environmental particulates of mass median aerodynamic diameter ≤ 2.5 μm  before decomposition of the ammonium ions. The zeolite was cooled to room temperature, and solid phenol (0.21 g phenol/g zeolite) was added. A weak vacuum was pulled on the system, and the temperature was raised slowly to 60°C. Solid paraformaldehyde  was added to the zeolite/phenol material, and the temperature was raised very slowly to 120°C in a nitrogen environment. At this point the solid material turns red as the phenol/paraformaldehyde cross-linked polymer forms. The solid material was held at 120°C for 5 hr to allow for complete polymerization. The zeolite/polymer mix was pyrolyzed at 800°C for 19 hr under flowing argon. The zeolite template was removed by etching in 48% hydrofluoric acid for 6 hr. The size of the particles was confirmed by scanning electron microscopy (SEM). Bulk analysis of C/Fe particulates found aluminum content of 1.38%, silicon 0.33%, iron 0.46%, and the rest carbon.The procedures followed were adapted from a previously published procedure for the synthesis of a carbonaceous negative image of a zeolite . The syn7 cells/well (2 mL/well), and incubated at 37°C in a humidified atmosphere of 10% CO2/90% air for 48 hr to allow adherence of monocytes. Nonadherent cells were then removed, and adherent monocytes were washed twice with Seligman’s buffered saline solution (SBSS) and supplied with fresh complete M-CSF–free leukocyte culture medium. Cells were incubated for an additional 10–12 days with medium changes at 48-hr intervals to allow differentiation into the macrophage phenotype and were labeled MDMs.Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation from fresh heparinized blood collected by venipuncture from healthy, nontransfused consenting volunteers, as previously described . This st2/90% air, after which nonadherent cells were recovered for use in experiments.T-lymphocytes were isolated from PBMCs by negative selection with a commercially available cocktail of monoclonal antibodies and complement , using methods based on those of + cells). T-cells routinely marked 90–95% positive for CD3 and 25–35% positive for CD8, with the remainder positive for CD4, and had undetectable levels of monocyte contamination (as indicated by the absence of CD14+ cells).To assess homogeneity of populations prepared in this manner, samples were suspended in SBSS, stained with fluorescein isothio-cyanate–conjugated monoclonal antibodies specific for CD3, CD4, CD8, and CD14  as previously described , and ana6 cells in 2 mL/well). Particulates were sonicated and immediately added to cultures at a non-toxic concentration of 5 μg/cm2 surface area. Trypan blue dye exclusion was used to demonstrate that the exposure of 5 μg/cm2 particles was not toxic to the cells. Plates were centrifuged for 10 min at 300 × g immediately after addition of particulates and then incubated at 37°C in a humidified atmosphere of 10% CO2/90% air. After various periods of incubation (2–24 hr), MDMs were washed twice with PBS and harvested for fixation by gentle scraping after 15 min of incubation in 0.01% EDTA/PBS at 4°C. Concurrently, free particulates were separated from T-cells by density gradient centrifugation through Ficoll-Hypaque , which allows sedimentation of free particulates but not T-cells. T-cells recovered from the gradient were washed twice in PBS before fixation.C/Fe particulates 1 μm in diameter as well as similarly sized C particulates (without iron) were sterilized by steam autoclave and suspended in serum-free DMEM. MDMs differentiated as described above were washed with phosphate-buffered saline (PBS) and supplied with fresh complete DMEM (2.0 mL/well). T-cells were likewise washed and suspended in fresh medium in six-well culture plates , and then dehydrated in graded ethanol washes. Specimens were embedded in Medcast . Blocks were cured for a minimum of 12 hr at 60°C. Thin sections (~100 nm) were cut from cured blocks using an ultramicrotome  and mounted on 2-mm 300-mesh copper grids. Grids were heavy metal stained using a standard two-step uranyl acetate/lead citrate technique and then examined and photographed at 60 kV with a Philips 300 transmission electron microscope .2, four replicate wells per treatment. As negative controls, each experiment included 4 replicate wells containing MDMs, medium, and luminol but no particulates. Plates were sealed with UV-sterilized Top Seals  and centrifuged at 4°C . Luminescence was immediately measured (time 0) with a Top-Count Microplate Scintillation and Luminescence Counter (Packard), after which plates were placed in a 37°C incubator. Subsequently, luminescence was measured at 10 min intervals, with plates being incubated at 37°C between counts.Human PBMCs were plated in 96-well Optilux culture plates  and allowed to differentiate into MDMs as described above. Immediately before assay, culture medium was removed, and cells were washed twice with PBS before addition of ice-cold serum-free RPMI 1640 , 100 μL/well. A stock solution of 100 mM luminol was prepared with 20 mg luminol /mL dimethyl sulfoxide and stored at −20°C until use. Immediately before assays, luminol stock solution was thawed, diluted 1:100 (1 mM) in ice cold RPMI, and added to culture wells (100 μL/well). Particulates (C or C/Fe) were sonicated and added to culture wells at a concentration of 5 μg/cmLuminescence indices were calculated by dividing the mean luminescence counts per minute (cpm) of four replicate particulate-treated wells by the mean cpm of four negative control wells at each time point.N-oxide  was added, along with 50 μL of a 30% hydrogen peroxide solution . The solution was shaken in the dark for 15 min. The particles were removed by centrifugation, and the solution was analyzed in a capillary column by electron spin resonance (ESR) spectroscopy . ESR was performed with a modulating frequency of 100 kHz with a modulating amplitude of 2.090 G. The microwave power was 0.632 mW. The samples were scanned 10 times with the average taken as the representative spectra.Five milligrams of synthetic C or C/Fe particles were weighed and suspended in 500 μL of PBS (pH 7.4) solution. To this suspension, 100 μL of 5,5-dimetheyl-1-pyrolline-For the untreated cells (controls), the nucleus showed the expected rim of heterochromatin. The cytoplasmic organelles appeared normal. The microvilli showed expected formations. Nucleoli were ultrastructurally normal.An SEM image of the C/Fe synthetic particulates is shown in To assess bioactivity related to these synthesized particulates, we performed luminol assays to measure the release of ROSs on phagocytosis . These eData presented in To examine the kinetics of cell/particulate interaction, we fixed the cells 2–4 and 24 hr after exposure to particulates.At 2 hr postexposure, ultrastructural signs of macrophage activation were evident with generalized dilatation of endoplasmic reticulum (ER). Numerous C particulates were seen within intact lysosomes . At 24 hBy the first sampling (4 hr), particulates of similar size and contours as the stock preparation of C/Fe particulates (as seen by SEM) were present within cells. Many particulates were in lysosomes with a detectable membrane surrounding them . In otheBy 24 hr, the particulates were sometimes partially or markedly aggregated  and 7. AThe control T-cells and T-cells exposed to the C or C/Fe particulates showed no detectable ultrastructural changes. Random fields revealed healthy appearing cells. No evidence of internalized C or C/Fe particulates could be seen in the experimentally exposed group or controls. In both categories, the nuclei were generally oval and had the expected abundant heterochromatin. No ultrastructural changes were detected between the two categories.To determine the role of serum proteins in the agglomeration of C/Fe particulates, free particulates were incubated for 24 hr in complete or serum-free leukocyte culture medium, washed in distilled water, and examined by SEM. The C/Fe particulates, which had been suspended in the serum-free medium, showed no tendency to agglomerate . In contWe observed the propensity of the particles to catalyze the formation of hydroxyl radicals from hydrogen peroxide by ESR spectroscopy. The procedure involved trapping of the hydroxyl radical with DMPO . The fouThe physical properties of both the C and C/Fe particulates were such that sections for ultrastructural evaluation could be prepared without sectioning artifacts. The occasional absence of lysosomal membranes around particulates e.g.,  might beEpidemiologic studies have demonstrated that there is a direct correlation between exposure to small airborne particulates and human disease. However, the mechanism(s) by which these particulates exacerbate pulmonary or cardiovascular disease is not known. To begin to address the role of iron in these processes, we synthesized carbon-based particles with or without iron. It was interpreted that cell features associated with activation seen in the MDMs exposed to the C particulates only were a consequence of the activation brought about by the extensive phagocytosis of particulates. There was no lysis of organelles in cells treated with C particles.Consider the functional significance of some of the C/Fe-exposed cells having intact lysosomal membranes and ER, whereas others did not appear to be related to the length of time of exposure: At the 4-hour sampling, both structures seemed to be morphologically intact (but with dilated ER), whereas at 24 hr, the lysosomal membranes around the agglomerates were often ruptured and there was extensive ER dilation and vacuole formation (approximately in proportion to distance away from the agglomerate). Thus, both time and formation of the large agglomerate were positively correlated with the intracellular lesions. From a functional standpoint, it seems likely that sequelae from generation of ROSs could require time as well as sufficient concentration in an agglomerate to reach the threshold to alter the ultrastructural morphology.We also observed the following regarding the phenomenon of intracytoplasmic agglomeration of C/Fe particulates: At 4 hr postexposure, numerous particulates had become internalized. These were interpreted as being within phagosomes because the membrane could generally be followed around these variably sized particulates. At 24 hr, the particulates were often seen to be forming into much larger clusters but still generally within the lysosomal membranes. At an apparently more advanced phase, the particulates had sometimes formed a dense spherical cluster, surrounded by a zone of nearly total organelle lysis. The cause of such an intracellular lesion is not certain, but it would appear to be compatible with that resulting from reactive oxygen metabolite generation from the C/Fe particles.To explain the propensity of the C/Fe particulates to form spherical clusters within cells, we tested cell-free medium with and without serum. The agglomerates formed after the addition of C/Fe particulates (but not with C-only particulates) providing that serum was present. The spherical clusters were often approximately 10 μm in overall diameter whether in the cellular or noncellular system. The reason why the individual particulates in the intracellular clusters were smaller than particulates initially ingested by the macrophage is not apparent. One explanation may be that the large C/Fe conglomerate formation underwent a fracturing process associated with the cutting effect by the microtome blade. Given that during synthesis of the C/Fe particulates, the material is subjected to extreme acidification procedures, the acidification reached in a lysosome would not be sufficient to decompose the particle. Hence, it seems unlikely that degradation on a purely chemical basis could have occurred in the biologic environment of the lysosome and that the apparent smaller particle size is an artifact of the preparation associated with the slicing of a dense agglomeration of graphitic particles.2.5 to bring about particle aggregation  postexposure, the morphologic evidence of ultrastructural change was not detected until much later. The lesions were not seen at the 4 hr sampling but were seen at the 24 hr sampling. The reason the morphologic changes were not evident until later is not clear. One possibility is that cell injury may have occurred early but did not manifest morphologic changes until later. Another possibility is that the lesions were a consequence of the concentration of the possibly toxic products resulting from the coalescence of multiple small phagolysosomes with their contents. Still another possibility is that the physical compression on adjacent organelles as a consequence of the mass of the agglomerate might be considered. Of these, it seems most feasible that the lesions were related to concentration of possibly toxic secondary products produced by a chemical reaction from the agglomerate. The C/Fe particulates contain iron on the surface, and the cell has been demonstrated to undergo an oxidative burst on phagocytosis of the particulates. A likely oxidant produced by the cell during the oxidative burst is hydrogen peroxide. The C/Fe particle can promote the formation of hydroxyl radicals via the Fenton reaction:The ultrastructural changes noted in this study are consistent with macrophage activation and damage, which could be explained by the uncontrolled formation of ROSs.Synthetic C and C/Fe particulates (1 μm) were given to cultures of human T-cells and MDMs. The T-cells failed to ingest either particles and showed no ultrastructural changes. The MDMs avidly ingested both type of particles. In contrast, those receiving C particulates showed only ultrastructural changes associated with cell activation. Those receiving C/Fe particulates by 24 hr showed evidence of clustering and coalescence of particulates. A highly discrete, concentrated mass of particulates was sometimes surrounded by a zone of total organelle lysis. Evidence that the C/Fe particulates were bioactive was demonstrated by a nearly 5-fold increase in oxidative burst by treated MDMs. Similar cells exposed to C particulates showed no increase in this regard. The synthetic C/Fe particulates also produced hydroxyl radicals on exposure to hydrogen peroxide. We hypothesize that the formation of intracellular ROSs is responsible for the ultrastructural changes observed. Results of these studies demonstrate that particle-induced ultrastructural changes depend on phagocytosis and suggest that, among respirable particulates of similar size, biologic activity can vary profoundly as a function of particulate physicochemical properties.


Cobalt is classified by the IARC as a possible human carcinogen (group 2B). Although there is evidence for in vivo and in vitro toxicity, the mechanisms of cobalt-induced lung toxicity are not fully known. The purpose of this work was to identify potential signatures of acute cobalt exposure using a toxicogenomic approach. Data analysis focused on some cellular processes and protein targets that are thought to be relevant for carcinogenesis, transport and biomarker research.It has been estimated that more than 1 million workers in the United States are exposed to cobalt. Occupational exposure to 59 Co. A group of 29 of these genes, representing the main biological functions, was assessed by quantitative RT-PCR. The expression profiles of six of them were then tested by quantitative RT-PCR in a time-dependent manner and three modulations were confirmed by Western blotting. The 85 modulated genes include potential cobalt carriers , tumor suppressors or transcription factors  and genes linked to the stress response . We also identified nine genes coding for secreted proteins as candidates for biomarker research. Of those, TIMP2 was found to be down-regulated and this modulation was confirmed, in a dose-dependent manner, at protein level in the supernatant of exposed cells.A time course transcriptome analysis was performed on A549 human pulmonary cells, leading to the identification of 85 genes which are repressed or induced in response to soluble Most of these genes have never been described as related to cobalt stress and provide original hypotheses for further study of the effects of this metal ion on human lung epithelial cells. A putative biomarker of cobalt toxicity was identified. Cobalt  workers ,4. A stu workers ,8. As wi workers ,11. Some workers . A 19-mo workers .Following absorbtion by inhalation, cobalt is eliminated in the urine. Biological monitoring of accidental exposure mainly involves measuring the concentration of metal in the urine. This might be inadequate for several reasons. Firstly, the quantity of metal excreted (exposure marker) does not necessarily reflect organ damage, which varies from one person to another. Secondly, depending on the chemical form, excretion does not necessarily reflect the level of metal in the body because some forms are retained in the lungs. Thirdly, depending on its solubility, clearance can be very rapid and the cobalt may have left the body by the time samples can be taken. Therefore, a key issue in monitoring occupational exposure is the availability of adequate biomarkers.Although the chemical toxicity of cobalt has been proven, the molecular mechanisms of its toxicity are not all known. Cobalt is genotoxic ,15, and Since the lung is the main target organ of cobalt toxicity, the human A549 lung cell line was chosen as a model for this study, to evaluate cobalt toxicity. Noteworthy, this cell line has been widely documented in molecular toxicology, including hypoxia mimicked by cobalt ,20.Microarrays are currently used for large scale gene profiling, measuring sensitive cell changes in response to xenobiotic exposure. Such investigative studies may help identify new molecular targets for toxicants or provide new hypotheses about their mechanisms of action . We usedA549 is a stable tumor cell line, obtained from human lung carcinoma, with properties of type II alveolar epithelial cells . The resThe global transcriptional response was monitored using CEA microarrays GPL4263) . A549 ce63 . A549We used DAVID  web-acceAXL, IFNA4, DLG1, TNFRSF9 and SIN3A). Some discrepancies between the two techniques may be due to technical artefacts even if the primers were designed within the microarray cDNA sequence. For further analysis of gene modulation, we performed time-course quantitative RT-PCR on several genes: AXL, UBC, FBXL2 and SLC12A5 , or being induced from 2 h (UBC), or finally, early and continuous diminishing (FBXL2). For genes displaying temporal modulation, down-fluctuation quickly followed by up-regulation, as with AXL  in some specific functional classes: development, differentiation and proliferation, signal transduction and trafficking, cell defense and finally gene transcription and modification. We also selected genes coding for secreted proteins as potential hits for biomarker research. Tables ALDOA), glucose transporter 1 (SLC2A1), glyceraldehyde 3-phosphate dehydrogenase (GAPD), lactate dehydrogenase A (LDHA), proapoptotic factor (BNIP3L), phosphoglycerate kinase 1 (PGK1) and transferrin receptor (TFRC), (table TFRC which was repressed at 30 min), therefore our results agree with those already published and validate our system relative to previous reports, since they represent an appropriate internal positive control. Recently, two transcriptomic studies of hypoxia compared the action of low oxygen with that of metal ions such as cobalt or nickel on embryonic mouse fibrobasts [Cobalt is known as a "hypoxia-simulating" agent and mostter 1 SLCA1, glycebrobasts  and humabrobasts . 21% andbrobasts . CompariHSPCB gene coding for heat shock protein 90, was induced by a factor of 2.2 at 24 h  [ST13 and DNAJA2.The  h table . HSP90 p Cr, Cd) . However Cr, Cd) . Two othUBB, ratio 1.94, UBC, ratio 5.6) while UBC gene induction was observed in the kinetics from 4 h with qRT-PCR , is a transcription factor involved in both the initiation and termination of target gene transcription. It binds zinc and can act as a tumor suppressor [BHC80 (BHC80 protein), NT5E (CD73) and SDCCAG33 (Teashirt homolog 1). Cobalt ions have been shown to substitute for zinc in zinc finger protein domains which control the transcription of several genes and also in zinc-DNA repair proteins, inhibiting DNA repair [The protein encoded by ppressor . This geA repair ,36,37.DLG1 (disk large homolog 1) has been identified as a tumor suppressor gene in drosophila, with its mutation inducing the loss of cell polarity and neoplastic tissue growth. This gene is highly conserved. The molecular mechanism which regulates cell proliferation by DLG1 is not well known but there are arguments for implicating epithelial cell polarization in this regulation [DLG1 (disk large homolog 1) gene was strongly repressed in qRT-PCR. The anti-DLG1 antibody revealed clear depletion of DLG1 protein in Co+ samples (figure gulation . The DLGs figure . This coMAZ and DLG1) were repressed by cobalt, and in one case (DLG1), this repression was also observed at protein level.Two tumor suppressors . The three genes, FBXL2, ZNT1 and SLC12A5, are therefore appropriate candidates for research into cobalt transport proteins in human cells and will be studied further using targeted biological approaches.The ICAM1 (CD54), SERPIN C1 (antithrombin-III), IFNA4 , TFRC (transferrin receptor), PLA2R1 (phospholipase receptor A2), TNFSF6 (FAS ligand), WFDC2 (WAP four-disulfide core domain protein 2), LTBP3 (latent transforming growth factor beta binding protein 3) and TIMP2 . To further evaluate the interest of these genes, we checked the secretion modulation of the corresponding proteins. A549 cells were exposed to cobalt in a dose-response manner, to 0.2, 1 and 2 mM, and the supernatants analysed using available immunoassays. The levels of FAS ligand, WFDC2 [TIMP2 mRNA is described as being suppressed by hypoxia in endothelial cells [Biological monitoring of exposure to cobalt is mainly based on its concentration in urine. This method may be inadequate for two reasons. Firstly, measuring the quantity of excreted metal (exposure marker) does not necessarily reflect organ damage, which varies from one person to another. Furthermore, depending on the chemical form, excretion does not necessarily reflect the concentration of metal in the body; indeed, certain forms are eliminated in several phases; only 40% of cobalt oxides inhaled are excreted within 72 h . In thisd, WFDC2 , ICAM1, d, WFDC2 . Endogend, WFDC2 ,49, regual cells . Exposural cells . Therefoal cells . Since ial cells , TIMP2 mTIMP2 protein modulation in cobalt-exposed cell supernatant corroborated the gene down-regulation observed on the microarrays and with qRT-PCR. TIMP2 modulation has never been associated with metal stress. This result is innovative and will be studied further in the biological fluids of rats exposed to different chemical forms of cobalt by inhalation.FBXL2, ZNT1, SLC12A5) and tumor suppressors or transcription factors . Some of these genes provide new hypotheses for elucidating the mechanisms of cobalt intracellular chemical toxicity. Targeted biological approaches might confirm their biochemical role in the cobalt response.This study provides the first toxicogenomic analysis of human lung cell response to acute cobalt exposure. We have confirmed that genes involved in the cobalt hypoxia response and apoptosis are modulated. We have also revealed genes linked to heat shock response and proteasome function that have already been described in other metal stress responses. Newly identified genes linked to cobalt acute toxicity include potential cobalt carriers . The viability rate was determined as the ratio between the ATP in treated cells and control cells. Cobalt concentration in cell pellets was measured using flame atomic absorption spectroscopy .Total RNA was isolated using the Quiagen RNeasy miniprep kit according to the manufacturer's instructions. mRNAs were prepared using the Oligotex mRNA Quiagen kit, the purification process being repeated once to eliminate any contamination. RNA concentration was determined by OD measurement (260 nm/280 nm), purity and integrity were assessed using an Agilent 2100 Bioanalyser.Microarrays were obtained from the Service de Genomique Fonctionnelle . Two types of DNA collections were used for preparing the cDNA arrays: a collection of 5760 full length cDNA clones from the human infant brain 1NIB library, kindly provided by Genethon, and a collection of 2304 human PCR products (400–600 bp) corresponding to specific key-words selected by direct query in the Unigene database. A complete description of the microarrays used in this study, including the protocols for slide production, has been submitted to the GEO database  under acFor each microarray experiment, 20 μg of total RNA were reverse transcripted and indirectly labelled using the FairPlay Microarray Labelling Kit (Stratagene). Amino reactive Cy3- and Cy5- dyes  were then chemically bound to cobalt and control cDNA respectively. Reverse labelling (or dyeswap) was performed for each experiment. Microarrays were pre-treated for 20 min with an N-methyl-pyrrolidinone solution containing 20 g/L succinic anhydride and 20 mM sodium borate, pH 8, immersed quickly in ethanol and dried by centrifugation for 6 min at 500 rpm. Following a quick rinse in RNase-free water, the slides were prehybridized in 30 ml 5X SSC, 1% SDS, 1% BSA (w/v) at 50°C for 40 min. The microarrays were then rinsed in RNAse-free water, immersed quickly in isopropanol and dried by centrifugation for 6 min at 500 rpm. The hybridization solution was prepared using 100 μl formamide, 10 μl SDS 10% and 30 μl RNase-free water. Labelled cDNA was solubilized in 17.5 μl of this solution plus 7.5 μl of 20 × SSPE and heat-denatured. Hybridization was performed overnight at 42°C. The microarrays were quickly rinsed in 0.1 × SSC, 0.01% SDS, washed twice for 10 min in 0.01 × SSC, then dried quickly in a stream of nitrogen and scanned with GenePix 4000B . Cy3 and Cy5 spot fluorescence intensities were quantified after local subtraction of background using Genepix Pro 4.0 software (Axon Instrument Inc.). For each time point the result files were submitted to GeneSpring software 6.2 (Agilent Technologies) as follows. The data were first converted to take into account the results of the dyeswap reverse labelling, then normalized using the Lowess method, applying robust locally-weighted regression to smooth the intensity-dependence of the log ratios. The normalized data were then filtered on a quality test basis. This involved selecting spots detected on at least half of the microarrays with at least 70% pixels above threshold intensity (set to the median background plus two standard deviations).From these remaining spots, we selected those with fluorescence ratios  above 1.5 fold with p-value < 0.05 using a t-test statistical analysis on Genespring software and performing a Benjamini and Hochberg false discovery rate multiple testing correction.® 2 system. The amplification program consisted of 1 cycle at 95°C with 10 min-hold followed by 40 cycles at 95°C with 15 sec-hold, 60°C with 1 min-hold, and a reading step at 60°C for 1 sec. Amplification was followed by melting curve analysis between 65°C and 95°C. RNA was used as a negative template for the absence of residual genomic DNA and a negative control without cDNA was used to control overall specificity. House- keeping genes, which are generally used as reference genes, are often modulated by stress. For example, GAPDH is strongly induced by cobalt (ratio 3). To find an invariant gene at each time point, several candidates were selected for their invariance from all the microarray data, and tested as invariant genes in qRT-PCR. FIGF (c- fos induced growth factor) was selected for time points 0.5 h, 2 h and 4 h. For samples at 24 h, mRNA had to be purified from total RNA to detect TUBA3 as an invariant reference gene. Differential analysis was performed on cDNA templates obtained from cobalt-treated or untreated cells in sextuplets for each gene, in the same way as for the reference gene . The results were processed using REST-MCS software [Specific primers were designed with Primer3  using cDsoftware  and testWestern blots were performed as previously described  after 24Immunoassays were performed on culture supernatants from cells exposed to various concentrations of cobalt over a 24 h period. The supernatants were tested crude or at 10 fold concentration. TIMP2 and FASL proteins were detected using ELISA kits (Raybiotech and R&D systems respectively) according to the manufacturer's instructions. The WFDC2 protein, was analysed in Dr Hellström's laboratory because they developed the WFDC2 ELISA . Other iVM designed the study and drafted the manuscript.FB carried out data acquisition and analysis and prepared the microarray results for tables and figures.OP was involved in the design stage, qRT-PCR analysis and microarray experiments.SR carried out qRT-PCR.GS carried out the RNA extraction, labelling and microarray hybridization.EQ was involved in the design stage and revised the manuscriptAll authors read and approved the final manuscript.


Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39×10−4 substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89  ParvoviridaeLitopenaeus stylirostris and L. vannamei. This viral pathogen is known to cause mortalities of up to 90% in juvenile and subadult individuals of L. stylirostrisL. vannameiL. stylirostris populations in the Gulf of California L. stylirostris fishery landings have increased since that time L. stylirostris has developed resistance to IHHNV and/or the virus has reached an equilibrium with the host in terms of genes related to virulence Effective implementation of monitoring and control measures for viral epizootic outbreaks requires an understanding of the factors that underlie molecular evolution and population dynamics. This is also true for introduction of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) into shrimp populations of the northern Pacific coast of Mexico. IHHNV is a single-stranded DNA-containing virus belonging to the family To better understand the molecular evolution, population structure and dynamics of IHHNV in the Pacific coast of Mexico, we analyzed capsid protein gene sequences from wild shrimp populations in the region. With sample sizes much larger than in previous studies, we were able to make inferences about levels of variation, population structure and recent population history in this virus, leading us to question whether it should be regarded as a static evolutionary lineage.L. stylirostris, L. vannamei and Farfantepenaeus californiensis) were collected from seven sites in the northwestern Pacific coast of Mexico from November 2004 to January 2005  and CP2R (5′- CGG GTA TAT ATT GCA CAT CGA A-3′) that were designed for this study based on a published IHHNV sequence (GenBank accession number AF273215) 2, and 1µl of extracted genomic DNA (∼40 ng) in a 25 µl final reaction volume. Amplification parameters included an initial denaturation of 94°C for 2 min., followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. A final extension of 72°C for 7 minutes was used. The amplified DNA was electrophoresed in a 1% agarose gel, and gel purified using a Qiaquick gel purification kit . Amplified DNA samples were directly sequenced in both directions using the CP1F and CP2R primers. Sequencing was performed at the High-Throughput Genomics Unit of the University of Washington .Genomic DNA was extracted from 50–100 mg of shrimp tail muscle using DNAzol™  following manufacturer's instructions. Extracted DNA was resuspended in 100 µl of tris EDTA buffer and stored at −20°C. The IHHNV capsid gene was amplified by PCR using primers CP1F  for the initial analyses. Arlequin v.3.0b L. stylirostris and P. monodon in different geographic locations were downloaded from GenBank and included in phylogenetic analyses . We first constructed a haplotype network using statistical parsimony in the program TCS v1.21 To determine evolutionary relationships among the Mexican IHHNV isolates obtained in this study and compare them to other geographic isolates, 14 IHHNV capsid protein sequences isolated from analyses . Eight P6 generations, and sampling every 1000 generations. Convergence was acknowledged when the standard deviation of the split frequencies dropped below 0.01; parameter files from the Bayesian analysis were analyzed in TRACER To perform the Bayesian phylogenetic analysis, the data set of 61 unique IHHNV capsid sequences (47 unique haplotypes obtained in this study and 14 sequences from GenBank) was aligned using ClustalX To assess the influence of multiple convergent mutations  on the inferred IHHNV phylogenetic tree, the data set used in the Bayesian analysis was analyzed to calculate the number of parsimony informative characters with homoplasy using the computer program MacClade v.4.08 Lightner and colleagues documented the initial history of IHHNV outbreaks in Mexico and noted that the virus was first detected in the southern region of the Gulf of California 6 generations, with 10% eliminated as burnin, which seemed appropriate based on the visual inspection of the run trace, and effective sample sizes were >200.The rate of nucleotide substitution and historical effective number of infections were estimated using the computer program BEAST eN can be inferred from the BSP 7. The MCMC algorithm was run for 30×106 generations, with sampling every 3000 generations and 10% burnin. Visual examination of parameter and likelihood plots in TRACER v.1.4 Historical changes in effective population size 6 generations, with sampling every 1000 generations. The remainder of the parameters were the same as described above.To determine whether inferences from the Bayesian skyline plot might be biased by IHHNV population subdivision, we also performed separate BSP analyses for each region . Bahia Magdalena (BM) was not included in the analysis due to its geographical separation from the Gulf of California. The constant skyline model included 4–8 size groups depending on the data set size, and the MCMC algorithm was run for 10×10a priori. In contrast, the FEL method estimates dN and dS directly from the data at each codon site. The SLAC method calculates these parameters from reconstructed ancestral sequences and compares observed dN and dS against the expected corresponding values. For all codon-based analyses, the 89 IHHNV sequence alignment was in-frame, with the first nucleotide corresponding to the first codon position.The 89 IHHNV capsid sequence alignment was analyzed for evidence of recombination breakpoints using the Genetic Algorithms for Recombination Detection (GARD) tool We also inferred whether positive selection has acted in specific IHHNV lineages, leading to differential selective pressure across the IHHNV phylogeny. This was performed using the Genetic Algorithm (GABranch) method L. stylirostris (n = 83) followed by L. vannamei (n = 5) and one from F. californiensis  and 64 mutations . Of the 64 substitutions, 28 corresponded to synonymous and 36 to nonsynonymous substitutions . No inseThere were 47 unique haplotypes, with only 2 shared among sites. One haplotype was present in seven individuals each from San Felipe and Golfo de Santa Clara, and a second was found in two shrimp from Empalme and four from Bahia Kino.ST = 0.369, p<0.0001). Exact tests of haplotype distributions showed significant differentiation between all pairs of sites , except San Felipe vs. Golfo de Santa Clara. Pairwise ΦST values also showed that all seven populations were genetically different from each other  than in the central  and southern regions . Both measures of genetic diversity decreased with increasing latitude , of which 194 were parsimony informative. Although this data set contained 13 distantly related sequences from outside of the study area, only 48 sites (5%) showed any degree of homoplasy  on the final Bayesian tree, and only 17 sites were inferred to have 3 or more mutations.The IHHNV haplotype network produced four distinct clades . The claA 50% majority-rule tree obtained from the Bayesian analysis is shown in Based on statistical parsimony assumptions of haplotype frequency and connectedness, a northern haplotype was inferred to be the ancestor of the sampled IHHNV lineages, since it is present in 14 individuals and is only 1 to 2 mutational steps away from 10 other haplotypes in the region . The IHH−4 substitutions/site/year. Comparison of the marginal likelihoods calculated by Bayes factors showed that the expansion growth model had the largest marginal likelihood value, and is therefore the best-fit population growth model for IHHNV. The expansion growth model had a mean mutation rate of 1.39×10−4 (95% HPD 3.34×10−5–2.91×10−4). The rate of substitution under strict molecular clock assumptions and constant population size was very similar, with a mean mutation rate of 1.81×10−4 (95% HPD 2.61×10−5–3.39×10−4).The mean rate of nucleotide substitution estimated from dated IHHNV capsid sequences under assumptions of both strict and relaxed molecular clocks were comparable and unexpectedly high for a DNA virus such as IHHNV. The relaxed molecular clock was favored over the strict clock, as the 95% highest posterior density interval (HPD) of the standard deviation of the lognormal marginal posterior distribution excluded zero . The relaxed molecular clock model also had a larger marginal likelihood according to the Bayes factors comparison (−2696.62 for the strict molecular clock compared to −2690.78 for the relaxed molecular clock). Mean mutation rates obtained with the five demographic models under the relaxed molecular clock ranged from 1.39 to 4.89×10−4 substitution/site/year for the 85 sequences obtained in this study (excluding the 4 used in the molecular clock estimate). The analysis suggested that IHHNV may have been present in Mexican penaeid shrimp population in the early 1970's, has been slowly increasing since the mid 1980's, and began an exponential phase of growth in approximately the early 1990's (−5) and 75% (2.23×10−4) estimated mutation rates (data not shown). However, the timing of the population expansion differed by approximately 10 years . Our analysis suggests that the common ancestor of the sampled IHHNV isolates dates to the early 1970's, which coincides with the assumed time of IHHNV introduction into the region Historical IHHNV population demography in the northwestern Pacific coast of Mexico was inferred from a Bayesian skyline plot incorporating the mean rate of 1.39×10y 1990's . These dNo evidence of recombination was found at any codon site in the IHHNV nucleotide alignment. Signals of natural selection were found in some codons, the number of which varied depending on the genetic algorithm used. The overall mean dN/dS ratio of the IHHNV capsid region analyzed as estimated by the SLAC method was 0.493. Only codon 7 was found to be positively selected under the three methods . The REL method found five additional positively selected codons . Negatively selected codons under the three algorithms included codons 66, 151 and 213. Both FEL and REL algorithms detected additional negatively selected codons . Other negatively selected codons detected by the REL method were 80, 91, 92, 118, 121,163, 180, 227, 233, 249, 262.The GABranch analysis suggested surprisingly high levels of positive selection in 13 lineages of the IHHNV genealogy. Specifically, three lineages found in the northern region, six in the central and four in the south showed the highest possible dN/dS values , supported by at least 98% of the models tested.The history of IHHNV in the northwestern Pacific coast of Mexico provides a unique opportunity to study the molecular evolution and population dynamics of a marine parvovirus after its introduction into a new environment. We found that IHHNV haplotype and nucleotide diversity is higher than previously reported. Tang and colleagues analyzed the capsid gene of 14 IHHNV isolates from the Americas that included only 4 samples from the Gulf of California, and reported 1.3% segregating sites in the capsid protein gene We found that genetic diversity is not uniform across geographic regions in the northwestern coast of Mexico. In fact, IHHNV localities in the central and southern regions of the Gulf of California are an order of magnitude more variable than those in the northern region. The statistical parsimony network and Bayesian phylogenetic tree also show more geographic structure in the southern and western regions, with the central region representing an admixture of isolates of different geographic origins. These differences may be explained in light of the marine and biogeographic features present in the Gulf of California. The northern area of the Gulf is characterized by a unique cyclic current system that limits interactions with the central and southern regions of the Gulf The mixture of haplotypes evident in the haplotype network and Bayesian analysis suggests high genetic exchange among the central and southern regions and across the Baja California Peninsula. Interestingly, Bahia Kino and Bahia Magdalena haplotypes are closely related despite being separated by the strip of land that represents Baja California Sur. Natural movement of aquatic organisms across the coasts of the Baja California Peninsula is highly unlikely due to the large geographic distance. However, association of geographically distant haplotypes could be due to human-mediated transport of IHHNV infected broodstock and post-larvae between these two regions. In shrimp aquaculture, a common practice is the capture of wild shrimp in different life stages for seed, spawners and broodstock The statistical parsimony haplotype network provided a clear indication that the geographic location of IHHNV introduction is in the northwestern coast of Mexico. The rooting algorithm used by statistical parsimony assumes that gene pools contain multiple, identical sets of haplotypes that mutate periodically producing a mixture of coexisting old and new haplotypes −4 substitution/ site/ year, which is unexpectedly high for a single-stranded DNA virus, and comparable to RNA viruses which evolve at a rate of 10−3 to 10−5 substitutions/site/year −5–2.91×10−4) also excludes slow, large DNA virus-like rates of evolution. It is generally assumed that DNA viruses evolve at a similar rate to that of their hosts due to viral dependence on the host's cellular machinery for replication −4), feline panleukopenia parvovirus (FPLV) (9.4×10−5) −4) −4) We estimated a rate of IHHNV nucleotide substitution of 1.39×10Penaeus monodon for aquaculture at that time has previously been suggested as the source of IHHNV in Mexico Additional support to our estimated substitution rate comes from close correspondence between IHHNV population growth inferred by the Bayesian skyline plot and the time of first detection and viral epizootics in the northwestern coast of Mexico. It is possible that the BSP was influenced by selection in some of the lineages and population subdivision . However, both the mean and the 25% estimated rates of nucleotide substitution produced population dynamics that fit reasonably well with the IHHNV epizootic history in the Gulf of California, although the 75% estimated rate suggests a younger population history inconsistent with the historical records. The BSP traces the IHHNV most recent common ancestor back to the early 1970's, and the introduction of The high levels of diversity we found in IHHNV contrast with previous suggestions of IHHNV low variation and stability IHHNV is a dynamic entity, with genetic differentiation among geographic regions in the Gulf of California. The presence of several quickly evolving lineages in the central and southern regions of the Gulf of California might be viewed as geographic “hotspots” of IHHNV variability that could lead to the emergence of virulent strain(s) following changes in the host or environmental conditions. The potential for an economically devastating IHHNV outbreak is further enhanced by current global aquaculture practices, with the spread of viral diseases in shrimp aquaculture attributable to the movement of infected animals among countries and even continents IHHNV dynamics in the Gulf of California should, therefore, warrant frequent genetic monitoring in both wild and farmed shrimp populations, as well as stricter enforcement of biosafety measures. Because our data show evidence for regional isolation of IHHNV in shrimp populations, restriction of shrimp movement is likely to be an effective management tool in limiting spread of the virus. The assumption that IHHNV is a stable, static virus understates the potential for a new virulent strain to arise, leading to epizootics similar to those observed in the early 1990's.Table S1Details and GenBank accession numbers of the IHHNV capsid sequences used in this study.(0.11 MB DOC)Click here for additional data file.


The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21cip1 wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma. Osteosarcoma is the most common primary bone malignant tumor occurring mainly in children . After iHedgehog (Hh) pathway has been implicated in different aspects of animal development, acting through several components, including the transmembrane proteins PATCHED (PTCH1) and SMOOTHENED (SMO), to activate the GLI zinc-finger transcription factors ,6. Hh paSMO shRNA.To explore the involvement of Hh pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of the Hh pathway genes in osteosarcoma and examined the effect of inhibition of SMO by cyclopamine, a specific inhibitor of SMO  or SMO sSonic Hedgehog (SHH) 2.1- to 18.8-fold . The reason for GLI1 down-regulation could not be determined. One possibility is that the GLI1 promoter is inactivated in human osteosarcomas by epigenetic modification. We found that GLI1 promoter contains a CG-rich region. Wong et al. reported that Hh pathway activity down-stream of SMO is mediated by GLI2 . Cells were then analyzed by flow cytometry using a FACS Vantage SE . Data were gated using pulse width and pulse area to exclude doublets, and the percentage of cells present in each phase of the cell cycle was calculated using FlowJo software .t-test, and differences in frequencies by Fisher's exact test. Differences were considered significant at P < 0.05.Each sample was analyzed in triplicate, and experiments were repeated three times. In all figures, error bars are standard divisions. All statistical analyses were performed using Microsoft Office Excel  and STASTISCA . Differences between mean values were evaluated by the unpaired (Hh): Hedgehog; (SMO): SMOOTHENED; (PTCH1): PATCHED; (SHH): Sonic hedgehog; (DHH): Desert hedgehog.The authors declare that they have no competing interests.TS was involved in the design and execution of the experiments, drafted the manuscript and contributed to the overall experimental design. MH conducted most of the experiments. HS was conducted a most of experiments. HG was conducted a part of experiments. YM was conducted a part of experiments. HN was conducted a part of experiments. OK was conducted a part of experiments. SK contributed to the overall experimental design. All authors read and approved the final manuscript.A, Immunohistochemical examination revealed that SMO was expressed on cytoplasm of 143B and GLI2 was localized in the nucleus of 143B. B, MTT assay showed that Sonic hedgehog promote proliferation of osteosarcoma cells. The experiment was triplicate with similar results.Click here for fileReal-time PCR was performed to quantify mRNAs of cell cycle related genes.SMO shRNA reduced levels of cyclin D1, cyclin E1, SKP2, and E2F1 transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. The experiment was triplicate with similar results.Click here for fileWe performed real-time PCR using formed tumors. Real-time PCR revealed that transcription of GLI1, GLI2, and PTCH1 was decreased in tumors formed by SMO shRNA-transfected 143B. In addition, SMO shRNA reduced levels of Cyclin E1, SKP2, and E2F1 transcription. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. The experiment was triplicate with similar results.Click here for filein vivo.Cyclopamine prevents proliferation of osteosarcoma  Immunohistochemical examination of ki67 was performed in xenograft tumors. Ki67 staining revealed that proliferation of osteosarcoma cells was decreased by cyclopamine treatment. The numbers of Ki67-positive cells was decreased to 50% of control revel by cyclopamine administration at day 14 (error bar means S.D.).Click here for file


The aim of this study was to confirm recent results from a previous study focussing on the development of a method to measure the bacterial translocation through puncture holes in surgical gloves under real surgical conditions.An established method was applied to detect bacterial migration from the operating site through the punctured glove. Biogel™ double-gloving surgical gloves were used during visceral surgeries over a 6-month period. A modified Gaschen-bag method was used to retrieve organisms from the inner glove, and thus-obtained bacteria were compared with micro-organisms detected by an intra-operative swab.In 20 consecutive procedures, 194 gloves  were examined. The rate of micro-perforations of the outer surgical glove was 10% with a median wearing time of 100 minutes (range: 20-175 minutes). Perforations occurred in 81% on the non-dominant hand, with the index finger most frequently (25%) punctured. In six cases, bacterial migration could be demonstrated microbiologically. In 5% (5/98) of outer gloves and in 1% (1/96) of the inner gloves, bacterial migration through micro-perforations was observed. For gloves with detected micro-perforations (n = 10 outer layers), the calculated migration was 50% (n = 5). The minimum wearing time was 62 minutes, with a calculated median wearing time of 71 minutes.This study confirms previous results that bacterial migration through unnoticed micro-perforations in surgical gloves does occur under real practical surgical conditions. Undetected perforation of surgical gloves occurs frequently. Bacterial migration from the patient through micro-perforations on the hand of surgeons was confirmed, limiting the protective barrier function of gloves if worn over longer periods. During surgery, intact surgical gloves act as a physical barrier against the transmission of blood-borne pathogens from hospital staff to patients and vice versa . SeveralIn a previous study, we established a method to evaluate the quantity of bacteria passing through undiscovered glove punctures under real surgical conditions . We demoThe purpose of this prospective investigation was to confirm our previously published results on bacterial translocations .® Indicator™, Mölnycke, Gothenburg, Sweden). This patented indicator system gives visual warning of glove puncture in the presence of fluid by showing a dark green patch around the site of puncture and has been shown to effectively indicate punctures [During a period of six consecutive months from December 2007 to May 2008, gloves from a total number of 20 elective and emergency surgical laparotomies in the Department of General Surgery at the Ernst-Moritz-Arndt University, Greifswald, were analyzed. The indications for laparotomy included: perforations and resections of the gastrointestinal tract n = 17) and lavages for underlying peritonitis (n = 3). Detailed demographic data of the procedures included are summarized in table 7 and lavunctures .There was no directive to surgeons on maximum glove wearing time. Gloves were changed and examined when a perforation was detected by the indicator system or at the end of a procedure. The impermeability of all inner and outer gloves worn was tested according to DIN EN 455-1 immediately after sampling. Gloves with obvious macro-perforations at any time were excluded. The person wearing the glove, their role within the surgical team, the type of surgery, date and the wearing time were documented. Surgical sites were examined by taking one swab from an area inside the situs and at the moment assumed to be most heavily contaminated, and was further processed following standard microbiological methods.To investigate bacterial migration from patients to the hand of the surgical staff through micro-perforations in gloves, the modified and standardized Gaschen-Bag method was used as previously described . BrieflyMicrococcus luteus, enterococci and E. coli were identified.In 20 consecutive surgical operations, 194 gloves  were examined [Figure The intact surgical glove is the most important barrier to protect the patient from microorganisms from the hand of the surgical team and vice versa. The preoperative surgical hand preparation can reduce but not eradicate the resident flora on the surgeon's hands, therefore reducing but not eliminating the risk of transmission of resident organisms into the wound. Conversely, blood-borne pathogens can be transmitted from the patient to the surgeon and endanger surgical team members .The role of glove perforation as risk factor of surgical site infection (SSI) is still not well understood. In a recently published study conducted by Misteli et al., macro-perforations of surgical gloves were found to be a significant risk factor for the development of SSI in cases where prophylactic antibiotics were not administered .Neither the question if of micro-perforations compromise the aseptic barrier nor as a risk factor for SSI has been well studied yet. To our knowledge, Harnoss et al. were the first who described the translocation of microorganisms through undetected micro-perforations under real conditions . Our stuInterestingly, only the surgeon was affected in this study, with his index fingers and thumbs/middle fingers at major risk for bacterial migration Table . ResultsAs we showed previously, the incidence of micro-perforations in surgical gloves depends on the duration of wear . We demoThe results from that study clearly indicate that the risk of micro-perforations and, as a consequence, the loss of protection increases with wear duration. Based on these data, a glove change for the surgeon and the first assistant after 90 minutes and after 150 minutes for the second assistant and the scrub nurse was recommended by the authors. In the meantime, these recommendations have been adopted be the Association of the Scientific Medical Societies in Germany (AWMF) .While our study has several limitations, it encourages safety measures to lower the risk of glove perforation. The possible transmission of pathogens shown here adds only indirect evidence to the role of micro-perforations as a risk factor for SSI. Because this was a single-center study using a single glove brand and including only one type of surgery, the results may not be fully transferable to other settings or glove brands. Essentially, the frequency of micro-perforations, the percentage of translocations, and the relation between wear duration and the number of perforations may vary. Further research should include multicenter studies with clinical endpoints to confirm our results. Nonetheless, high frequencies of perforations in surgical gloves have been repeatedly described in literature, and our findings are supported by results from other groups and settings, as well as by our own group ,8,12-17.Due to ethical and safety considerations, the recommendations given by Partecke et al. and the AWMF for the daily routine in surgical settings should be followed even before final proof is available. These recommendations include a change of gloves after 90 minutes. While our findings support an earlier change, this seems to be a good compromise between safety and feasibility ,18.To improve protection of the whole surgical team and the patient, an alternative for changing gloves after 90 minutes might be an improvement of glove material or the application of double gloving . This beThis study confirms previous results that bacterial migration through unnoticed micro-perforations in surgical gloves does occur under real practical surgical conditions. Undetected perforation of surgical gloves occurs frequently. Bacterial migration from the patient through micro-perforations onto the hand of surgeons was confirmed, limiting the protective barrier function of gloves if worn over longer periods. Preventive measures for lowering the risk of glove perforation can include a change of gloves at least every 90 minutes, the use of double-gloving, or the specific strengthening of predilection sites for punctures, and are therefore strongly recommended.The authors declare a financial competing interest:Mölnycke Healthcare, Gothenburg, Sweden accepted the costs of the study including the article-processing charge.NOH had the idea for the study and planned and supervised the experiments, drafted the manuscript, and analyzed and interpreted the data. AMG participated in the design of the study and helped to draft the manuscript. NS performed most of the microbiological methods and helped to draft the manuscript. OA participated in the design of the study and helped to draft the manuscript. CDH participated in the study design and coordination, and helped to draft the manuscript. AK participated in the study design and coordination, and helped to draft the manuscript. LIP participated in the design of the study, supervised the experiments, analyzed and interpreted the data, and helped to draft the manuscript.All authors have been involved in drafting the manuscript or revising it critically for important intellectual content and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/192/prepub


Tumour stromal neovascularization was investigated in 114 invasive and 20 in situ carcinomas of the uterine cervix by staining representative sections with the specific endothelial marker anti CD31 . A digital image analyser was used to measure the immunoreactivity. The following parameters were determined in the 'hot spots': vessel counts, vessel perimeter and endothelial stained area (expressed per mm2). The results were correlated with clinical and histopathological data. There was no significant relationship between the histopathological findings  and the median vessel count. In a univariate analysis all angiogenesis parameters had prognostic value: a higher vascularity was associated with worse prognosis (P < 0.05). Multiple regression analysis showed that vascular permeation (P < 0.001) and the median vessel count (P = 0.005) were the most important prognostic indicators. In the future these criteria may be used for selection of patients for anti-angiogenesis therapy.


Germ cells arise from a small group of cells that express markers of pluripotency including OCT4. In humans formation of gonadal compartments  takes place during the 1st trimester (6–8 weeks gestation). In the 2nd trimester germ cells can enter meiotic prophase in females whereas in males this does not occur until puberty. We have used qRTPCR, Westerns and immunohistochemical profiling to determine which of the germ cell subtypes in the human fetal gonads express OCT4, DAZL and VASA, as these have been shown to play an essential role in germ cell maturation in mice.OCT4 mRNA and protein were detected in extracts from both 1st and 2nd trimester ovaries and testes. In ovarian extracts a marked increase in expression of VASA and DAZL mRNA and protein occurred in the 2nd trimester. In testicular extracts VASA mRNA and protein were low/undetectable in 1st trimester and increased in the 2nd trimester whereas the total amount of DAZL did not seem to change. During the 1st trimester, germ cells were OCT4 positive but did not express VASA. These results are in contrast to the situation in mice where expression of Vasa is initiated in Oct4 positive primordial germ cells as they enter the gonadal ridge. In the 2nd trimester germ cells with intense cytoplasmic staining for VASA were present in both sexes; these cells were OCT4 negative. DAZL expression overlapped with both OCT4 and VASA and changed from the nuclear to the cytoplasmic compartment as cells became OCT4-negative. In males, OCT4-positive and VASA-positive subpopulations of germ cells coexisted within the same seminiferous cords but in the ovary there was a distinct spatial distribution of cells with OCT4 expressed by smaller, peripherally located, germ cells whereas DAZL and VASA were immunolocalised to larger (more mature) centrally located cells.OCT4, DAZL and VASA are expressed by human fetal germ cells but their patterns of expression are temporally and spatially distinct. In the 1st trimester OCT4 was detected in most germ cells. In the 2nd trimester the onset of expression of VASA was associated with the formation of oocytes and spermatogonia both of which were OCT-4 negative. Relocation of DAZL from nucleus to cytoplasm paralleled the down regulation of OCT4 and the onset of expression of VASA. These data reveal similarities between the expression of key regulatory proteins within germ cells as they mature in male and female fetal human gonads suggesting that in the female these maturational changes are not determined by entry into meiosis. Germ ceiewed in ). Withiniewed in ) and NANiewed in  and humaiewed in ,7. We harimester . In prevrimester ; howeverDazl, an RNA binding protein that is a member of a conserved gene family members of which include BOULE and DAZ [Mvh (mouse Vasa homologue) a gene that encodes an RNA helicase which is specific to the germ cell lineage [Dazl mRNA is first detectable in post-migratory germ cells on e11.5 [Dazl gene results in germ cell loss in both males and females [Dazl -/- females germ cell loss occurred in fetal ovaries at the time of meiotic entry and adult ovaries did not contain oocytes [Dazl -/- males the pattern of germ cell loss is variable and in some studies has been reported to occur during fetal life [Studies in knockout mice have identified a number of genes the expression of which is critical for germ cell survival and functional maturation in both ovary and testis. One such gene is  and DAZ ,11. A se lineage . In miceon e11.5  and the on e11.5 . Targete females . In Dazl oocytes ,15. In Dtal life ,16 or ental life .VASA is a member of the DEAD box family of genes first identified in Drosophila where it was shown to be essential for female germ cell development [Mvh ovarian function appeared normal but males were infertile with demise of germ cells at the zygotene stage of meiosis [elopment . In mice meiosis . Mvh has meiosis ,19,20. I meiosis . First tIn the present paper we have extended our studies on the germ cell subpopulations within the human fetal ovaries and testes to determine when and where DAZL and VASA proteins are expressed and have performed co-staining for OCT4 as this is commonly used to delineate the germ 'stem cell' population ,22,23. TOCT4 mRNA was detected in both the 1st and 2nd trimester samples with a slight, but non-significant decrease in the older samples . In the testis, OCT4 and DAZL mRNAs were detectable in both 1st and 2nd trimester with no significant difference between ages  although this was less marked than that seen in ovarian samples  which was particularly striking in the ovarian samples . In the 2nd trimester, OCT4 positive germ cells were located at the periphery of the ovary whereas the most intense immunoexpression of VASA was detected in the cytoplasm of OCT4 negative germ cells (oogonia) located in nests Figure . As in tThe above data suggested that expression of DAZL and VASA was similar but not identical, in both ovary and testis. This was directly investigated by dual immunohistochemistry for DAZL and VASA in the 2nd trimester Figure . This idIn the ovarian samples the pattern of expression was further investigated by measuring germ cell diameter at 3 representative gestations  as our previous studies have established that cell diameter provides an indication of the maturational status of the human fetal female germ cell . This dein vitro can spontaneously enter meiosis emphasising the common developmental potential of the germ cells in the two sexes and the critical role played by the testicular environment in modifying germ cell fate [Germ cells within the male and female gonads develop from an apparently homogeneous bi-potential population of PGC. In both male and female human fetal gonads germ cells initially proliferate but thereafter their fates diverge with female germ cells entering meiotic prophase as early as 11 weeks gestation  whereasell fate ,26.DAZL and VASA was compared to OCT4 as studies in knockout mice have reported that both genes are essential for germ cell maturation in that species [in vitro remains unresolved . In theiewed in ,30. In trm cells .Co-incident and overlapping patterns of expression of these germ cell specific proteins were revealed by fluorescent co-immunolocalisation on fixed specimens from a range of gestational ages. Previous studies have immunolocalised DAZL in a small number of samples recovered in the 2nd trimester -34 but wDAZL is important for normal functioning of the human germ line. The first study reported a strong association between several common single nucleotide polymorphisms (SNPs) and age at menopause in a sample population of 324 women [DAZL (Arg to Gly at 115) in a region of the protein critical for RNA binding [DAZ gene on the Y chromosome, deletions of which have been frequently documented as a cause of male infertility [DAZL (Asn 10 Cys) was reported to be azoospermic [Two studies on human populations have provided preliminary evidence that expression of 24 women . In a se binding . The sitertility . Howeverospermic .VASA between 9 and 14 weeks gestation suggested to us that this was coincident with the entry of female germ cells into meiosis. However increases in both protein and mRNA were also demonstrated in male germ cells suggesting that the onset of expression of VASA was associated the maturation of the gonocytes into prespermatogonia rather than meiotic entry per se. In the ovary VASA was detected in the cytoplasm of in slightly larger, more mature germ cells than DAZL, and was also present in oocytes within primordial follicles. Previous studies that have detected expression of VASA in cytoplasm of germ cells within the fetal ovary and testes at 17 weeks gestation [st trimester and provides a useful method for identifying the different populations of germ cells present within the 2nd trimester gonads.The marked increase in expression of estation ,37. Co-sMvh mRNA has revealed a number of putative Dazl-binding sites that are conserved between human, rat and mouse [VASA although further studies are required preferably using isolated human germ cells. DAZL has also been implicated in the regulation of other conserved germ-cell RNA-binding proteins including PUM2 [Pumilio that is required for maintenance of germ line stem cells in Drosophila and Caenorhabditis elegans.Examination of the 3' UTR of the nd mouse . Our dating PUM2 , the humOCT4 expression in the less mature germ cells  to VASA expression in the more mature germ cells . In testicular samples germ cells at different stages of maturation co-exist within the same seminiferous cords. Both male and female germ cells showed a similar pattern of change with development, despite the onset of meiosis in the female but not in the male, suggesting that although increases in expression of DAZL and VASA in the ovary may be related to meiotic entry there are underlying developmental patterns common to both sexes.By documenting the differential and partially overlapping patterns of expression of OCT4, DAZL and VASA proteins in the human fetal ovary and testis we have gained new insight into parallels in the maturation of germ cells in these two organs. Our findings clearly demonstrate changes in overall germ cell maturation with increasing gestation, and indicate a switch from SRY gene as detailed in [Human fetal gonads were obtained following termination of pregnancy during the first  and 2nd trimesters . Women gave written consent according to national guidelines  and the ailed in . Testes Total RNA was extracted from fetal gonads using the RNeasy Mini Kit  for mid trimester gonads and the RNeasy Micro Kit for 1st trimester gonads  The RNA was primed for reverse transcription with oligo(dT) primer  at 65°C for 10 min. The entire reaction was added to a total volume of 38 μl containing dNTP to 1 mmol/l, dithiothreitol (DTT) to 10 mmol/l, 1× Expand Reverse Transcriptase (RT) buffer and 120 IU RNasin ribonuclease inhibitor . One half (19 μl) of this reaction was added to 1 μl water (RT negative reaction) to act as a negative control to confirm the effiency of the DNase treatment. Fifty IU of Expand Reverse Transcriptase (Roche Diagnostics Ltd) was added to the remaining 19 μl (RT positive reaction) and both reactions were incubated for 1 h at 42°C. Reactions were stored at -20°C until required.®SYBR Green qPCR SuperMix-UDG . Amplification was continued for 45 cycles with signal acquisition at 84°C after each round of extension. Following amplification, continuous melt curve analysis was performed to ensure product accuracy and samples were analysed by agarose gel electrophoresis (data not shown) to confirm product size. Primer sequences are given in Table GAPD, OCT4, DAZL and VASA were derived by making a series of dilutions (1 in 5 to 1 in 10000) of first-strand cDNA from a mid trimester ovary. The number of cycles needed to yield a fluorescent signal above background  at each dilution was plotted against the log of relative concentration using LightCycler Software . The dilutions yielded a straight line for each product, confirming that Cp is a good indicator of target concentration across at least 2 orders of magnitude. The slopes of these curves are a measure of the efficiency of the PCR, which gave an amplification rate of 1.8-fold/cycle for GAPD and DAZL, and 1.7-fold/cycle for OCT4 and VASA. All gene amplification reactions were performed in triplicate. Calculations of mRNA concentration were made relative to GAPD.Quantitative real-time RT-PCR was performed using the Lightcycler (Roche Diagnostics Ltd) as described previously . ReverseFetal gonads were homogenised in 1× RIPA buffer containing: 25 mM Tris, 1% Triton, 0.05% sodium deoxycholate, 0.1% SDS and 150 mM NaCl. Total protein was measured using the Protein Assay DC Kit from Bio-Rad . Samples were denatured in 1× reduced sample buffer containing: 625 mM Tris (pH 6.8), 5% glycerol, 2% SDS, 0.0025% bromophenol Blue, 2.5% β-mercaptoethanol and 5 μg of total protein was loaded onto individual wells in a SDS-PAGE gel made with 10% (w/v) acrylamide; samples of pre-stained protein size markers were run on each gel . Following separation of proteins they were transferred onto PVDF Immobilon-Fl membranes ; non-specific binding sites were blocked by incubating membranes in Odyssey blocking buffer . All membranes were incubated with primary antibodies diluted in Odyssey blocking buffer overnight at 4°C. Proteins were detected using rabbit-anti-VASA  diluted 1 in 500 together with mouse anti-β tubulin  diluted 1 in 300, mouse anti-DAZL  diluted 1 in 500, with rabbit anti-β-tubulin  diluted 1 in 1000, or with goat anti-OCT4 (Santa Cruz) together with rabbit anti-β-tubulin (Santa Cruz) diluted 1 in 1000. Membranes were washed in PBS containing 0.1% Tween and bound antibodies were detected using fluorescently labelled secondary antibodies: goat anti-rabbit 680  and goat anti-mouse 800  for VASA and β-tubulin respectively; goat anti-mouse 680  and goat anti-rabbit 800  for DAZL plus β-tubulin; while OCT4 and β-tubulin, were detected using donkey anti-goat 680  and goat anti-rabbit 800 . Secondary antibodies were all diluted 1 in 10000 in Odyssey blocking buffer and incubated for 1 h at room temperature; bound fluorescent secondary antibodies were visualised using a LI-COR-Odyssey Infrared Imager .Immunolocalisation was carried out using standard methods . BrieflyThree representative non-adjacent tissue sections from ovaries at gestational ages 14, 16 and 19 weeks immunostained for DAZL and VASA were analysed. The cell diameter of all immunopositive germ cells on each section was calculated as the average of two orthogonal measurements and classified as expressing DAZL, VASA or both. Data were analysed by analysis of variance with Tukey-Kramer post hoc testing.RAA and PTKS designed the study and wrote up the manuscript. GC and NF performed QRTPCR, Western and immunohistochemical analysis and acquired images on the confocal microscope. SC performed tissue collections. All authors read and approved the final manuscript.