Despite recent progress, investigating the impact of targeted therapies on Head and Neck Squamous Cell Carcinoma (HNSCC) remains a challenge. We investigated whether short-term culture of tumour fragments would permit the evaluation of tumour sensitivity to targeted therapies at the individual level.We cultivated tumour slices prepared from 18 HNSCC tumour samples obtained during surgical resection. The samples were treated for 48 h with a panel of 8 targeted therapies directed against selected oncogenic transduction pathways. We analysed the cell proliferation index (CPI) of tumour cells using Ki67 labelling and the activation status of the RAF-MEK-ERK cascade through ERK phosphorylation analysis.Fourteen tumours were successfully analysed after short-term culture of tumour samples, revealing a striking individual heterogeneity of HNSCC in terms of tumour cell sensitivity to targeted therapies. Using 50 % inhibition of CPI as threshold, sorafenib was shown to be active in 5/14 tumours. Cetuximab, the only approved targeted drug against HNSCC, was active in only 2/14 tumours. A more than 50 % inhibition was observed with at least one drug out of the eight tested in 10/14 tumours. Cluster analysis was carried out in order to examine the effect of the drugs on cell proliferation and the RAF-MEK-ERK cascade.In vitro culture of tumour fragments allows for the evaluation of the effects of targeted therapies on freshly resected human tumours, and might be of value as a possible guide for the design of clinical trials and for the personalization of the medical treatment of HNSCC.The online version of this article (doi:10.1186/s12885-016-2318-x) contains supplementary material, which is available to authorized users. Head and neck squamous cell carcinomas (HNSCC) are tumours characterized by great phenotypic, aetiological and biological heterogeneity among individuals . StandarWhile every individual HNSCC bears a unique load of genomic alterations, therapeutic targeting is limited by the fact that the current level of genomic analysis does not translate into clear information regarding tumour sensitivity to most drugs . New comThis study was conducted in compliance with the French legislation and the declaration of Helsinki regarding ethical principles for medical research involving human subjects. The use of surgically-resected solid tumours for research purposes in the laboratory of Biochemistry of the University Hospital of Amiens was approved by the Comité de Protection des Personnes Nord-Ouest (CPP NO ref. 2009/14). A written consent was obtained from the patients. No samples were obtained from any patients that were minor or physically or mentally unable to understand and give their consent to the use of surgical samples.Head and neck tumour specimens were obtained from 18 adult patients who had been referred to the Head and Neck department of Amiens University Hospital (France) for a surgical procedure between September 2013 and September 2014. Surgery was the first line of treatment, without previous radiotherapy or chemotherapy. Different localisations and stages of squamous cell carcinoma from the upper aero-digestive tract are represented . Slices were maintained for 48 h in 24-well culture plates in Dulbecco’s Modified Eagle Medium (DMEM) culture medium, supplemented with 10 % fetal calf serum (PAN-Biotech), penicillin / streptomycin, and 1 % glutamine at 37 °C in a 5 % CO2 atmosphere.About 1 cubic cm of non-necrotic tumour was selected by the pathologist from each fresh surgical specimen. Tumour samples were prepared as 300 Eight targeted therapies  were selected on the basis of their distinct reactivities toward oncogenic kinases Table . Drug cohttp://jvsmicroscope.uta.fi/sites/default/files/software/immunoratio-plugin/index.html). The total number of pictures analyzed was 20 per experimental condition, representing in total a minimum of 1000 tumour cells. Mean CPI was determined by calculating each time the average, using the results from all slides.Tumour slices were fixed in formalin and then paraffin-embedded; 3 μm sections were cut and stained with hematoxylin phloxin saffron (HPS) to select non-necrotic and non-fibrotic areas with a high density of tumour cells. The monoclonal antibody MIB1  was used for the immunostaining of Ki67. Two tumour slides were analyzed for each experimental condition. Ten microscopic pictures were taken from each slide focusing on non-necrotic and non-fibrotic tumour areas, as defined by a senior pathologist (J.-F.I.). The cell proliferation index (CPI) was calculated from tumour cells only, excluding cells from the matrix and vessels. The ratio of Ki67-positive cells  was automatically determined by using ImmunoRatio, an Image J plugin adapted to automated image analysis  and transferred to nitrocellulose membranes . Antibodt-test was used for individual biological analysis and a value of p < 0.05 was considered as threshold for significance. Pearson’s r test was used for correlation analysis. Hierarchical cluster analysis was performed with the software R3.02 (http://www.r-project.org/). The hclust function in R was used, using the agglomeration method “complete”. The algorithm proceeds iteratively. At each stage distances between clusters are recomputed by the Lance–Williams dissimilarity update formula.The Student’s In order to analyse the impact of targeted therapies against HNSCC, we designed a panel consisting of eight targeted therapies that have reached at least phase II/III in clinical trials for the treatment of various solid tumours. This panel included the drugs cetuximab, sorafenib, erlotinib, tivaninib, masitinib, ponatinib, afatinib and rapamycin, and was selected on the basis of the distinct reactivities of these compounds against important oncogenic kinases Table . Tumour We focused our analysis on the 14 other tumour samples. The effect of each treatment was analysed by exploring the % of tumour cells labelled with Ki67 by immunohistochemistry Fig. . When weIn order to explore the efficacy of each drug at the level of the entire tumour population, we performed a dendrogram analysis of the measurements obtained in the proliferation assays. The cluster analysis was performed using the values measured with Ki67 labelling Fig. . HoweverIn order to further explore the use of short-term culture of tumour fragments, an immunoblot analysis of the expression levels of the markers ERK and phospho-ERK (pERK) was performed on samples obtained in the same conditions as previously Fig. . The levr2 = 0.2018, p = 0.0277 using Pearson’s r test)   Fig. . No such80) Fig. . We concHere, we explored the individual sensitivity of HNSCC tumours that were exposed in culture to various drugs, using a procedure that has been described by us and others and applied to a variety of human solid tumours in the past –13. The n = 14), which does not allow us to draw any definitive conclusions regarding the frequency with which tumours are resistant or sensitive to different drugs. Having in mind these limitations, it is nevertheless interesting to note that cetuximab, the only approved drug for the treatment of HNSCC that was tested in the panel, significantly inhibited the proliferation of only two tumour samples. An anti-proliferative effect was more often observed with sorafenib (n = 5), masitinib (n = 3), and afatinib (n = 3), thus opening the possibility that some HNSCC tumours might be individually sensitive to these drugs. At this stage, we conclude that short-term culture of tumour samples is a technique that could be useful for exploring the tumour-intrinsic determinants of drug sensitivity at the individual level.We observed that proliferation was inhibited by at least one drug in 10/14 HNSCC tumours in our experimental conditions. At this stage however, the aim of the present study was not to raise any definitive conclusions regarding the sensitivity of HNSCC to each of the drugs that was tested. Indeed, there are three major limitations to this study: i) the first limitation is the fact that a single pharmacological concentration of each drug was tested. Future studies should ideally assess a range of concentrations to determine an optimal inhibitory concentration for each drug. ii) The second major limitation of the use of tumour slices maintained ex vivo is that they reflect the consequences of the direct inhibition of signal transduction pathways inside tumour cells. Short-term culture of tumour slices cannot be used to evaluate anti-angiogenic or immunotherapeutic procedures, whose effects on tumour cells are indirect and require mid- to long-term exposure. ii) The third limitation of the present study is the limited number of tumours analysed  optimize the conditions for the assessment of tumour sensitivity in individual patients; ii) compare the results of the assay with the clinical response of patients with advanced / metastatic HNSCC. Together with other functional assays that explore complex aspects of cancer cell physiology, such as the functional state of the cell death machinery  and geno


Fluid bolus administration is widely recommended as part of the initial treatment of paediatric sepsis, though the physiological benefits and harms are unclear. The primary aim of this study is to determine the effect of fluid bolus administration on cardiac index (CI). Secondary aims are to determine the effect of fluid bolus administration on extra-vascular lung water (EVLW), whether fluid responsiveness can be predicted by inferior vena cava (IVC) collapsibility, and whether fluid responsiveness correlates with changes in vital signs.A prospective observational study of children presenting to the Emergency Department of The Royal Children’s Hospital with clinically diagnosed sepsis requiring fluid bolus administration. Prior to fluid bolus administration, an echocardiogram, lung ultrasound, and IVC ultrasound will be performed, and vital signs recorded. These will be repeated 5 min after and 60 min after fluid bolus administration. Recorded echocardiograms and lung/IVC ultrasound will be evaluated independently by a paediatric cardiologist and paediatric emergency physician, respectively, blinded to the patient identity and time of examination relative to time of fluid bolus administration. Fifty patients will be enrolled in the study based on a precision based sample size calculation. Results will be analysed for change in CI and change in EVLW 5 min after and 60 min after fluid bolus administration compared to baseline, IVC collapsibility as a predictor of fluid responsiveness, and the relationship between fluid responsiveness and changes in vital signs.This study will explore assumptions about the effect of fluid boluses on CI in children with sepsis, and will provide evidence for secondary effects on other organ systems. This may lead to novel methods for assessment and decision making in the initial resuscitation of paediatric sepsis in clinical and research settings, and will likely influence the design of future interventional studies in this arena.The study is registered with the Australian and New Zealand Clinical Trials Registry . Sepsis is the final common pathway and mode of death for many paediatric infectious processes, globally accounting for over 6 million childhood deaths per year, mostly in underdeveloped countries . Case fath century [Fluid resuscitation was initially described in patients with severe dehydration during cholera epidemics in the early 19 century , and sub century . Both of century . This ap century , and the century –19. In p century . This raAggressive FRT with the aim of increasing stroke volume, with the assumption that this will improve end-organ perfusion and cellular oxygen use, may be physiologically flawed . FRT intp < 0.001) [Limited published literature on the effect of FRT on cardiac output (CO) suggests that any effect is modest and transient. In a study of 20 adults with circulatory shock, the mean increase in CO immediately following FRT was 16 %, which decreased to 6 % above baseline after sixty minutes . In a se< 0.001) . There i< 0.001) . In the < 0.001) . The rem< 0.001) . In spon< 0.001) . No studWe set out to determine the effect of FRT on CI 5 min after and 60 min after fluid bolus administration in paediatric sepsis. Secondary aims include: the effect of FRT on extra-vascular lung water (EVLW), whether fluid responsiveness can be predicted by inferior vena cava (IVC) collapsibility, and whether fluid responsiveness correlates with changes in vital signs.The study is designed as a prospective observational study of children with sepsis. It will be conducted at a single paediatric ED in The Royal Children’s Hospital (RCH), Melbourne, Australia. RCH is a tertiary children’s hospital with an annual ED census of >85,000 children less than 18 years of age.Inclusion and exclusion are listed in Table Patients who fulfil the eligibility criteria and whose parents provide written informed consent will be entered into the study. Prior to administration of a standardised fluid bolus, transthoracic echocardiography, lung and IVC ultrasound will be performed, and vital signs recorded Fig. . Five miThe primary outcome measure is the percentage change in CI 5 and 60 min after standardised fluid bolus administration compared to baseline.Secondary outcome measures include the change in EVLW after standardised fluid bolus administration, whether IVC collapsibility is predictive of fluid responsiveness, and whether the observed change in cardiac output following standardised fluid bolus administration is correlated with changes in vital signs and clinical observations. We also collected the following outcome data: ICU admission, requirement for inotrope/vasopressor infusion, requirement for non-invasive or invasive ventilation, requirement for renal replacement therapy, requirement for extracorporeal membrane oxygenation (ECMO), final microbiological diagnosis, and mortality.Cardiac index will be measured by trans-thoracic echocardiography using a Zonare Z.one  and a 3–10 MHz phased array (cardiac) transducer. An apical 5 chamber view of the heart will be obtained and the left-ventricular outflow tract visualised. A 3 mm pulsed-wave doppler sample volume will be gated at the level of the aortic valve and the resulting waveform recorded. The mean velocity-time integral (VTI) over three respiratory cycles will be recorded as the final VTI measurement. Left-ventricular outflow tract (LVOT) diameter will be measured from a parasternal long-axis (PLAX) view in systole. Stroke volume will be calculated as (πr2) x velocity-time integral (VTI), and CO as stroke volume SV x HR. CI will be calculated as CO/BSA. Attempts to minimise the impact of inter and intra-observer variability on the study results include: blinding of the ultrasound assessor to the patient identity and timing of the study relative to the timing of fluid bolus administration; and assessment of intra and inter-rater reliability between the PI and the PI and blinded assessor, respectively, for all ultrasound-based assessments.Extravascular lung water will be measured on lung ultrasound using a Zonare Z.one and an 5–14 MHz linear array probe set at a depth of 5 cm. Eight views will be obtained and recorded as described in the Fluid Administration Limited by Lung Sonography (FALLS) protocol [protocol . Lung ulprotocol , 42.Inferior vena cava collapsibility will be measured using a Zonare Z.one and a 1–4 MHz curved array transducer. A sub-costal long-axis view of the IVC will be obtained including the IVC-right atrial (RA) junction and confluence of the hepatic veins [ic veins . A cine-ic veins . IVC colFor a precision based sample size estimation based on the continuous outcome variable of change in cardiac output following standardised fluid bolus administration, the following assumptions have been made: a mean increase in cardiac output of 20 % with a standard deviation of 10 %, a precision of the study tool (echocardiogram) of +/− 2 %. For a precision estimate of 5 %, a sample size of 50 would be required. This would also allow for regression analysis of several suspected confounding variables, including age, underlying illness aetiology (respiratory vs non-respiratory), and total volume of fluid previously administered.Ultrasound-based assessments are user-dependent. As such, they are prone to variability both within and between sonographers. Ultrasound-based assessment of EVLW and fluid responsiveness based on IVC collapsibility have been validated in adult patients but not in children. Age-based physiological differences in children may limit their utility in the study population. Some vital signs recorded  are qualitative and prone to bias. Efforts to minimise the impact of potential bias on the study results include video recording of the patient at each time point and subsequent assessment blinded to the timing of the video recording relative to the timing of fluid bolus administration. At the time of assessment, enrolled patients are undifferentiated and are being treated empirically for sepsis. They may have an alternate final diagnosis. At the time of enrolment, patients are receiving multiple interventions, some of which are distressing and make ultrasound assessment challenging. Interventions other than fluid bolus administration  may confound the study results. To minimise the effect of other therapies on study results, the study will focus only on the first hour of resuscitation. Any other interventions performed during the study period will be recorded. The study will only recruit patients when the PI is available in the ED. This limits the total number of patients recruited and the time of day when patients are recruited.Four major questions remain unanswered regarding fluid resuscitation for paediatric sepsis: what is the ideal fluid content, what is the ideal fluid volume, over what time period it should be administered, and what therapeutic targets should be used to titrate therapy. This study will help answer the questions of fluid volume and therapeutic targets. Current paediatric fluid resuscitation guidelines and planned trials are based on the concept of empiric fluid loading using vital signs as a marker of response to treatment. This is despite evidence that the host response to sepsis in children is highly variable and dynamic during the disease course –47, and This study may inform the design of future interventional studies in paediatric sepsis. The use of fluid-responsiveness based resuscitation limited by EVLW observed on lung ultrasound may allow targeted and individualised treatment . This maBP, blood pressure; CI, cardiac index; CO, cardiac output; CVP, central venous pressure; ECMO, extracorporeal membrane oxygenation; ED, emergency department; EGDT, early goal-directed therapy; EVLW, extra-vascular lung water; FEAST, fluid expansion as supportive therapy; FRT, fluid resuscitation therapy; GCS, Glasgow Coma Scale; HR, heart rate; IVC, inferior vena cava; PI, principal investigator; RCH, The Royal Children’s Hospital; RR, respiratory rate; ScvO2, central venous oxygen saturation; SIRS, systemic inflammatory response syndrome; SMD, septic myocardial dysfunction; SV, stroke volume


Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for  Arabidopsis thaliana, as a regulator of meiotic gene expression. We found that DUET is required for proper expression of JAS and TDM1. These genes function in male meiosis, and regulate spindle organization during meiosis II and cell cycle transitions, respectively. Expression of DUET at the end of prophase coincides with the onset of TDM1 expression, and DUET directly binds TDM1, indicating TDM1 is a direct target of DUET. Our results provide an initial framework for further elucidating the developmental and molecular controls of meiotic gene expression in plants.Meiosis is a critical event in sexual reproduction. During meiosis, chromosomes recombine and segregate twice consecutively to produce haploid daughter cells, which differentiate into gametes. In humans, errors in meiosis are the leading causes of congenital birth defects. In plants, bypassing the meiotic program can lead to production of clonal seeds that retain hybrid traits that otherwise segregate. Thus, understanding the controls of meiosis has major implications for both health and crop improvement. How meiotic gene expression is regulated in multicellular eukaryotes to promote entry into and progression through the meiotic program is poorly understood. Here we identify DUET, a protein essential for male meiosis in the model plant  Reproductive development in sexual organisms culminates in the production of highly specialized haploid cells, the gametes, which fuse to produce the zygote. An essential event in the production of gametes is meiosis, which is directly responsible for producing haploid cells. Meiosis is a complex process during which homologous chromosomes recombine, synapse, and segregate in two successive rounds without an intervening S-phase. Coordination of meiotic events is essential for successful production of haploid daughter cells.S. cerevisiae, entry into meiosis activates Ime1, a transcription factor that is responsible for expression of early genes that are required for premeiotic S-phase, synapsis and recombination. Among ImeI targets, Ndt80 is a transcription factor that activates middle genes, which are required for progression through meiotic divisions and spore formation, followed by late genes which control spore development [S. pombe. However, the key transcription factors Ste11 and Mei4 are not homologues of their S. cerevisiae counterparts ImeI and Ndt80 respectively, indicating poor conservation of these factors even though the transcriptional cascades they control are conserved [In yeast, temporal control of meiotic gene expression plays a critical part in coordinating meiotic events with meiotic progression. Entry into meiosis in yeast triggers a transcriptional cascade resulting in sequential expression of meiotic genes. In onserved .Arabidopsis, mutants displaying abnormal expression of meiotic genes in ovules have been recently described. ACTIN RELATED PROTEIN 6 (ARP6) encodes a subunit of the H2A.Z histone variant loading complex SWR1, and KLUH (KLU) encodes a putative cytochrome P450 monooxygenase [ARP6 and KLU result in somatic expression of the recombination factor DMC1, which is normally restricted to meiocytes, but have a limited impact on DMC1 expression in female meiocytes. The relationship, if any, between these genes are unknown, and the mechanisms by which they regulate meiotic expression of DMC1 remain to be fully elucidated. Thus, the controls of meiotic gene expression in plants are largely unknown.In contrast to yeast, few factors controlling meiotic gene expression have been identified in multicellular eukaryotes, and how they contribute to normal meiosis and participate in specifying distinct male and female meiotic programs remain unclear –6. To daxygenase ,9. MutatDUET  encodes a PHD finger protein essential for male meiosis [DUET results in cytoplasmic collapse of meiocytes, defects in chromosome condensation, delay in progression and arrest at metaphase I, absence of an organelle band at interkinesis, and formation of aberrant meiotic products including dyads and triads, which do not survive. The relationship between these phenotypes, and the function of DUET during meiosis are unknown. However, DUET was shown to be expressed during male but not female meiosis, suggesting a sex-specific function [duet lines, with a PHD finger that had reduced binding affinity for H3K4me2. We found that meiosis I defects were rescued, but that meiosis II still produced aberrant meiotic products, with defects in chromosome segregation and cytokinesis. Analysis of microtubule organization in duet and duet partially complemented lines, revealed a parallel spindle phenotype during meiosis II consistent with formation of dyads and triads in duet. The parallel spindle phenotype in duet correlated with the loss of expression of JAS, a gene required for meiosis II spindle organization in male meiosis [TDM1, a gene required for meiotic cell cycle transitions [duet. Remarkably, we found that DUET expression was restricted to the diplotene stage of prophase I. We further show by chromatin immunoprecipitation experiments (ChIP) that DUET binds TDM1 promoter, indicating that TDM1 is a direct target of DUET. Consistent with this result, we found that the onset of TDM1 expression coincided with the timing of DUET expression. Moreover, TDM1 was not expressed during female meiosis, indicating a possible role of DUET in regulating male meiotic gene expression.In Arabidopsis,  meiosis ,11. The function ,11. Here meiosis . Furthernsitions , was alsArabidopsis, and that binding of its PHD finger to H3K4me2 is critical for function. DUET expression was detected only at diplotene, and the functions of the targets we identify in meiosis I and II processes, are reminiscent of the control of middle meiotic gene expression by transcription factors in yeast. Our results also suggest a role for histone modifications in the regulation of meiotic gene expression in plants.Our results reveal that DUET functions as a transcriptional regulator during male meiosis in Arabidopsis gene DUET/MMD1 is essential for male meiosis and encodes a plant specific protein with a Plant Homeo Domain (PHD) finger from amino acid 606 to 656 , a mutant in a residue outside the aromatic cage S20A), and a triple mutant of structural residues predicted to be required for folding  , and tesf pollen ,11. The A, and a entation . In compentation , and theentation . Overallduet include cytoplasmic collapse of meiocytes, delayed progression, formation of dyads and triads, and absence of pollen [DUET prevents analysis of the relationships between these phenotypes. However, we found that a complementing construct carrying the S20A mutation in DUET PHD finger frequently resulted in partial complementation  in addition to dead pollen  duet triad of spores with two small and one large nucleus. (K) duet dyad of spores containing two small nuclei each. (L) dyad of spores containing one large (white arrow) and two small nuclei, respectively, in duet;S20A. (M) dyad of spores containing two small nuclei each, in duet;S20A. Compare with (K). (N) Quantification of the tetrad phenotypes described above. Top graph: frequency of spore numbers per tetrad irrespective of number of nuclei. Tetrad = three spores, triad = three spores, dyad = two spores. Bottom graph: frequency of nuclei per spore, irrespective of spore number. Single = similar to WT; double = two nuclei per spore; large = large nucleus, arrow in (J) and (L). Numbers above columns indicate the number of analyzed tetrads (top graph), and spores (bottom graph) respectively.(H) WT tetrad with four spores containing each a single haploid nucleus. (I) duet;S20A line to explore the function of DUET during meiosis II. In Arabidopsis, mutants affecting spindle organization in meiosis II have been shown to produce dyads and triads [duet and duet;S20A result from spindle defects in meiosis II, we analyzed microtubule organization by immunostaining of male meiotic squashes. At interkinesis, microtubules form arrays emanating from the nuclei and interrupted by the organelle band  between adjacent nuclei . In Arabduet meiocytes, indicating the antibody is specific  and PARALLEL SPINDLE 1 (PS1), affect meiosis II spindle organization and produce high frequencies of dyads and triads as a consequence [duet, we examined JAS and PS1 expression. We found that expression of PS1 and MUT-L HOMOLOGUE 3 (MLH3), a meiotic specific gene required for completion of recombination [duet. However, expression of JAS was reduced by about 80% in duet , OMISSION OF SECOND DIVISION 1 (OSD1), and THREE DIVISION MUTANT 1 (TDM1). OSD1 encodes a UVI-like protein which functions as a putative APC/C inhibitor [OSD1 skip meiosis II and produce dyads of diploid spores [TAM encodes a cyclin A required for proper meiotic cell cycle progression [OSD1, mutants in TAM skip meiosis II and produce dyads, as well as triads occasionally, indicating cell cycle defects can also result in triads in Arabidopsis [TDM1 encodes a tetratricopeptide repeat protein required for cell cycle exit after meiosis II [TDM1 mutants, meiocytes undergo a third round of division without intervening S phase, and the attempt to segregate haploid chromatids results in polyads. When we examined expression of OSD1, TAM and TDM1 in duet, we found that expression of TDM1 was severely reduced, whereas TAM expression was similar to WT, and OSD1 expression was mildly reduced , we used single mutants from the segregating population as controls for each double mutant combination. Each single mutant produced dyads, triads, tetrads and polyads, similarly to their parent  < 0.001). We used meiotic anther cDNAs to validate our microarray results by qPCR on a subset of downregulated individual genes, and all the genes we tested showed downregulation  from WT inflorescences using DUET antiserum. We failed to detect enrichment of DUET at JAS relative to a control locus (At4g03870), but found that DUET showed a 15-fold enrichment at TDM1 relative to the same control locus. We selected three regions for a more detailled analysis of DUET binding to TDM1 . We detected pTDM1::NLS-GUS expression in anther meiocytes  besides OSD1 might result in epistasy over the loss of TDM1 expression in duet.nsitions ,28,36,38 mutants . HoweverJAS, which is required for spindle organization in meiosis II [duet. Consistent with this, we observed spindle defects similar to jas mutants during meiosis II in duet and duet:S20A. These results further suggest that the pleiotropic meiotic phenotype of duet results from the loss of expression of several genes. Indeed, DUET is likely to control expression of several additional genes, since the cause of many phenotypes in duet, including cytoplasmic shrinkage, partial cytokinesis and elimination of meiotic products, cannot be explained by the loss of JAS and TDM1 alone. In support of this view, we identified several genes with meiotic preferred expression, whose levels of expression were affected by the loss of DUET. It is possible that some of the phenotypes in duet might result from the simultaneous loss of expression of several genes. Overall, our results are consistent with the view that DUET is likely to function as an activator of gene expression during male meiosis.The loss of expression of iosis II , provideDUET, and the functions of the target genes JAS and TDM1 that depend on DUET for expression, define DUET’s function in meiotic processes post-prophase during meiosis I, meiosis II and cytokinesis. We also found that the onset of expression of TDM1, which we show to be a direct target of DUET, is at late diplotene/diakinesis. Similarly, expression of BRP4, a DUET target gene identified by microarray analysis, was shown to be in late meiosis [S. cerevisiae and S. pombe, meiotic genes performing key functions during meiosis I division, meiosis II, and meiotic exit are called “middle genes”, and constitute a group of co-expressed genes under the control of the transcription factors Ndt80 and Mei4 respectively [The phenotypes resulting from the loss of  meiosis . In S. cectively ,40. The ectively ,42. LikeDrosophila and mouse, DNA binding transcription factors have been identified as key regulators of meiotic gene expression, pointing to a DNA sequence based recognition mechanism [The essential requirement of the PHD finger of DUET for its function, suggests that recognition of H3K4me2 plays an important role in the regulation of meiotic gene expression by DUET. In contrast, in yeast, echanism . DUET PHArabidopsis and other eukaryotes, H3K4me2 accumulates at promoters and 5’ genic regions, but contrary to H3K4me3, does not correlate with active transcription. Rather, it is enriched on genes with tissue specific expression and with developmental functions [TDM1 promoter has been found to be marked with H3K4me2 in seedlings [TDM1 expression in young anthers is limited to meiotic cells.While most PHD fingers characterized to date have been shown to recognize H3K4me3 , we foununctions ,34,43–46eedlings ,34, and TDM1 and JAS. Mutations in TDM1 and JAS both result in male meiotic phenotypes. Moreover, we find that TDM1, whose expression in male meiosis directly depends on DUET, is not detected in female meiosis. Thus, a critical function of DUET might be to activate expression of male meiotic genes that contribute to sexual dimorphism particularly in the regulation of meiotic cell cycle transitions and spindle organization.Previous studies showed that DUET is expressed specifically in anthers, suggesting a male specific function. We identified here two genes depending on DUET for expression during male meiosis, Arabidopsis male reproduction. In Drosophila and C. elegans, H3K4me2 appears to be associated with male reproductive development. Mutations in Drosophila PHF7, which encodes an H3K4me2 PHD finger reading protein, result in loss of male germ cell identity [C. elegans, increase in H3K4me2 due to loss of an H3K4me2 demethylase, results in ectopic expression of spermatogenesis genes [Our results also suggest a developmental function for H3K4me2 in identity . In C. eis genes . Thus, Hps1-4 (SAIL_1164_C09 in Col), jas-3 (SAIL813_H03 in Col), and tdm1-8 (SALK_124300 in Col) from the Arabidopsis Biological Ressource Center. We obtained osd1-2 seeds (in Ler) from Raphael Mercier. The duet allele (in Ler) has been described previously [After 3 days at 4°C in the dark, seeds were germinated on MS media for 8–10 days. Seedlings were then transferred to soil and grown in a growth chamber under long day conditions. We obtained For histone pull-downs, 5 μg of purified GST, GST-DUET-PHD and GST-BPTF-PHD recombinant proteins were incubated with 10 μg calf thymus histones (Amersham) in binding buffer , and protease inhibitor) at 4°C overnight. Samples were then incubated with streptavidin beads for 1 h at 4°C. The beads were washed 5 times with 20 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol 1 mM PMSF and protease inhibitor. Pull-downs were analyzed by western blot with core histone antibodies anti-H2A (1:1000), anti-H2B (1:4000), anti-H3 (1:1000) and anti-H4 (1:250). For histone peptide pull down assays, 1 μg of WT or mutated purified GST-DUET-PHD fusion protein were incubated with 1 μg biotinylated histone peptides (Upstate Millipore) were incubated together in binding buffer  at 4°C for 4 h followed by addition of streptavidin beads (Amersham) and incubating for 1h at 4°C. The beads were washed 4 times with 10 mM Tris-Cl pH 7.4, 0.05% Tween 20, 300 mM NaCl, 1 mM PMSF and protease inhibitor, and analyzed by western blot with an anti-GST antibody (Sigma).Two DNA fragments, corresponding to amino acid 303–340 and 651–704 respectively, were PCR amplified from DUET cDNA with AccuPrime Pfx DNA polymerase (Life Technologies) using primers DUET-AbA, DUET-AbB, and DUET-AbC and DUET-AbD respectively , and using primers listed in pTDM1::NLS-GUS, we amplified TDM1 promoter from genomic Ler DNA, and recombined the fragment with pDONR41 by BP reaction (Initrogen). The resulting entry clone was cloned into destination vector pB7m34GW along with entry clones containing a 3xNLS and a GFP-GUS tag, respectively by LR reaction (Invitrogen). All clones were verified by sequencing and plants were transformed the floral dip method. Primary transformants (T1) were obtained by selecting seeds obtained from transformed plants on MS supplemented with 10 μg/ml BASTA. T1 plants were PCR genotyped for the construct and for the Ds transposon inserted in the DUET gene.DUET PHD finger was amplified by PCR using primers DUETphdBamHI and DUETphdEcoRI was cloned as a Arabidopsis microarray from Agilent. The entire processing, including RNA extraction, procession and hybridization as well as the array analysis, was conducted by Genotypic  according to the Agilent’s instructions.For microarray analysis, about 150 0.5 mm-0.6 mm buds of 5–6 week old plants were collected in a 1.5 ml tube in liquid nitrogen. We purchased custom made ACT11 from threshold points  in the log-linear range of amplification plots using the 2- ΔCt method.Anthers were dissected from 0.5 mm-0.6 mm buds of 5–6 week old plants and RNA was extracted with the plant RNeasy kit (QIAGEN), with on column DNA digestion with RNase free DNase (QIAGEN), according to the manufacturer’s instruction. 500 ng RNA was used for reverse-transcription using the Superscript III First-Strand Synthesis System (Invitrogen). Quantitative realtime PCR was performed in triplicates using Syber Green Master Mix (Life technologies) in an ABI Light Cycler. Dissociation curves were performed at the end of each run to confirm absence of genomic DNA contamination. Quantification of mRNA was calculated relative to pTDM1::NLS-GUS expression pattern in prophase was done on segregating WT and duet T2 plants of 3 independent lines in which the construct segregated as a single locus. After 4 days incubating at 37°C in GUS staining solution, infloresences were fixed in 4% PFA, 1x PBS. Anthers were dissected out in 2 μg/ml DAPI solution and squashed to release and spread meiocytes. Images were acquired with a Zeiss Imager Z.2 mounted with a Zeiss AxioCam MRC color camera with differential interference contrast and with either an oil 63x or 100x Plan Apochromat DIC objective. All images were processed with Zeiss Axiovision software and figures were assembled with Adobe Photoshop CS5 extended.Inflorescences with open flowers removed were fixed in 4% paraformaldehyde, 1x PBS, 0.1% Triton-X, for 2 hours under vacuum. Samples were rinsed at least 3 times with 1x PBS. For direct observation, anthers were dissected from meiotic buds in 2 μg/ml DAPI in 50% glycerol on a slide and squashed under a coverslip. For immunostaining, buds were digested with a mixture of cell wall degrading enzymes  for 30 min at 37°C. Anthers were dissected in 1x PBS on a slide, squashed under a coverslip and plunged in liquid nitrogen. The coverslips were removed, and the slides left to dry. Cells were covered with a thin layer of 1% gelatin, 1% agarose, then left to dry, and cells were digested again as described above for 30 min at 37°C. Cell membranes were permeabilized in 1% Triton-X, 1x PBS for 30mn, and slides were rinsed 3 x 5 min in 1x PBS, 0.1% Triton-X. Immunostaining was performed as described . PrimaryChromatin immunoprecipitation (ChIP) was done using the Low Cell ChIP Kit from Diagenode according to manufacturer’s instructions. For each ChIP experiment, 200 inflorescences were collected from four to five week old WT Ler plants and crosslinked in Buffer A . Chromatin was isolated according to manufacturer’s protocol. The suspended chromatin was sonicated for 15 cycles with low intensity on a Bioruptor UCD-200 sonicator, in which each cycle comprised of five sonication pulses of 15 seconds each with a one minute pause. The size of fragmented chromatin DNA was estimated to be between 0.5 and 1 kb by gel electrophoresis. Each sample was then split into two and 10% input was kept aside. Out of two one sample was incubated with rabbit anti-DUET antiserum and another with Rabbit IgG. Immunoprecipitations were performed according to manufacturer’s protocol and analysed on ABI 7900HT fast real time system using primers listed in S1 Fig(A) Histone peptide pull-down with the indicated peptides and followed by anti-GST western blot. (B) Alignment of DUET and characterized PHD fingers from animal proteins. (C) Phylogenetic tree made from the alignment in (b). DUET belongs to a group of H3K4me2/3 binding PHD fingers.(TIF)Click here for additional data file.S2 Fig(A) DUET immunostaining (green) on meiotic cells at interkinesis and telophase II. Only a single meiocyte at diplotene shows DUET signal. (B) DUET immunostaining on male meiotic squashes performed with a rat anti-DUET polyclonal serum.(TIF)Click here for additional data file.S3 FigFrequencies of meiotic stages were established from anthers collected from buds measuring (A) 0.4–0.5 mm, (B) 0.5–0.6 mm, (C) 0.6–0.7 mm. The numbers indicate the total number of meiocytes that were counted.(TIF)Click here for additional data file.S4 Fig(A) Alexander staining of the indicated genetic backgrounds. (B) DAPI (blue) and DIC overlay images of meiosis I in the indicated backgrounds. Scale bar 10 μm. (C) DAPI (blue) and DIC overlay images of meiotic products in the indicated backgrounds. Scale bar 10 μm. (D) Meiotic progression analysis in the indicated backgrounds. The total number of meiocytes counted is indicated between brackets.(TIF)Click here for additional data file.S5 FigpTDM1::NLS-GUS expression during female meiosis. (A) late prophase, based on overall ovule and meiocytes morphology and (B) tetrad stage. Arrows point at meiocytes. Scale bar 10 μm.(TIF)Click here for additional data file.S6 FigPatterns of expression of DUET homologues in the indicated plant species.(TIF)Click here for additional data file.S1 Tableduet. Sheet 2 –set of meiotically preferred genes from Libeau et al., 2011 showing differential expression in duet. Sheet 3 –qPCR validation of a subset of differentially expressed meiotically preferred genes. Sheet 4 –GO term analysis for downregulated meiotic genes. Sheet 5 –GO term analysis for upregulated meiotic genes.Sheet 1 –genes showing 2-fold or greater differential expression in (XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


In general, oral fluids provided higher S/P results than serum, but the patterns of response were highly similar, especially for the sow groups. Thus, the use of oral fluids in endemic farms is effective and economical, particularly for large herds. In conclusion, the authors strongly recommend the use of oral fluids for PRRS monitoring in endemic farms.Porcine reproductive and respiratory syndrome (PRRS) is a disease that is both highly contagious and of great economic importance in Malaysia. Therefore, reliable and improved diagnostic methods are needed to facilitate disease surveillance. This study compared PRRSV antibody responses in oral fluid versus serum samples following PRRS modified live (MLV) vaccination using commercial antibody ELISA kits . The study involved two pig farms located in Perak and Selangor, Malaysia. Both farms were vaccinated with PRRS MLV 1 month prior to sample collection. Thirty-five animals were used as subjects in each farm. These 35 animals were divided into 7 different categories: gilts, young sows, old sows, and four weaner groups. Oral fluid and serum samples were collected from these animals individually. In addition, pen oral fluid samples were collected from weaner groups. The oral fluid and serum samples were tested with IDEXX PRRS Oral Fluid Antibody Test Kit and IDEXX PRRS X3 Antibody Test Kit, respectively. The results were based on sample to positive ratio (S/P ratio of the samples). Results revealed a significant and positive correlation between serum and oral fluid samples for both farm A ( Porcine reproductive and respiratory syndrome (PRRS) is one of the major threats to the pig industry and can cause great economic loss due to reproduction failure in sows and preweaning mortality of up to 60 %. This will also cause significant economic loss to the pig farmers as they are not able to increase the production of the farm. Based on a recent survey, it is estimated that losses due to PRRS problems in the USA are as high as $668.58 million annually , porcine circovirus type-2 infection (PCV2), and classical swine fever (CSF). Hence, when the clinical signs and postmortem findings are suggestive of PRRS, detection of viral antigens, viral genomic material, or isolation of virus from clinical specimens is necessary to confirm the diagnosis. Besides that, rising serum antibodies against PRRSV can also be used to support the diagnosis, provided that the time frame is compatible with the clinical episode.There are several ways to monitor PRRS status, including detection of serum antibodies using commercial PRRS enzyme-linked immunosorbent assays (ELISAs), reverse-transcriptase polymerase chain reaction (RT-PCR) assays, frozen tissue section fluorescent antibody (FA) assay, and immunohistochemistry (IHC). Detection of pathogen-specific antibodies by ELISA is one of the most common methods for detecting PRRSV infection. The ELISA format makes it possible to test and analyze a large number of serum samples, which can reduce cost and labor involved. However, blood sampling in pigs is laborious, time consuming, and requires restraining of individual pigs. Therefore, a novel method of detecting PRRS in pigs is needed.Oral fluid is collected by placing an absorptive device, e.g., cotton rope, in the mouth. The use of cotton rope to collect oral fluid samples from pigs has been done successfully under experimental and field conditions  sows ≥six parities, (2) sows two to five parities, (3) gilts, (4) 10-week-old pigs, (5) 15-week-old pigs, (6) 20-week-old pigs, and (7) 25-week-old pigs.Oral fluid samples were collected using three-strand twisted undyed cotton rope. Both individual and pen oral fluid samples were collected. For individual oral fluid samples, pigs were identified based on their age group and record. After subjects were identified, the cotton rope was offered and the pigs were allowed to chew for 20 min. Individual oral fluid samples were collected from all seven groups, with five subjects from each category selected for collection. For individual oral fluid sampling, other pigs were prevented from chewing the same rope to avoid cross contamination. For pen oral fluid sampling, the ropes were hung inside the pen for 20 to 30 min. Pen oral fluid samples were collected from groups 4 to 7, only. Pen oral fluid samples were collected three times as replicates. Oral fluid collection was done by inserting the wet end of the rope into a clean plastic bag. The rope was squeezed slowly to remove the fluid into the collection tubes. Blood samples were collected from pigs via the jugular vein. Serum was then extracted from the collected blood and stored under −20 °C for further processing.® software).Serum samples were tested using the IDEXX PRRS X3 Antibody Test Kit, and oral fluid samples were tested using the IDEXX PRRS Oral Fluid Antibody Test Kit. Both test kits use the indirect ELISA format. Test results were expressed by calculating the sample-to-positive control (S/P) ratio for each sample using commercial software  sows ≥six parities, (2) sows two to five parities, (3) gilts, (4) 10-week-old pigs, (5) 15-week-old pigs, (6) 20-week-old pigs, and (7) 25-week-old pigs. Both blood and oral fluid samples were taken from all individual pigs from all seven categories. In addition, pen oral fluid samples were collected from categories 4, 5, 6, and 7.Results showed that for farm A, oral fluid had higher S/P ratios than serum samples Fig. . Similarp = 0.0001, r = 0.681) using Pearson’s correlation test  of 0.464. This means that about 46.4 % of the total variation in S/P values of oral fluid samples can be explained by variation in S/P values of serum samples.Both oral fluid and serology samples show positive, significant, and strong correlation  with a coefficient of determination (r2) equal to 0.369   Fig. .Fig. 7AvPen oral fluids for the following age groups were also collected: 10 weeks old, 15 weeks old, 20 weeks old, and 25 weeks old. Three replicates were taken and tested with PRRS OF Ab Test Kit. These results were compared with individual samples.p = 0.094). This means that there is no difference between these two samples at 95 % confidence interval. On the other hand, analysis of farm B determined that the results of pen and individual samples of same age group were the same, with the exception of week 20 (p = 0.05)  Table .Table 1SFrom this experiment, there are significant, positive, and strong correlations between oral fluid and serum samples for both farms A and B. This positive and significant correlation indicates that increase in S/P ratios of serum samples will also cause increase in S/P ratios of oral fluid samples. This proves that oral fluid samples can be used as PRRS monitoring tool instead of serum samples as they are closely correlated to each other. A comparison had also been done on matched samples from individual boars, and it revealed that oral fluid was equal to serum for the detection of PRRSV at DPI 7 and more likely to be more positive than serum on DPI 14 and 21 , and porcine circovirus type 2 (PCV2) infection which cause huge economic loss to the farmers as well.As a conclusion, oral fluid is a powerful tool to be used for the purpose of disease monitoring, and the data collected is crucial to improve health management in the herds. Thus, the author strongly suggested the usage of oral fluids for PRRS monitoring instead of serum samples in endemic farms.


According to Guyton and colleagues , expansiPatients admitted to the ICU were monitored with a calibrated LiDCOplus  and Navigator  to estimate Pmsf analogue (Pmsa). Then 250 ml Hartmann's solution or Volplex was infused over 5 minutes. Data were recorded automatically from baseline to 10 minutes after the end of fluid infusion. The time to maximum response and percentage change between baseline and last value of different haemodynamic parameters are also reported.P = 0.05) but not in other haemodynamic variables. Time to maximal response on CO was 2 minutes after the end of the infusion . In 22 events (37%), CO increased more than 10% (responders). The change between baseline and last value was greater in nonresponders for heart efficiency (Eh) (-9.2% ± 9.7 vs. -3.1% ± 13.9, Stress relaxation damps down the effect of a fluid challenge after 10 minutes except in terms of heart efficiency. The effect of a fluid challenge should be assessed up to 2 minutes after the end of fluid infusion. Failure to do so may mislead clinicians about the patient's fluid responsiveness.


Some of the funders listed in the Funding statement did not contribute financially to this study. The publisher apologizes for the error. The correct funding information is as follows: This work was supported by a grant from Kyung Hee University 2013 [KHU-20130438].


While dissemination and implementation (DI) science has become a clear area of research focus with associated methodologies, grant programs, journals, and training programs, these are relatively recent developments which have created a gap in training and career development. Many large institutions with major research programs have some investigators who are carrying out DI research but these tend to be limited and many investigators whose work has relevance to the field are not trained adequately to take advantage of existing grant mechanisms. The University of Pennsylvania has taken the strategy of creating the Implementation Science Working Group (ISWG) with the goal of providing an opportunity for idea development, feedback, grant review, and review of key methodologic approaches and conceptual frameworks for the field. A key element of the ISWG has been to support pilot studies by faculty from across the university which focus on DI topics and which have potential for translation into NIH grant applications. In this presentation, we discuss current efforts of the Penn ISWG as an exemplar for other institutions, and present examples of pilot grant receipts who have received funding to conduct DI related work within the context of the university efforts to support DI capacity.


Aging is characterized by a profound remodeling of the epigenetic architecture in terms of DNA methylation patterns. To date the most effective tool to study genome wide DNA methylation changes is Infinium HumanMethylation450 BeadChip (Infinium 450k). Despite the wealth of tools for Infinium 450k analysis, the identification of the most biologically relevant DNA methylation changes is still challenging. Here we propose an analytical pipeline to select differentially methylated regions (DMRs), tailored on microarray architecture, which is highly effective in highlighting biologically relevant results. The pipeline groups microarray probes on the basis of their localization respect to CpG islands and genic sequences and, depending on probes density, identifies DMRs through a single-probe or a region-centric approach that considers the concomitant variation of multiple adjacent CpG probes. We successfully applied this analytical pipeline on 3 independent Infinium 450k datasets that investigated age-associated changes in blood DNA methylation. We provide a consensus list of genes that systematically vary in DNA methylation levels from 0 to 100 years and that have a potentially relevant role in the aging process. In the last two years the Infinium HumanMethylation-450 BeadChip (Infinium 450k)  has beenUsing the Infinium 450k, researchers have identified several CpG sites that either get hypermethylated or hypomethylated during aging in different tissues , and a sAlthough defining a list of CpG sites whose methylation status is age-dependent is an essential step in aging research, the real challenge is to identify biologically relevant DNA methylation changes and their relative contribution to the aging process.The difficult task of extracting relevant information from microarray data can be made easier if the number of microarray features is reduced on the basis of a biologically meaningful criterion. In this way the top ranking groups of features are more likely to be functionally linked to the phenotype under study than the single features. For expression microarrays this task has been successfully addressed by grouping genes that share common biological functions , but thist EXON, GENE BODY, 3′ UTR, ILSAND, NSHORE, SSHORE, NSHELF, SSHELF) and calculates their mean or median methylation values, which are then compared between the samples under analysis [At present, different region-centric approaches have been proposed. Illumina Methylation Analyzer (IMA) defines for each gene 11 region categories .The main point is that the Infinium 450k probes are not evenly distributed across the genome, but they are enriched in specific regions while others are underrepresented. To deal with this issue, a methodology in which differentially methylated regions (DMRs) are defined as regions in which at least two contiguous probes within 1-kb distance have a significant differential statistic was recently proposed  and usedHere we implemented an alternative pipeline for the analysis of Infinium 450k data that is based on a careful description of CpG probes distribution within the array. The proposed methodology: 1) classifies CpG probes based on their genomic localization 2) defines groups of adjacent CpG probes based on their density in the region 3) depending on the previous classifications, applies a single-probe or a region centric analysis which considers the concomitant variation of a group of adjacent CpG probes.As a proof of principle, we used our approach to conduct a meta-analysis on 3 independent datasets in which the Infinium 450k was used to investigate age-associated variations in blood DNA methylation profiles. From this meta-analysis, we extracted a short list of genes that potentially have a biologically relevant role in the aging process.i) Class A, including probes in CpG islands and CpG islands-surrounding sequences (shores and shelves) that map in genic regions; ii) Class B, including probes in CpG islands and CpG islands-surrounding regions (shores and shelves) which do not map in genic regions; iii) Class C, including probes in genic regions which are not CpG rich; iv) Class D, including probes in non-genic regions which are not CpG rich. These four classes have different epigenetic functions, as their methylation status can affect gene function and chromatin structure in different ways [The method we propose focuses on grouping CpG probes into clusters, hereafter referred as “blocks of probes” (BOPs). CpG probes were grouped taking into consideration not only their contiguity in DNA sequence, but also their genomic localization, which represents a critical aspect for data interpretation . Using Ient ways . Class AThen, we defined BOPs as follows Fig. : for proClass A, B and C included 77202, 34448 and 20273 BOPs respectively  to test for general changes in methylation of a genomic region. However, as described above, Class A and Class B BOPs contain a largely variable number of CpGs, spanning from 1 to 75 for Class A BOPs and from 1 to 41 for Class B BOPs. We considered BOPs containing 1 or 2 CpG probes not informative enough for a region-centric approach but more suitable for a single-probe approach. On the contrary, the remaining BOPs  are analysed by applying a sliding-window MANOVA Fig. . Indeed,p-values and, if present, associated genes, is provided as output. Then, authors can choose to select significant BOPs containing at least 2 adjacent CpG sites for which the DNA methylation difference between the considered groups  or two selected ranges of values of the continuous variable is higher than a set threshold.We implemented the proposed pipeline in R software environment. Starting from a table containing the beta-values of interest , the script annotates the probes and divides them in Class A, Class B, Class B and Class D. Class A and Class B probes are grouped in BOPs and BOPs containing 3 or more probes are analysed by the region-approach (MANOVA on sliding windows of 3 CpG probes within the same BOP). The remaining CpG probes are analysed by ANOVA. Both categorical and continuous covariates can be used. A list of significant CpG probes is outputted from the single-probe analysis. For the region-centric approach, a list of BOPs ranked by nominal or FDR-corrected Significant BOPs can be ranked according to this criterion allowing authors to identify “bubbles” of different methylation between the conditions under study, which are likely to be biologically meaningful. The pipeline provides as outputs several useful plots, including schematic diagrams in which the chromosomal position of the probes within the selected BOPs is plotted against the methylation level of each probe  includes data from 656 subjects ranging from 19 to 101 years (average 64 ± 15 years) . The secIn each dataset, probes containing missing values in at least one sample and probes on X and Y chromosome were removed. Age-associated DMR were identified in D1 and D3 using the age as a continuous variable and in D2 the group (newborns or nonagenarians) as a categorical variable. Ethnicity (Caucasian-European or Hispanic-Mexican) was used as covariate in the D1 analysis. As the relative proportions of the different types of blood cells can vary significantly with age, we inferred blood cell counts from methylation data and use them as covariates in the analysis.q-values. Based on these criteria 21083, 517 and 2736 BOPs were identified as age-associated DMRs in D1, D2 and D3 respectively.Here we present results only for the region-centric analysis performed on Class A BOPs containing at least 3 CpG probes. MANOVA results were corrected for multiple comparisons through Benjamini-Hochberg False Discovery Rate correction; 0.05 was used as significant threshold for The considerably lower number of BOPs identified in D2 is ascribable to the effect of blood cell types correction, as cord blood and whole blood significantly differ for this parameter.To validate our approach, we compared the results of the region-centric approach with the results of a single-probe approach by analysis of variance (ANOVA). As shown in Figure To confirm that our region-centric approach reduces spurious results and is more likely to identify biologically relevant regions, we compared the results from the three datasets.q-value < 0.01) and that showed a difference in average beta-values between newborns and nonagenarians greater than 0.20. We considered only the CpG probes belonging to Class A probes, that is 45 and 800 probes in D1 and D2 respectively, and we matched them with the corresponding BOPs. In this way, we achieved a list of 34 and 472 BOPs identified by Hannum et al. and by Heyn et al.. Only one BOP was shared by the two lists  and CpG probes  and we calculated the number of common DMRs between the three datasets. As shown in Figure Finally, we provided a short list of genomic regions whose methylation levels vary according to age in D1, D2 and D3  or of bisulfite-treated genomic DNA (BS-seq) provide a more comprehensive picture of DNA methylome  but, desA recent comprehensive review has collected the bioinformatic tools developed to specifically analyze Infinium 450k data . Apart fHere we reasoned that the statistical analysis of the Infinium 450k microarray should take into account the specific architecture of the microarray. Indeed, we clearly showed that different genomic regions present different level of coverage in terms of probes distribution. We grouped probes in blocks (BOPs) based on microarray annotations and verified that only in CpG islands and in the surrounding regions (shores and shelves) the mean distance between probes in a BOP was within 500 bp, a range in which the methylation values of CpG sites are usually correlated . On the We used multivariate ANOVA (MANOVA) to test for general changes in methylation in the region-centric analysis. This approach has been previously adopted for the analysis of methylation data , 30 as iOverall the strength of our approach is that not only microarray features are grouped in biologically meaningful groups, but also that the ranking criterion is based on a multivariate approach. Additionally, significant BOPs can be ranked also on the basis of the “bubble” of differential methylation, defined as at least two adjacent probes whose mean methylation in the groups under investigation differs of at least a minimum value. As a confirm of the validity of the approach we demonstrated that, when considering the top ranking BOPs, our method increased the number of common genes identified in all three studies compared to a single-probe analysis, indicating that it is likely to provide a more informative overview of biologically relevant results.The analysis pipeline we propose is implemented in R software environment and is therefore freely available. Researchers just need to define the analysis parameters, such as the covariates to be used in the ANOVA/MANOVA, the FDR correction method, the significance threshold and the minimum difference between mean methylation values between the groups under investigation.et al. and by Heyn et al. or was included in Horvath's epigenetic clock. Inferring how the methylation of these regions can contribute to the aging process is out of the scope of this paper, although the life-long variations in DNA methylation that we described are suggestive of a profound link between development and aging [We used our analysis pipeline to perform a meta-analysis on 3 Infinium 450k datasets that investigated age-associated changes in DNA methylation. The 3 datasets are considerably different in terms of both samples number and age range. Nevertheless, we were able to identify a core of genomic regions whose methylation profiles systematically vary with aging, from newborns to nonagenarians. To our knowledge, this is the first report describing a relatively large number of genomic regions with such characteristics. Only a subset of these genes was identified by Hannum nd aging . Many gend aging . Moreovend aging –38. COL1nd aging  in whichnd aging .Collectively the above observations indicate that this short list of genes, selected by means of an analytical pipeline that is tailored on the architecture of the microarray and that is more likely to provide biologically relevant findings, can be used as the basis for deeper investigations to shed light on the molecular basis of the aging process.http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE40279 and GSE30870 respectively. D3 includes 32 mother–offspring couples and is part of a larger datasets submitted to GEO with accession number GSE52588.D1 and D2 are publicly available at NCBI Gene Expression Omnibus (GEO)  Definition of Classes and BOPs; 2) DMRs identification 3) FDR correction, selection of significant DMRs/probes, sorting of probes and plots. Step 2 and step 3 are separately provided for Class A BOPs containing 3 or more probes, Class A BOPs containing 1 or 2 probes, Class B BOPs containing 3 or more probes, Class B BOPs containing 1 or 2 probes, Class C probes and Class D probes. Detailed explanations of input and output files are provided. A general description of the 3 analytical steps is provided below:The analytical pipeline is implemented as an R script freely available at p-value greater than 0.05 in more than 75% samples were removed, together with those localized on sexual chromosomes. Probes that contained SNPs were annotated as previously described [Step 1: Probes containing missing values and probes with a detection escribed . For procar. For sliding windows MANOVA, the function is applied on sliding windows of 3 consecutive CpGs within the same BOP. For each BOP, the lowest p-value among those calculated for the different sliding windows is kept. Both MANOVA and ANOVA analysis support the use of both categorical and continuous covariates. Parallel processing can be used if the computational environment supports it.Step 2: DMRs identification is based on MANOVA and ANOVA functions from the R package p-values for multiple testing using the correction methods implemented in the R package multtest. BOPs/probes can be selected on the basis of a significance threshold on either nominal or FDR-corrected p-values. In the case of BOP analysis, BOPs can be ranked on the basis of a user-defined minimum mean methylation difference between adjacent CpG probes. MDS plots, heatmap plots and line plots are generated as outputs.Step 3: The analytic pipeline allows to correct MANOVA/ANOVA


Cutaneous basal cell carcinoma (BCC) is a common malignancy amongst the elderly. BCC rarely metastasises, and fewer than 300 cases of metastatic BCC have been reported in the literature. Here, we report a case of sequentially developed BCCs located adjacent to each other. We discuss that these BCCs were considered to have developed independently rather than due to metastasis, after referencing previous studies on metastatic BCC. An 80-year-old woman presented at our department with two tumours in the right axillary area. A medical interview revealed that the tumour located on the axillae appeared first. The patient reported that the tumour was sometimes accompanied by excoriation and serous discharge. Four years after the appearance of the first tumour, the second one developed on the right side of the chest and contacted the first tumour when the right shoulder joint was adducted Figures . PhysicaBCC is the most prevalent form of skin cancer which develops in the basal parts of the epidermis and rarely metastasizes –3. PathoThe routes of metastasis of skin cancers are classified into the following four categories: lymphatic, haematogenous, disseminative, and direct invasion . Direct The diagnostic criteria of metastatic BCC were advocated by Lattes and Kessler . AccordiThen, how do we interpret this phenomenon? In a survey of 732 patients with BCC in Japan, 52 (7.1%) showed multiple lesions and >50% of these lesions were one-sided and located close to each other [


In the Funding section, the grant number 61370109 was omitted from the National Science Foundation of China. Please view the complete, correct Funding statement here.This work was supported by the National Science Foundation of China under Grant Nos. 61272339 & 61370109, the Natural Science Foundation of Hubei Province under Grant No. 2012FFB04204, and the Key Project of Anhui Educational Committee, under Grant No. KJ2012A005. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


To the Editor: O’nyong-nyong virus (ONNV) is a mosquitoborne RNA virus of the Togaviridae family. The virus was first isolated in June 1959 from serum samples from febrile patients in the northern province of Uganda . Full blood count and results of liver function tests were within reference ranges. Thin and thick blood films were examined again and were negative for e et al. . Serologe et al. . The pree et al. . Ten day,Aedes aegypti, found in some parts of Europe (We report the laboratory-confirmed case of an ONNV infection imported into Europe. This patient most likely was infected in the eastern part of Kenya (Kisumu region), where she had stayed during the 2 weeks before symptom onset. The case highlights the fact that ONNV infections, which occur sympatrically with CHIKV infections in East Africa, lead to symptoms resembling CHIKV infection. The clinical and laboratory findings emphasize the importance of a careful diagnostic and clinical assessment of travelers with suspected arboviral disease, and consideration of the well-known serologic cross-reactions in the alphavirus group. Because of the serologic and clinical similarities of ONNV and CHIKV infections, it remains unclear how many true ONNV infections in travelers have been diagnosed as CHIKV infections. Similar to other arboviruses, especially CHIKV and dengue viruses (


Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5′-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk−/−) cells should kill tumour cells from ‘ATP crisis’. We show that Ampk−/−-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk+/+ and Ampk−/− cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing ‘ATP crisis’. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity.Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with  Dividing cancer cells rely extensively on glycolysis to maintain a high rate of proliferation -3. Despi+/lactate bidirectional symporters with different affinities and tissue distribution. MCT1 is ubiquitous, whereas MCT4, a HIF-1-target gene [As a consequence of exacerbated glycolysis large amounts of lactic acid are produced. Our laboratory has been evaluating the anti-cancer approach of inhibiting lactic acid export. This approach leaded to intracellular accumulation of lactic acid associated with a severe drop in the intracellular pH (pHi), which results in inhibition of glycolysis , 17. Lacget gene , is stroget gene -24. We aget gene , 26. Howget gene -27. The get gene , 27, 28.get gene , metabolget gene , or muscget gene , 32. Wheget gene , glycolyget gene , mitochoget gene , and mitget gene , while sget gene -40, glycget gene  and fattget gene , 43. How−/−Ampk), Ampkα1 and Ampk2 [Mct4 (−/−Mct4) in Ras-transformed MEFs, lead to inhibition of glycolysis and proliferation in vitro. However, −/−Ampk MEFs preserved viable levels of ATP following acute inhibition of glycolysis. Moreover, AMPK was not capable of providing a survival advantage following severe inhibition of ATP production by glycolysis and OXPHOS. This unexpected finding suggested that AMPK is dispensable in regulating the plasticity of bioenergetic pathways. Finally we showed, using a xenograft tumour model, that the knockout of Ampk or Mct4 (−/−Ampk or −/−Mct4) delayed tumour development while −/− Mct4−/−Ampk MEFs severely impacted on tumour establishment. These in vivo studies suggest that combined inhibition of AMPK and MCT4 could be exploited as an anti-cancer strategy.We therefore hypothesized that genetic disruption of AMPK in a context of strong inhibition of glycolysis (MCT blockade) should sensitize tumour cells to death as a result of a deficit in maintenance of the ATP level. As no specific inhibitor for AMPK exists, a model of Ras-transformed murine embryonic fibroblasts (MEFs) genetically knocked out for the two catalytic subunits of AMPK  or deficient for the expression of both the AMPKα1 and AMPKα2 catalytic subunits (−/−Ampk) were transformed with oncogenic RasV12 , the index of lactic acid export and thus glycolysis, was also identical in cells with and without functional AMPK, in the absence or presence of glucose   in +/+ and Ampk−/−Ampk MEFs. Knockout did not alter expression of MCT1  abolished lactate transport in hypoxia  or the absence (−/−Mct4) of MCT4, and after addition or not of MCTi , we observed that the ATP level increased slightly and reproducibly, although not significantly, after 48h of MCTi treatment in normoxia. As we previously observed, using different cell lines [Mct4 in +/+Ampk MEFs increased basal levels of ATP in normoxia and hypoxia, probably due to inhibition of proliferation. MCT1 inhibition in +/+ Mct4−/−Ampk MEFs reduced the ATP level in normoxia and hypoxia by about 23%±3% and 47%±3%, respectively. The most important effect of MCT inhibition in hypoxia was due mostly to the metabolic shift toward glycolysis, which made the cells more dependent on this pathway for ATP production [−/−Ampk MEFs in normoxia was similar to that of +/+Ampk MEFs but not modified by inhibiting MCT1 or by hypoxia. Similar to +/+ Mct4−/−Ampk MEFs, Mct4 knockout in −/−Ampk MEFs (−/− Mct4−/−Ampk) increased the basal level of ATP. However, cells were more sensititive to MCT1 inhibition. Indeed, the level of ATP was reduced by about 16%±3% in normoxia and 28%±3% in hypoxia for the first clone  or 48h   but less in MEFs that did not express functional AMPK. At 72h, both inhibition of MCT1 and knockout of MCT4 led to a stronger inhibition of proliferation in +/+Ampk in normoxia (76%±0.6%) than that in −/−Ampk MEFs (32%±3%). In hypoxia, +/+Ampk and −/−Ampk MEFs proliferated more slowly than that in normoxia and MCT1 inhibition had no significant effect on these cells  and +/+ Mct4−/−Ampk MEFs (62%±0.8%)  Figure . When Mcs Figure . In cont) Figure . Cell gr) Figure  and in h) Figure  were detAmpk+/+ and Ampk−/− MEFs were found, all these results suggest that after blockade of lactate export and inhibition of glycolysis, MEFs did not require the presence of AMPK to maintain a viable level of ATP in vitro.Although small differences between +/+Ampk and −/−Ampk MEFs expressing MCT4 (+/+Mct4) or not (−/−Mct4) were treated in hypoxia with MCTi combined with phenformin (MCTi/Phenf) an inhibitor of mitochondrial respiratory chain complex 1. Combination of MCTi/Phenf did not alter ATP levels in MEF expressing MCT4 (+/+ Mct4+/+Ampk et −/− Mct4+/+Ampk MEFs) , 39%±4% (+/+ Mct4Ampk−/−), 30%±6% (−/− Mct4+/+Ampk) and 28%±3.8% (−/− Mct4Ampk−/−)  and −/− Mct4+/+Ampk (33%±2) MEFs  Figure  without s Figure . Howeverty assay .Taken together these results showed that MCT4 knockout sensitized MEFs to the MCTi/Phenf combination, which reduced substantially the ATP level, viability and proliferation. Knockout of AMPK was neither beneficial nor unfavourable to these parameters.+/+Ampk or −/−Ampk MEFs,. This level was gradually restored, and even reached values above the basal level of ATP 1h after treatment  . In the absence of 10 mM glucose±2-deoxy-glucose, oligomycin collapsed ATP production, independently of the AMPK activity . The only differences observed were obtained under conditions of low glucose concentrations (0.1 and 1 mM).Taken together these results suggest that AMPK is not required to maintain a viable level of ATP in response to inhibition of glycolysis +/+ Mct4Ampk+/+ control MEFs showed rapid tumour growth, up to 200mm3 after 12 days (±3) and greater than 1000mm3 after 21 days of injection  post-injection. However, after this period, the rate of tumour growth was identical for +/+Ampk and Ampk−/− MEFs  and 40 days (±6), respectively  did not drastically change the ATP level or the characteristics of growth in vitro, it showed a marked decrease in the tumour incidence. These results lead us to ask the following questions: (i) What are the differences, from an energy point of view, between inhibiting glycolysis by restricting lactic acid export versus decreasing availability of glucose? (ii) Is AMPK a tumour suppressor or an oncogene in our model? (iii) Why did the double knockout of AMPK and MCT4 reduce substantially tumourigenicity while no phenotypic differences were found in vitro?AMPK is widely recognized as the guardian of the balance in energy and as a critical regulator of intracellular ATP level. However, in the present study, we showed for the first time, that AMPK was dispensable for maintenance of the ATP level and viability of fibroblasts when glycolysis was limited by MCT blockade. However, we confirmed that Our approach to reducing tumour growth by blocking the last step of fermentative glycolysis (export of lactic acid)  was receMct4−/− MEFs in normoxia rapidly led to inhibition of glycolysis, which induced a stress in energy as observed from the activation of AMPK in Ampk+/+ MEFs  overexpressing c-Myc [Ampk−/− MEFs after tumour establishment. However, the question of the role of AMPK in initiating solid tumours remains open. Indeed, both models developped by Faubert or our selves failed to clearly define the positive or negative effect of AMPK during establishment of solid tumour. However, we provided additional evidence that AMPK is not a key oncogene in tumour growth after their establishment. In contrast to Li et al. [et al. in [AMPK and tumour development. The role of AMPK in cancer is controversial . AMPK ise et al. , confereng c-Myc . This rei et al.  or Zhou t al. in  who studMct4 knockout alone severely affected tumour establishment of both Ampk+/+ and Ampk−/− MEFs in subcutaneous xenografts. However, it did not affect the tumour growth rate suggesting that in this cellular setting MCT1 did not ensure sufficient export of lactate on its own. It also established that the function “lactate transport” is crucial when establishing solid tumours, at least in the model of murine tumour xenografts. These results are consistent with those obtained with the Ras-transformed fibroblast CCL39-derived mutants impaired in mitochondrial respiration [MCT4 and AMPK, a dynamic duo for tumour growth. In contrast to studies that have largely focused on MCT1 , 47, 64,piration  and exprpiration . These rpiration . Forced piration .Mct4 knockout in a Ampk−/− context severely affected both tumour establishment and tumourigenicity, demonstrating an important role for AMPK and MCT4 in tumour development. How do we explain the effect of Ampk /Mct4 double-knockout on tumourigenicity when we observed no or little phenotypic differences of these cells in vitro, compared to single Ampk−/− MEF knockout? Could MCT4 be involved specifically in proliferation in a 3D context, in angiogenesis, or in metabolic interactions with the tumour microenvironment? A recent study demonstrated that Mct4 knockdown by shRNA reduced both the stem cell population (CD133+) and the glioblastoma tumour growth by an unknown mechanism, independently of its function of lactate transporter [−/−Mct4 tumours. Moreover, the absence of AMPK could also amplify this phenomenum.Our study showed that nsporter . Such anin vivo results suggest that these molecules could have some therapeutic interest in the context of restricted AMPK activity. Unfortunately, currently there is no specific inhibitor of AMPK. However, as LKB1, the upstream kinase of AMPK, has been found to be mutated in several tumour types [Molecules targeting MCT4 are currently under development, and our ur types , one couvia inhibition of mitochondrial complex I.Finally, this study revealed that the bioenergetic plasticity in regulation of glycolysis and OXPHOS could occur in AMPK-null cells. We propose that intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major ATP consuming pathway, therefore preventing killing by ‘ATP crisis’. In line with cell killing we are left with the option of targeting MCTs with acute treatment with the anti-diabetic drug phenformin , 26, 27.Wild-type (Ampk+/+) and double knockout for Ampkα1 and Ampkα2 (−/−Ampk) murine embryonic fibroblasts (MEFs), were previously described [V12 (plasmid #1768 from Addgene) following the Retrovirus Production and Infection from the Rockefeller University protocols. MEFs and the human colon carcinoma LS174 cell line were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL) supplemented with 10% foetal bovine serum (FBS). LS174-−/−Mct4 were previously described [escribed  and wereescribed . PenicilFor hypoxic cultures the Bug-BoxTM anaerobic workstation  was set at 1% or 0.1% oxygen, 94% or 94.9% nitrogen, respectively and 5% carbon dioxide was used.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Centre National de la Recherche Scientifique (CNRS). All efforts were made to minimize suffering. Our experiments were approved by the “Comité national institutionnel d’éthique pour l'animal de laboratoire (CIEPAL)”. The project is registered under the reference: NCE/−165.mct4 gene, Colony-forming assay, Proliferation and Cell survival Assay, Immunoblot analysis, Metabolic measurements, ATP determination, Glucose consumption and Lactate measurement. The corresponding methodology is given in the ZFN-mediated gene knockout of the t-test and P values indicated .All values are the means ± SD of the indicate number of determinations (n) and significant differences are based on the Student's


Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip.A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for  In particular microarrays have allowed the detection of mutations in specific genes that are recognized as diagnostic “markers” for the onset of particular diseases  The rate-limiting step in the establishment of a diagnostic biochip is the evaluation of its probes. These probes have to satisfy several requirements such as a uniform melting temperatures (Tm) and being a similar size so that their signal intensities are comparable under the same hybridization conditions. The theoretical profiles of probes, however, are often inconsistent with experimental data, and this can generate biochips with irregular signal responses. It has been estimated that between 21–34% of probes do not match their intended targets Toxoplasma gondii, Chlamydia trachomatis, Rubella, Cytomegalovirus, Herpes virus). The matrix used for the biochip was a three-dimensional one suitable for depositing a large number of probes The cassette approach we adopted for probe evaluation allowed multiple sets of oligonucleotides for different genes to be assessed simultaneously under standard hybridization conditions. The cassettes comprise a tandem arrangement of probes, which act as the target DNA, permitting copy number control in standard reactions, so that poorly performing probes can be rapidly identified. As the model for fine-tuning the binding affinity of probes to their target DNA we have used a biochip designed for the identification of 5 pathogens involved in TORCH infections  gene and a cryptic plasmid sequence of Chlamydia trachomatis; the envelope protein E1 gene of Rubella virus (RV); the immediate early gene (IE) and the phosphorylated matrix protein pp65 gene of human Cytomegalovirus; and the DNA polymerase gene of the Herpes Simplex Viruses type 2 (HSV-2). The characteristics of probes are shown in the 4, producing dialdehyde groups that couple with hydrazine groups in the gel matrix The biochip (0.6×0.6 cm) consisted of 5×5 circular polyacrylamide gel pads, 0.8 mm×0.04 mm, separated by 0.7 mm and fixed on a glass plate. The matrix comprised 9 mM N,N-dimethylacrylamide, 1 mM 2-hydroxyethylacrylate and 0.2 mM N,N methylene–bis-acrylamide, TEMED, glycerol and methylene blue, which was polymerized after UV light treatment and activated by hydrazine hydrate treatment Chlamydia trachomatis; two for the major outer membrane protein (CT1 and CT2) and one for the cryptic plasmid (CT3). Cassette N2 (139 bp) had the probe sequences for the immediate early protein (CMV1) and the phosphorylated matrix protein pp65 (CMV2) of Cytomegalovirus, DNA polymerase of Herpes simplex virus type 2 (HSV), the B1 gene of Toxoplasma gondii (TOXO1), and the envelope protein of Rubella virus (RV). Cassette N3 had the same probe sequences as cassette N2 but in a different order. Cassette N4 (125 bp) contained four different probe sequences for gene B1 of Toxoplasma gondii . Cassette N5 (141 bp) contained five probes  for detection of each pathogen. All ssDNA cassettes contained the sequencing T7 primer at 5′-end (sense orientation) and the sequencing SP6 primer at 3′-end (antisense orientation).Five cassettes were synthesized to evaluate the different probes see . Cassett5′-GTA ATA CGA CTC ACT ATA GGG-3′) and reverse SP6 (5′-TAC GAT TTA GGT GAC ACT ATA G-3′) primers. The SP6 primer was modified by the attachment of a fluorescent dye, Cy3, to its 5′ ends, for detection purposes. The high-fidelity DNA polymerase  was used to amplify all cassettes. The reactions were initiated by denaturation at 98°C for 30 seconds followed by 35 cycles of 10 seconds at 98°C, 20 seconds at 55°C, 10 seconds at 72°C and a final extension for 10 minutes at 72°C.The cassettes were amplified from ssDNA using forward T7  were suspended in the hybridization buffer  at a concentration of 100 pmol, heated at 95°C for 5 minutes then quickly quenched on ice and hybridized against the biochip at 45°C in the presence of 0.2 mg/ml bovine serum albumin for 3 hours. The biochips were washed twice with 2X SSC +0.2% SDS at 25°C for 5 minutes, followed by a single wash in 2X SSC at 25°C for 1 minute. The fluorescent patterns were recorded using a Portable Imager 5000 . The negative controls for all hybridization reactions were those probes present on the biochip that were not being evaluated because they were absent in the cassette. In addition to this, DNA from  control  e5. Unde control  c5, d5. Chlamydia trachomatis probes were assessed using cassettes N1. All of the probes exhibited comparable hybridization capacities (Five cassettes (N1 to N5) were used to evaluate the hybridization potential of each probe. The number of probes included in each cassette ranged from 3 to 5, and the size varied from 105 to 141 bp. The evaluating power of the system can be increased by synthesizing cassettes with a greater number of probes of variable size. Hybridization reactions with the activated biochip were performed under conditions where target DNA and oligonucleotide concentrations were similar, so that the influence concentration on the reaction was negliable. The hybridization behaviours of the pacities , indicatCytomegalovirus genes coding for the immediate early protein (CMV1) and the matrix protein pp65 (CMV2), the DNA polymerase of Herpes simplex virus (HSV), the B1 protein of Toxoplasma gondii (TOXO1), and the envelope protein E1 of Rubela virus (RV) were evaluated using cassettes N2  with the other probes. Each of these probes behaved in a similar fashion when hybridized to their target under the same hybridization conditions .In this short communication we describe a practical, emprical approach to the evaluation of probes, ensuring that they share hybridisation characterisitcs so that they can be included in a biochip. The main advantage of the approach is the ability to distinguish the hybridisation capacities of different probe sequences present in equimolar proportions in one long DNA fragment. The method was assessed using five variants, hybridised under the same conditions. Variation in hybridisation intensity is to be expected if the probes have different denaturation/reassociation kinetics. Variation in kinetics behaviour is defined by many factors, including the concentration of the target DNA and the immobilized oligonucleotides, duplex length, G+C content and secondary and tertiary structures in DNA and oligonucleotides DNA microarray development comprises two fundamental areas: the identification of suitable target probes and the determination of optimal hybridization conditions. The cassette approach to probe screening, described in this paper, decreases the time needed to evaluate individual oligonucleotides because it allows them to be processed as a batch in a single reaction. This saving is not insignificant. It can take weeks, or longer, to evaluate and fine-tune the selection of a series of probes for inclusion in a biochip, whereas with our approach it takes hours, once the cassettes have been constructed and prepared. The development time of any biochip can be considerably shortened using this strategy, making it more comparable with that for other multiplex detection systems and other techniques such as probe free nucleic acid detection using isothermal amplification.The isothermal amplification techniques, which include Loop-Mediated Isothermal Amplification (LAMP) The detection of low abundant targets, important for clinical sample screening, presents a specific problem for microarrays in that pre-amplification steps (gene specific or whole genome) are necessary before hybridisation. This increases the time needed for target detection and opens the process to potential contamination issues. Furthermore, the amplification stages are reliant on PCR techniques with a requirement for a thermal cycling Data S1The composition and sequences of N1, N2, N3, N4 and N5 cassettes.(DOC)Click here for additional data file.