Transient episodes of ischemia in a remote organ  can attenuate myocardial ischemia/reperfusion injury but the underlying mechanisms of RIPC in the target organ are still poorly understood. Recent animal studies suggested that the small redox protein thioredoxin may be a potential candidate for preconditioning-induced organprotection. Here we employed a human proteome profiler array to investigate the RIPC regulated expression of cell stress proteins and particularly of thioredoxin in heart tissue of cardiosurgical patients with cardiopulmonary bypass (CPB).RIPC was induced by four 5 minute cycles of transient upper limb ischemia/reperfusion using a blood pressure cuff. Right atrial tissue was obtained from patients receiving RIPC (N = 19) and control patients (N = 19) before and after CPB. Cell stress proteome profiler arrays as well as Westernblotting and ELISA experiments for thioredoxin (Thio-1) were performed employing the respective tissue samples.Protein arrays revealed an up-regulation of 26.9%  of cell stress associated proteins in RIPC tissue obtained before CPB, while 3.8%  of the proteins were down-regulated. Array results for thioredoxin were verified by semi-quantitative Westernblotting studies which showed a significant up-regulation of thioredoxin protein levels in cardiac tissue samples of RIPC patients taken before CPB . Quantification of thioredoxin levels in tissue of RIPC and control patients by ELISA experiments further confirmed the Westernblotting results .We provide evidence for thioredoxin as a RIPC-induced factor in heart tissue of cardiosurgical patients and identified several cell stress associated proteins that are regulated by RIPC and may play a role in RIPC-mediated cardioprotection.The online version of this article (doi:10.1186/s12967-015-0403-6) contains supplementary material, which is available to authorized users. Ischemia/reperfusion injury is commonly found during cardiac surgery and can lead to myocardial and neurological dysfunction as well as increased mortality -3.In the last decades it has been shown that transient episodes of ischemia (ischemic preconditioning) if applied before prolonged ischemia/reperfusion injury have the potential to reduce myocardial infarction -9. SubseRIPC-mediated signalling events within the myocardial tissue possibly involve protein kinase C activation, phosphorylation of pro-survival kinases akt and erk1/2 -22 and pThe thioredoxin system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through thioredoxin reductase to thioredoxin, which reduces its target proteins using highly conserved thiol groups -31. InteHere we performed proteomic profiling with cardiac tissue obtained from cardiosurgical patients subjected to RIPC or sham intervention and investigated the expression levels of 26 cell stress associated proteins. We moreover focused on the potential role of thioredoxin by verifying the results of the proteome profiling approach using Westernblotting and ELISA experiments.The study protocol, patient information, and informed consent were approved by the Ethics Committee of the University Hospital Schleswig-Holstein, Campus Kiel, Germany (Reference number: A165/08). The study was performed in accordance with the fourth revision of the Declaration of Helsinki (1996) and is registered at ClinicalTrials.gov (NCT00877305). Employing patient sera and biopsy material, two experimental sub-studies have been published recently  data of the patients employed in the clinical trial and experimental sub-studies (NCT00877305) have been presented recently ,27. CardProteome profiling was performed using commercially available cell stress proteome profiler arrays  and the manufacturer’s protocol provided with the assay kit. Expression levels of 26 cell stress associated proteins were evaluated in cardiac tissue that was derived from cardiosurgical patients (control and RIPC) before as well as after CPB. Equal amounts of protein (15 μg) of each biopsy sample were pooled and applied to the respective array membrane. Densitometric analyses of the arrays were performed using the ImageJ 1.41o software . Only proteins with signal intensities ≥10% of the internal reference control protein spot were further analysed.Based on the results of the cell stress proteome profiler arrays, thioredoxin was chosen as a promising candidate for RIPC-mediated cardioprotection -34 and WHuman thioredoxin ELISAs  were performed using unpooled samples from each patient separately. Briefly, protein extraction from cardiac tissue biopsies was performed with RIPA buffer as described above. 50 μg of total protein from each patient were transferred into each well of a 96-well plate and all further steps were performed according to the manufacturer’s protocol. The amount of thioredoxin was relativized to the total protein within the respective sample. As suggested in the manufacturer’s protocol, hemolytic tissue lysates (HTLs) were excluded from further analyses .Statistics were performed using the software GraphPad Prism version 5.01 for Windows. Each data set was tested for normality using the D’Agostino and Pearson omnibus test. Two way ANOVA in combination with Bonferroni post-tests were employed for analyses of the Westernblotting results, while due to missing data points (excluded HTLs) in the control group, ELISA data were evaluated using unpaired T-tests. Variables are expressed as mean ± SEM.Proteome profiling employing cardiac tissue of cardiosurgical control patients and cardiosurgical patients undergoing RIPC revealed an increased expression of 26.9% (7/26) of stress associated proteins in RIPC tissue obtained before CPB , while expression levels of 3.8% (1/26) of the proteins were down-regulated compared to the control group [proteins down-regulated in the RIPC group: Sirtuin 2 (SIRT2)]. In cardiac tissue obtained after CPB, only one of the proteins 3.8% (1/26) was slightly up-regulated in the RIPC group [proteins up-regulated in the RIPC group: Phospho-p38 alpha (pp38α)] while compared to the control group, expression levels of 26.9% (7/26) of the proteins were down-regulated  are up-regulated in the RIPC group. This finding is of major interest as an increased expression of cell stress proteins may have a positive effect on the heart  and can Our findings that several cell stress associated proteins, including thioredoxin, exhibit a temporary upregulation by RIPC may be of value for future studies employing RIPC to reduce morbidity and mortality in cardiosurgical patients. In a clinical perspective, cardiosurgical patients seem to profit from an early and temporary induction of cell stress associated proteins by transient episodes of remote ischemia before surgery. However, the RIPC-induced response in the remote target organ (e.g. heart) most likely relies on a delicate balance between protective and potentially harmful RIPC mediators - circulating factors as well as local target tissue derived molecules. The expression of these mediators needs to be precisely adjusted to exploit the cardioprotective potential of RIPC and to avoid possible adverse effects of RIPC. Therefore, optimizing the currently available RIPC protocols ,55 by adThere are some limitations of the study which need to be considered: (i) Post-operative or clinical endpoints were not evaluated in the experimental study presented. However, clinical outcome parameters are described in the context of our previous pilot study (Additional file In the present study we performed proteomic profiling with heart tissue obtained from cardiosurgical patients that were subjected to RIPC or sham intervention. We provide evidence for thioredoxin as a RIPC-induced factor in the heart of cardiosurgical patients and identified several cell stress associated proteins that are regulated by RIPC and may play a role in RIPC-mediated cardioprotection.


Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA. The analysis of circulating cell-free DNA (cfDNA) shows exciting promise for the detection of genomic alterations associated with cancer. CfDNA is a natural phenomenon and is thought to originate from DNA released into the circulation from apoptotic cells deriving primarily from normal noncancerous tissue. However, in cancer patients the release of DNA from necrotic tumor cells (ctDNA) constitutes a significant portion of total cfDNAStrong correlations between ctDNA and disease prognosis have been reported in advanced colorectal cancerOvarian Cancer (OC) is one of the most common cancer deaths among patients with gynecologic malignancies, with approximately 21,290 new cases diagnosed and 14,000 deaths estimated for 2015Previously ctDNA studies in OC have focused on the identification of point mutations in TP53 15We report here an individualized, sensitive and specific approach for disease surveillance and therapeutic response monitoring in OC. A next-generation sequencing mate-pair protocol (MPseq) was used to identify somatic structural genomic alterations in primary tumors and a subset were quantitated in blood samples of each patient. Unique for each tumor, these aberrant DNA junctions involved genes with protein products that could be therapeutically targetable or were characteristic of significant clonal populations. A novel algorithm utilizing qPCR values for tumor-specific junctions and a housekeeping gene was applied to quantify the relative level of mutant DNA fragments to assess tumor burden. Thus, this proof-of-concept study describes a methodology that could serve as a useful tool to monitor disease regression or recurrence in a novel quantitative manner.1919Large genomic rearrangements were highly abundant in all cases analyzed . An averThe landscape of genomic rearrangements for representative sample OC067 is presented as a genome plot . The numPre- and post-surgical plasma DNA was processed for each case using the Qiagen circulating nucleic acid kit. CfDNA was successfully detected in all samples. Concentration ranged from 9 to 35 ng/ml . When coFrom rearrangements detected in each case, panels of PCR primers spanning the detected breakpoints were designed as unique tumor markers. In representative exemplar case OC049 a total of 229 chromosomal junctions were observed in the primary tumor B and 3A.For eight out of the ten patients tested, junctions identified in DNA of the primary tumor were also detected in the cfDNA derived from pre-surgical plasma . Of thesQuantitative PCR assays were developed and used to quantitate the amount of each aberrant chromosomal junction relative to the control beta actin housekeeping gene . Primersnearity) . The staτ, was subsequently calculated using the tumor fraction equation (nat) and the deleted, 1-copy state (Mdel). Example reads for case OC049 are plotted according to their frequency; with resulting peaks correlating with copy number status  center around value of 50  in the tumor: δ = 1 if βT loses one copy, δ = 2 if βT is normal, and δ = 3 corresponds to one copy gain in the effective correction equation  was derived for calculating the percentage of ctDNA present in cfDNA, with the more in-depth derivation presented in materials and methods.The Quantitative Circulating Tumor Percentage Equation was used to estimate ctDNA in all cases , Fig. 5.Qualitatively, our results matched clinical record data (when available) for the presence of disease at each draw. There was also a strong relationship between post-surgical CA-125 measurement and post-surgical qualitative qPCR results in our patients for whom comparisons could be made . Post-suWe report here a new algorithm, based on the quantification of specific somatic junctions in the blood of OC patients, to assess the fraction of ctDNA. Large numbers of chromosomal rearrangements were identified in the primary tumors of ten OC cases using a MPseq protocol. Individual somatic junctions were detected in the blood of eight (80%) patients . The relQuantification methods for the ctDNA fraction of cfDNA reported up to date vary tremendously, making cross-comparisons difficult. In one study, ctDNA was quantified using standard curves constructed from serial dilutions of the purified rearrangement productThe detection of chromosomal junctions in ctDNA offers several benefits over the detection of point mutations. First, it is extremely specific and robust, and performance is not affected by the abundance of fragments from normal DNA212223The recent explosion of ctDNA studies and the rapid increase in sensitivity and specificity of methodologies for ctDNA detection allude to a coming clinical integration, and point to the possibility of routine measurement of ctDNA in cancer patients in the near future. OC patients appear particularly suited to benefit from this approach, as they shed relatively higher amounts of ctDNA than those with other cancersWe envision performing sequencing of the tumor and creating a personalized monitoring panel after the commencement of treatment. Additionally, this may be the ideal time to identify potential therapeutic targets unique to the cancer. Thus, should a patient recur, as most patients with ovarian cancer willRecently, ctDNA increases in breast cancer were found to indicate recurrence earlier than rises in CTCs or CA 15-3. Furthermore, detection of ctDNA in half of these cases was possible an average of 5 months before radiologically confirmed disease progression was observed3Our novel algorithm helps to bring a liquid biopsy protocol closer to routine use in clinical practice. Following acquisition of tumor tissue via surgery or biopsy, the generation of individualized junction signature panels could be designed within three weeks. Our assay would provide clinicians with individualized tumor response information that could be used with great flexibility in case management.Cases were selected out of 100 consecutive patients with suspected epithelial serous stage IIIC-IV OC seen at the Mayo Clinic in Rochester, MN from 8/1/2010–4/30/2012 with available fresh frozen primary tumor and both pre- and post-surgical blood. Post-surgical draws were chosen as available for this retrospective study. Late stage OC patients were selected because of their high tumor burden thus greater ctDNA contentDNA from primary tumor was prepared using the Illumina MPseq Kit following the manufacturer’s instructions and sequenced as two libraries per lane on the Illumina HiSeq2000 platform19http://bioinformaticstools.mayo.edu/Panda/). The results were then cross-referenced with a database, which includes drugs approved for clinical use . After identifying the breakpoint for each junction of interest, new primers were designed to generate products <150 bps to be more suitable for qPCR and account for the fragmentation of ctDNAA panel of 2 to 4 unique junctions was selected from primary MPseq data for each case to be used for detection in the blood . SelectiDetection of ctDNA was accomplished using qPCR, performed as follows: 150 nm of primers were added to 2x Sybr Green  and 3–4 ul of template DNA. Serial dilutions of patient tumor DNA were used as positive controls and to allow quantification of the mutant product. Negative controls included NCcfDNA , pooled gDNA, and water as a No Template Control (NTC). Beta actin was amplified in each qPCR for normalization purposes . qPCR was run on Applied Biosystems 7900 HT using a standard protocol (40 cycles of 95 °C 15 seconds and 60 °C 1 minute). Products were resolved by 4% agarose gel electrophoresis for visualization purposes.φj and φβ are defined as the Ct values for individual tumor-specific junctions and for the housekeeping gene beta actin, respectively. The middle derivation additionally corrects for the difference in primer efficiencies by normalizing standard qPCR curves of tumor DNA for each individual case: where, The above formula presents the derivation of the final formula for quantitation of each aberrant tumor chromosomal junction relative to control housekeeping gene. Where ch ctDNA .EfCor variable correction for confounding factors of the percentage of normal cells, intratumeral heterogeneity and CNV of the house keeping genes:The final derivation incorporated the The initial derivation incorporates the tumor fraction and heterogeneity equations (Equations 4 and 5).τ):nat and Mdel are coverage for the normal 2-copy state and the deleted, 1-copy state, respectively.Fraction of tumor cells in tumor tissue (χ):Correction for intra-tumoral heterogeneity , and Mcov is the mode of the bridge coverage for a single allele.where, νeffective correlation equation (Equation 3) uses the value δ to adjust for any CNV of beta actin (βT) in the tumor: δ = 1 if βT loses one copy, δ = 2 if βT is normal, and δ = 3 corresponds to one copy gain.The final derivation of the How to cite this article: Harris, F. R. et al. Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers. Sci. Rep.6, 29831; doi: 10.1038/srep29831 (2016).


Preterm infants have a high risk to develop visual deficits due to retinopathy of prematurity (ROP), brain lesionsand prematurity per se . The posThe aim is to identify early signs of visual and motor-perceptual deficit in the first years in order to program a specific intervention before school age.Verypreterm infants born at Gestational Age (GA) <31 weeks, with and without brain lesions and ROP ≤ stage 2, were assessed at 35 and 40 weeks post-menstrual age using a visual assessment specifically designed for neonates; a structured follow up assessment, including fixing, tracking, visual acuity, visual fields and visual attention (using the Fixation Shift test) was used at 3, 5 and 12 months corrected age. Tractography of the optical radiation was performed in some consecutive infants in the neonatal period. Results at all the tests were compared with normative data on term born infants.P<0.001).At 3, 5 and 12 months preterm infants showed similar results to term born infants in all visual aspects but visual attention, with a high percentage of infants failing or refusing the test. Irrespective from the MRI findings, preterm infants with a normal neonatal assessment showed normal visual competences at 12 months corrected age.Ocular movements and tracking were more complete in preterm infants at 35 weeks than in full term infants, whereas reaction to a colored target, discrimination of stripes and attention at distance were more mature at term age both in preterm and term born infants. Tractography of the optical radiation showed that at term equivalent age visual assessment was significantly correlated with fractional anisotropy values (A structured visual assessment can be reliablesince the neonatal age . Some vi


Anas platyrhynchos). Combining observations in both the field and in mallards kept in captivity, we connected maternal AIV antibody concentrations in eggs to (i) female body condition, (ii) female AIV antibody concentration, (iii) egg laying order, (iv) egg size and (v) embryo sex. We applied maternity analysis to the eggs collected in the field to account for intraspecific nest parasitism, which is reportedly high in Anseriformes, detecting parasitic eggs in one out of eight clutches. AIV antibody prevalence in free-living and captive females was respectively 48% and 56%, with 43% and 24% of the eggs receiving these antibodies maternally. In both field and captive study, maternal AIV antibody concentrations in egg yolk correlated positively with circulating AIV antibody concentrations in females. In the captive study, yolk AIV antibody concentrations correlated positively with egg laying order. Female body mass and egg size from the field and captive study, and embryos sex from the field study were not associated with maternal AIV antibody concentrations in eggs. Our study indicates that maternal AIV antibody transfer may potentially play an important role in shaping AIV infection dynamics in mallards.Maternal antibodies protect chicks from infection with pathogens early in life and may impact pathogen dynamics due to the alteration of the proportion of susceptible individuals in a population. We investigated the transfer of maternal antibodies against avian influenza virus (AIV) in a key AIV host species, the mallard ( Maternal antibodies provide protection against pathogens in the early stages of a chick's life Larus delawarensis) ranged between 82 and 92%, with antibody prevalence in 3-wk-old chicks ranging between 5 and 30% Larus michahellis), 81% of females had circulating AIV antibodies and 51% of their eggs received these antibodies maternally Low-pathogenic avian influenza virus (AIV), a pathogen that circulates naturally in wild birds, is highly studied, though the role of maternal antibodies in AIV infection dynamics in wild birds has been largely neglected. The limited studies on maternal antibodies against AIV in wild birds are conducted in gulls Borrelia (Lyme disease agent) antibody concentrations in kittiwake (Rissa tridactyla) eggs and female blood sera correlated positively Many avian studies showed a strong correlation between maternal antibodies in egg yolk and/or chicks and circulating antibody levels in mothers Anas platyrhynchos) The aim of our study was to investigate maternal antibody transfer in a key AIV host species, the mallard , the Netherlands. Free-living mallards were caught under the Flora and Fauna permit (FF/75A/2010/011), issued by the Dutch Ministry of Economic Affairs, since the mallard is a protected species in the Netherlands. Handling and sampling of free-living mallards was approved by the Animal Experiment Committee (protocol CL10.02) of the Royal Netherlands Academy of Arts and Sciences (KNAW). The captive study was conducted in the outdoor aviary at the Netherlands Institute of Ecology (NIOO-KNAW) in Heteren , the Netherlands, who granted permission to perform this study. Approval from the KNAW Animal Experiment Committee (addendum protocol CL10.02) was given to handle and sample mallards in captivity. Free-living mallards were released into the wild after sampling, whereas captive mallards were set on an From April until June 2010, 67 free-living female mallards were caught from their nest with a sweep net. Females were caught during incubation to lower the chance of nest abandonment. To reduce the risk of including nests with eggs that had primarily been dumped there by other females, we only sampled females with a clutch of 13 eggs or less 1 and B2; nearest 0.01 mm) were taken to assess egg size. Egg yolks were separated on the day of collection. The size of each embryo was measured with a ruler (nearest 0.01 mm) to account for potential age differences affecting yolk AIV antibody concentration Per clutch, two randomly chosen eggs were collected to assess maternal AIV antibody concentration in egg yolk. Of each egg, the length  and breadth  or were bred at the NIOO-KNAW (n = 10). All birds had been kept in the outdoor aviary for at least a year prior to the study. The females were individually marked with colour rings to allow visual recognition.ad libitum and consisted of a mixture of commercial food pellets and seed-based mixed grains.The outdoor aviary was divided in five compartments: one large compartment (15×13 m) and four smaller compartments (6×13 m). In the large compartment, six females and three males were housed. The smaller compartments contained: two females and two males, three females and one male, three females and two males. Males were assigned to females according to pairs that had already formed before the start of the study. Each compartment was connected to a pond (34×1.5 m), with continuous flowing water for bathing and drinking. The outdoor aviary was surrounded by anti-bird nets and vermin proof mesh wire to prevent (egg) predation. To lower the chance that eggs were laid in a foreign nest, a surplus of nest boxes were provided in each compartment. Birds had access to shelter in the form of tall vegetation surrounding the aviary. Food was provided 1 and B2) were taken, egg yolks separated, embryos measured and samples frozen at −20°C until analysis.During egg laying, blood samples were collected from the brachial vein of females to measure concentrations of AIV antibodies. Analogous to the field study, serum was separated from red blood cells, and stored at −20°C until analysis. Female body mass, tarsus and head+bill lengths were measured (wing length was not scored as primary feathers were clipped to prevent flying). Once females started laying eggs, freshly laid eggs were numbered with a nontoxic pen referring to the position within the laying order. Per clutch, we collected four eggs (one fresh egg and three eggs during incubation) to assess a potential change in yolk AIV antibody concentration during the course of incubation. At the day of collection, egg length (L) and two breadth measurements  following manufacturer's instructions. Samples were tested in duplicate, with each plate containing two positive and two negative controls. The absorbance  was measured at 620 nm using an infinite M200 plate reader . Female serum and egg yolk samples were considered AIV antibody positive if the signal to-noise ratio  was <0.5.2 = 86.791, p<0.001; linear model: y = −0.55x+1.07, r2 = 0.73) and female sera  AIV antibody positive egg yolks and female sera from the field study. On two bELISA plates, the AIV antibody positive yolks and sera were diluted 1/3, 1/10, 1/30 and 1/100, together with positive and negative controls for each dilution except 1/3. Samples were tested in duplicate and the OD-value measured. Dilution and OD-value were log10-transformed before tested in a linear mixed model (LMM), with individual sample as random factor. There was a strong correlation between the dilution and the OD-value of egg yolk . A small amount of blood or tissue was transferred into a 1.5 ml tube containing 1000 µl of Cell Lysis Solution and 10 µl Puregene Proteinase K solution. After overnight incubation at 55°C, the remainder of the extraction protocol was completed according to the manufacturer's instructions. At the final step DNA was dissolved in 100 µl DNA Hydration Solution .Maternity was assigned using eight polymorphic microsatellite markers for mallards  and 40 ng genomic DNA. PCR was performed on a PTC200 . PCR product was digested with 1 µl EcoRI restriction endonuclease (20 U/µl) in a total volume of 10 µl The sex of embryos was determined for eggs collected in the field. Embryo gender was identified using the W chromosome-linked CHD-1 gene. Primers P2 and P8 were used following Griffiths et al. 3) was calculated following Hoyt 1×B2 .The field dataset contained 64 biological females  and their (non-parasitic) eggs (n = 115). For 13 clutches only one egg was included in the analysis, as the other eggs contained no embryo (n = 3), had insufficient yolk (n = 4) or the female caught on the nest was not the biological mother (n = 6). The dataset of the captive study contained eight egg laying females, where one female produced two clutches. A total of 33 eggs were collected. For three clutches, one egg during incubation was missing. As an index of body size, we used the first principal component (PC1) of a PC analysis of the biometric measurements. For the field study, PC1 explained 60% of the variance across tarsus, head+bill and wing lengths. Only tarsus and head+bill lengths were used in the PC analysis of the captive study, PC1 explaining 79% of the variance. To assess egg size, the volume (mm2 = 0.14), and body mass and egg volume were correlated .All covariates were log10-transformed to meet the assumption of normality. AIV antibody concentrations in egg yolk and female serum were minuslog10-transformed so that high values indicated high concentrations of AIV antibodies. Collinearity between the various covariates for the field and captive study were tested using Pearson correlation (r). In the field study, body mass and female AIV antibody concentration  . Full factorial models were tested and model selection was used to assess the better models on the basis of Akaike Information Criterion (AIC) corrected for small sample sizes AICc . The betAll analyses were conducted using R 2.14.1 We identified 12 parasitic eggs out of 127 (9%) in nine out of 67 clutches (13%), with both eggs parasitic in three clutches. Prevalence of AIV antibodies in breeding females was 48% (31 out of 64), with 43% (50 out of 115) of the eggs receiving maternal AIV antibodies.Of all investigated factors, only female AIV antibody concentration was correlated with AIV antibody concentration in egg yolk : high anAIV antibody prevalence in breeding females was 56% (5 out of 9) and in eggs 24% (8 out of 33). Similar to the field study, AIV antibody concentrations in egg yolk were positively correlated with female AIV antibody concentrations , Table 1In free-living mallards, nearly half of the eggs received maternal AIV antibodies. Although maternal AIV antibodies were not measured in chicks, several studies showed positive correlations between maternal antibodies in females, eggs and chicks In the captive study, only a quarter of the mallard eggs received maternal AIV antibodies, whereas at least half of the females had circulating AIV antibodies. This may be considered surprising, since females in captivity had unlimited access to food and thus resources to support immune function. Several studies showed that food supplementation may increase and food limitation may decrease the levels of maternal antibodies that are transferred into eggs Calonectris diomedea) chicks with maternal antibodies against Newcastle disease virus (NDV) did not increase their antibody levels when vaccinated with NDV, whereas chicks without maternal NDV-antibodies did.As hypothesized, the circulating AIV antibody concentrations in females correlated with the AIV antibody concentrations in egg yolk in both the field and the captive study. Our results are in accordance with earlier findings of maternal AIV antibody transfer in free-living gulls We found a positive correlation between egg laying order and maternal AIV antibody concentration in our captive study. An increase in maternal antibodies over the laying sequence may function as a maternal tool to mitigate the negative consequences for the last-hatched chick(s) According to evolutionary theory parental investment should be adjusted to the reproductive value of current eggs or offspring Coturnix japonica) that were fed low protein diets produced smaller eggs, but maternal antibody concentration in egg yolk was not affected. In contrast to our prediction, eggs with female embryos did not receive higher concentrations of maternal AIV antibodies than males. Despite lower survival in females In both field and captive study, female body mass was not correlated with yolk AIV antibody concentrations. Also in female gulls no correlation was found between their body mass and AIV antibody levels in their eggs In conclusion, by investigating transfer of maternal AIV antibodies in mallards as a key AIV host species, we demonstrated that in free-living mallards nearly half of the eggs received maternal AIV antibodies. Concentrations of maternal AIV antibodies in mallard eggs are positively correlated with circulating AIV antibody concentrations in females and laying order. With this study, we highlight the importance of studying maternal AIV antibody transfer in wild birds, which may play an important role in shaping AIV infection dynamics in host populations.Figure S1OD-values  as a function of dilution factor. (A) Egg yolk and (B) female serum. Lines represent significant least square regression lines.(TIF)Click here for additional data file.Table S1Correlation coefficients between the (continuous) covariates of interest for the field and captive study. Covariates that are significantly correlated are depicted in bold.(PDF)Click here for additional data file.Table S2Model selection to assess the better models to test the relationship between maternal avian influenza virus (AIV) antibody concentration in egg yolk and the covariates of interest for the field and captive study. The better models are shown in bold.(PDF)Click here for additional data file.


Venous thromboembolism (VTE) is the most widespread severe complication after total hip arthroplasty (THA) and total knee arthroplasty (TKA). We conducted this meta-analysis to further validate the benefits and harms of rivaroxaban use for thromboprophylaxis after THA or TKA. We thoroughly searched PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. Trial sequential analysis (TSA) was applied to test the robustness of our findings and to obtain a more conservative estimation. Of 316 articles screened, nine studies were included. Compared with enoxaparin, rivaroxaban significantly reduced symptomatic VTE (P = 0.0001) and symptomatic deep vein thrombosis  but not symptomatic pulmonary embolism (P = 0.57). Furthermore, rivaroxaban was not associated with an increase in all-cause mortality, clinically relevant non-major bleeding and postoperative wound infection. However, the findings were accompanied by an increase in major bleeding (P = 0.02). The TSA demonstrated that the cumulative z-curve crossed the traditional boundary but not the trial sequential monitoring boundary and did not reach the required information size for major bleeding. Rivaroxaban was more beneficial than enoxaparin for preventing symptomatic DVT but increased the risk of major bleeding. According to the TSA results, more evidence is needed to verify the risk of major bleeding with rivaroxaban. Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the most widespread severe complication after total hip arthroplasty (THA) and total knee arthroplasty (TKA)12456Low molecular weight heparin (enoxaparin), synthetic pentasaccharides (fondaparinux), or vitamin K antagonists (warfarin) are recommended and used for routine postoperative thromboprophylaxis91012The drawbacks of existing anticoagulants have driven the development of new oral anticoagulants11Several studies have assessed the benefits and harms of rivaroxaban for patients following THA or TKA. One study found that rivaroxaban was associated with a greater risk of major bleeding compared with enoxaparin171819202123The study characteristics of the included studies are presented in 2526272829303132252627282930312526272829303125262728303132The major bleeding rates described in the four RECORD  trials with rivaroxaban28293034One study252728293031322526272829303125272829303125262728293031Details regarding the Grading of Recommendations Assessment, Development and Evaluation (GRADE) evidence profiles for the outcomes are presented in 2 = 0%; Eight randomized controlled trials252627282930312 = 0%; Nine studies25262728293031322 = 0%; 2 = 12%; 2 = 0%; see 2 = 0%; see Rivaroxaban reduced the incidence of symptomatic DVT , allocation concealment (adequate or unclear), number of patients (<1000 or ≥1000), rivaroxaban dosage (a single dosage or multiple dosages), enoxaparin dosage (30 mg twice daily or 40 mg once daily), and surgery duration (<90 minutes or ≥90 minutes) did not exhibit noteworthy differences see . The decOn the whole, the sensitivity analyses did not change the findings; however, the exclusion of one studyMeta-regression analyses indicated that mean age was unlikely to be a source of heterogeneity in the results of this meta-analysis see  and S8.Because fewer than ten trials were included in our meta-analysis, we did not test the publication bias.The present meta-analysis demonstrated that compared with enoxaparin, rivaroxaban significantly decreased the rate of symptomatic VTE in patients undergoing THA or TKA; additionally, it reduced the risk of symptomatic DVT but not symptomatic PE. The evidence of efficacy was verified by most subgroup analyses and the TSA. In addition, rivaroxaban was associated with a significant increase in major bleeding and a higher clinically relevant non-major bleeding tendency. There was no difference in all-cause mortality and postoperative wound infection.et al.et al.252627171820This meta-analysis showed that rivaroxaban significantly increased the incidence of major bleeding, but not clinically relevant non-major bleeding, for patients with THA and TKA. Opina and colleaguesRivaroxaban appeared more efficient than enoxaparin in preventing symptomatic VTE and symptomatic DVT; however, there was no significant difference in the risk of symptomatic PE. This finding was consistent among numerous studies1718233738252627It has been confirmed that excessive anticoagulation may result in increased wound complications and postoperative infections1738There are some highlights in this meta-analysis. The methodology recommended by the Cochrane CollaborationOur study has several limitations. First, the trials were not consistent in terms of the duration of intervention. A short duration of treatment may affect the benefits and harms of rivaroxaban. Second, only two trials investigated the enoxaparin regimen of 30 mg daily. The inadequate number of patients in these two trials could not demonstrate a significant difference. Additionally, it was challenging to incorporate numerous trials within the same drug development program. However, because of the large number of participants and the fact that we included only randomized controlled trials, we believe that this analysis is convincing. Furthermore, because of the limited number of eligible studies, we did not assess publication bias. Our results should be interpreted with the consideration of the underlying publication bias. Finally, all of the studies were sponsored by pharmaceutical companies. This may also influence the robustness of our conclusions.In conclusion, rivaroxaban was more beneficial than enoxaparin for preventing symptomatic VTE and symptomatic DVT, but not symptomatic PE, after THA and TKA. Moreover, rivaroxaban was not associated with an increase in all-cause mortality, clinically relevant non-major bleeding or postoperative wound infection. However, rivaroxaban was associated with an increase in major bleeding. According to the TSA results, more evidence is needed to verify the incidence of major bleeding associated with rivaroxaban.We performed a thorough literature search of PubMed, EMBASE, and CENTRAL to identify randomized controlled trials that compared rivaroxaban with enoxaparin for patients undergoing THA or TKA. The literature search was completed on September 19, 2015. We combined MeSh terms with text words in the electronic search. The search terms pertaining to THA and TKA were combined with terms related to both rivaroxaban and enoxaparin. The details of the search strategies are presented in Participants: Only trials enrolling adult patients undergoing THA or TKA were included in this meta-analysis.Interventions: The intervention in the experimental group was, the new oral anticoagulant, rivoraxaban.Comparisons: The intervention in the control group was the approved treatment, enoxaparin 40 mg once daily (Europe) or 30 mg twice daily (North America).Outcomes: Studies were qualified when at least one of the following outcomes were described: symptomatic venous thromboembolism, major bleeding, all-cause mortality, clinically relevant non-major bleeding, and postoperative wound infection.Study design: Only randomized controlled trials were considered as qualified in the present study.For the included studies, two assessors independently carried out the data extraction. Study characteristics, including details regarding methodology, patients, experimental and control interventions, and outcomes were extracted. For the outcomes of interest, we extracted the number of events and the total sample sizes. If the data were not described in the text of the articles, we extracted data from the tables and diagrams, if availableThe primary outcome measures of interest were major bleeding and symptomatic VTE (symptomatic DVT or symptomatic PE). The secondary outcome measures consisted of all-cause mortality, symptomatic deep vein thrombosis, symptomatic pulmonary embolism, clinically relevant non-major bleeding, and postoperative wound infection.2, wound hematoma >100 cm2, a multiple site or extrasurgical site bleed, epistaxis >5 min, and macoscopic hematuria >24 hMajor bleeding was defined as clinically overt bleeding associated with a drop in the hemoglobin level of at least 2 g/dL; clinically overt bleeding resulting in the transfusion of ≥2 units of blood; fatal bleeding, bleeding into a critical organ ; or bleeding warranting treatment cessation or requiring re-operation. Clinically relevant non-major bleeding was defined as skin hematoma >25 cmFor studies with numerous intervention groups, we incorporated all pertinent experimental intervention groups (different rivaroxaban dosage regimens) into a specific group and combined all pertinent control intervention groups (different enoxaparin dosage regimens) into a specific control group. For binary results, both the sample sizes and the number of people with events can be totaled across groups. This method is recommended by the Cochrane CollaborationWe used the risk of bias tool to evaluate all of the included studies in accordance with the Cochrane Handbook for Systematic Reviews of Interventions (version 5.1.0)The quality of evidence for each pooled outcome was rated according to the GRADE system442 statistic2 > 50% was determined to indicate significant heterogeneity. Based on the type of surgery (THA or TKA), allocation concealment (adequate or unclear), number of patients (<1000 or ≥1000), rivaroxaban dosage (a single dosage or multiple dosages), enoxaparin dosage (30 mg twice daily or 40 mg once daily), and surgery duration (<90 minutes or ≥90 minutes), we conducted subgroup analyses for the primary outcomes. P < 0.05 indicated a statistically significant interaction between the estimates of the subgroupsIn this meta-analysis, RR and the corresponding 95% CI were calculated for all of the outcomes. The random-effect model was employed for the present meta-analysisIn a meta-analysis, the risk of false positive errors (type I error) may arise. This phenomenon may result from random errors when a small number of studies and participants is analyzed50515253www.ctu.dk/tsa)We estimated a diversity-adjusted information size in accordance with the diversity of the intervention effect estimates among the included studies. The TSA was conducted to maintain a type I error of 5% with a power of 80%. In the present meta-analysis, we calculated the required information size using the estimates of the intervention effects of trials with adequate allocation concealment535556How to cite this article: Ning, G.-Z. et al. Rivaroxaban for thromboprophylaxis after total hip or knee arthroplasty: a meta-analysis with trial sequential analysis of randomized controlled trials. Sci. Rep. 6, 23726; doi: 10.1038/srep23726 (2016).


Influenza A viruses (IAV) are zoonotic pathogens that pose a major threat to human and animal health. Influenza virus disease severity is influenced by viral virulence factors as well as individual differences in host response. We analyzed gene expression changes in the blood of infected mice using a previously defined set of signature genes that was derived from changes in the blood transcriptome of IAV-infected human volunteers. We found that the human signature was reproduced well in the founder strains of the Collaborative Cross (CC) mice, thus demonstrating the relevance and importance of mouse experimental model systems for studying human influenza disease. Recent studies comparing inflammatory responses in mice and humans have reported conflicting results regarding whether or not the transcriptome signature found in human patients can be replicated in mice1The similarity in overall gene expression changes following IAV infection in humans and mice can be best appreciated by a principal component analysis (PCA). Consistent with previous reportsNext, we analyzed changes in gene expression for the human signature genes in the peripheral blood transcriptome of four CC founder strains  infected with mouse adapted H3N2 IAV that were published previously by our laboratoryIFIT3 and IFIT44 expression after infection but only a relatively low increase in expression was observed in the resistant strain PWK/PhJ . In the human data set, all genes were contributing to PC1 (the polarity of infection response) whereas in the mouse, several genes were also contributing to PC2. We attribute this to the fact that the mouse strains exhibited a high level of genetic diversity whereas the genetic diversity of the human volunteers was fairly homogeneous and composed mainly of Caucasian background By evaluating the observed variables (genes) as vectors in a PCA biplot, the magnitude and polarity of each variable’s contribution to the first two principal components can be visualized (data not shown). It revealed that both mouse and human responses along PC1 (the infection response) are largely driven by interferon-response genes . The protocol used in these experiments has been reviewed by an ethics committee and approved by the ‘Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany’ (Permit Numbers: 33.9.42502-04-051/09 and 3392 42502-04-13/1234).et al.http://www.ncbi.nlm.nih.gov/geo/; GSE52428). For better visibility of the larger data set, we did not represent all groups but selected the most informative ones: one control group , an early time point (21.5 hr p.i.), a median (45.5 hr) and three late time points . The later time points represent the stages after infection where IAV signature genes best distinguished infected patients from controlsAs data set for the human blood transcriptomes, we used gene expression data from the blood of human volunteers which had been infected with H1N1 and H3N2 IAV by Woods The human influenza signature gene list has been taken from et al.A first analysis of the human gene expression profiles for the signature genes of the data set from Woods http://www.ncbi.nlm.nih.gov/geo/; GSE74077).The transcriptome data from the blood of four CC founder micehttp://www.ncbi.nlm.nih.gov/geo/; GSE52428), log2 transformed and quantile normalized before further analysis. Principal component analysis (PCA) was performed using the affycoretools packageArray data were analyzed using the R software packageHow to cite this article: Elbahesh, H. and Schughart, K. Genetically diverse CC-founder mouse strains replicate the human influenza gene expression signature. Sci. Rep.6, 26437; doi: 10.1038/srep26437 (2016).


Warm ischemia and reperfusion (WIR) causes hepatic damage and may lead to liver failure, however the mechanisms involved are largely unknown. Here we have characterized the microcirculatory status and endothelial phenotype of livers undergoing WIR, and evaluated the use of simvastatin in WIR injury prevention. Male Wistar rats received simvastatin, or vehicle, 30 min before undergoing 60 min of partial warm ischemia (70%) followed by 2 h or 24 h of reperfusion. Hepatic and systemic hemodynamics, liver injury , endothelial function (vasodilatation in response to acetylcholine), KLF2 and nitric oxide pathways, oxidative stress, inflammation (neutrophil and macrophage infiltration) and cell death were evaluated. Profound microcirculatory dysfunction occurred rapidly following WIR. This was evidenced by down-regulation of the KLF2 vasoprotective pathway, impaired vasodilatory capability and endothelial activation, altogether leading to increased hepatic vascular resistance and liver inflammation, with significant leukocyte infiltration, oxidative stress and cell death. Simvastatin preserved the hepatic endothelial phenotype, and blunted the detrimental effects of WIR on liver hemodynamics and organ integrity. In conclusion, WIR-induced injury to liver sinusoidal endothelial cells is mitigated by pre-treatment with Simvastatin probably through a KLF2-dependent mechanism. Ischemia/reperfusion injury is the phenomenon of interruption of blood supply followed by restoration of blood flow and the accompanying oxygen, nutrient supply and shear stress. Clinically, warm ischemia/reperfusion (WIR) injury is almost unavoidable in liver resection surgery, liver transplantation, and in blood transfusion for hemorrhagic shock, and may lead to delayed graft function and liver failure. Two different phases can be distinguished during reperfusion: an early phase (within 2 h after restoring reperfusion), which is characterized by the release of reactive oxygen species (ROS) and production of inflammatory mediators 1Kruppel-like factor 2 (KLF2) is a transcription factor predominantly expressed by the endothelial cell669Statins are HMG-CoA inhibitors originally designed to lower cholesterol levels, however they have shown other therapeutic effects independent on lipid lowering121314In the present study we have characterized for the first time the hepatic microcirculatory status and sinusoidal endothelial phenotype of livers undergoing warm ischemia and reperfusion injuries, and evaluated the applicability of simvastatin to improve or prevent warm ischemia/reperfusion injury.Livers undergoing warm ischemia and reperfusion (WIR) exhibited significantly impaired microcirculation evidenced by a marked increase in intrahepatic vascular resistance at 2 h and 24 h of reperfusion, associated with increased portal pressure and a reduction of portal blood inflow . FurtherIn addition, WIR caused a significant increase in the release of transaminases and LDH compared to sham operated animals especially at the early phase of reperfusion . At 24 h2− production and nitrotyrosine formation (caused by the scavenging of NO by O2−) were significantly increased at both time points of reperfusion  and VCAM-1 , which wSimvastatin administration 30 minutes before ischemia prevented the WIR-derived increment in intrahepatic vascular resistance, thus improving liver perfusion without changes in portal pressure . In addi2− production and O2−-mediated NO scavenging .LSEC treated with simvastatin showed a statistically significant 8-fold and 2.6-fold increase in KLF2 and eNOS mRNA expression respectively after reperfusion. This was associated with a decrease in the mRNA expression of the pro-inflammatory & capillarization markers iNOS (1.00 ± 0.16 vs. 0.76 ± 0.19) and VCAM-1 1.00 ± 0.06 vs. 0.85 ± 0.14). Furthermore, LSEC pre-treated with simvastatin exhibited lower levels of oxidative stress, measured as OLiver sinusoidal endothelial cells (LSEC) damage following ischemia reperfusion (IR) injury is the first event in the development of graft failure. Although this was already suggested in the late 1980 s1718In the present study, we demonstrate for the first time that a short period of warm ischemia is enough to cause striking deleterious effects on the hepatic microcirculation markedly increasing the intrahepatic vascular resistance (IVR), both at the early and late phases of reperfusion, leading to increased portal pressure and to a significant reduction in hepatic perfusion. Furthermore, animals suffering WIR develop acute endothelial dysfunction evidenced by a decreased response to the endothelial dependent vasodilator acetylcholine2123It has been described that blood flow-derived shear stress maintains a vasoprotective endothelial phenotype owing to the activation of the transcription factor KLF2, which mediates the transcription of several protective genes8942− produced during reperfusion. This scavenging causes the formation of the free radical peroxynitrite that may rapidly react with cell components such as proteins, lipids, and DNA further damaging the cellTo evaluate our hypothesis we measured KLF2 protein expression in liver tissue observing a significant reduction after WIR, both at the early and the late phases of reperfusion. Interestingly, this decrease was accompanied by a reduction in eNOS activation, one of KLF2 target genes. We further evaluated the bioavailability of the vasodilator NO by measuring the levels of cyclic guanosine monophosphate (cGMP) as a second messenger of NO and found a significant reduction in cGMP levels at the early phase of reperfusion. The reduction in NO bioavailability is explained by a combination of two factors: less synthesis due to reduced eNOS activity and; increased NO-scavenging by the high amount of O2− production at the late reperfusion phase. Microvascular dysfunction and parenchymal damage associated with WIR induced hepatic cell death.We further characterized the consequences of liver endothelial de-regulation during WIR by analyzing liver inflammation at two reperfusion time-points: early (2 h) and late (24 h). We evaluated hepatic VCAM-1 and P-selectin protein expression due to their implication in neutrophil adhesion and extravasation272830Statins or HMG-CoA inhibitors were primarily designed to decrease cholesterol levels but they have also shown to have beneficial cholesterol-independent effects such as up-regulating KLF2-derived transcriptional programs that improve endothelial function1434353637Our results demonstrate that simvastatin administration 30 min before ischemia prevents the increase in hepatic vascular resistance during reperfusion, and that is associated with better liver perfusion at early and late reperfusion periods. Simvastatin pre-treatment also improved liver endothelial function and prevented the peak in transaminases and LDH release observed at 2 h of reperfusion.Evaluation of the KLF2 pathway revealed that simvastatin is able to prevent liver KLF2 protein expression decay observed at the early reperfusion period, thus maintaining the activation of its target gene eNOS, enhancing NO bioavailability. Moreover, simvastatin-treated animals showed reduced levels of ROS. This might be explained by the antioxidant properties of KLF2 through activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and up-regulation of heme oxygenase 1 (HO-1)396in vivo were validated in vitro. LSEC cultured in an anoxia/reoxygenation system showed that simvastatin was able to partly abrogate the burst in oxidative stress and inflammation due to I/R, most probably due to the induction of the KLF2 protective pathwayThe results obtained in vivo after episodes of shock and/or hypovolemia, does not allow extrapolating what happens in liver resection surgery. On the other hand, the role of KLF2 in protection against liver WIR could be further delineated using genetic models where KLF2 expression would be knocked-down specifically.Despite the positive results achieved in this study using simvastatin in the setting of WIR injury, we have to acknowledge some limitations. First, the model of partial ischemia without hepatic resection, although representative of the WIR that occurs In summary, the results herein reported shed light on the pathophysiology of liver ischemia reperfusion injury demonstrating that a warm ischemia period, even of short duration, profoundly affects the liver endothelial phenotype, which becomes dysfunctional, leading to an immediate increase in the hepatic vascular tone, inflammation, polymorphonuclear cells infiltration, and hepatic cell death. WIR-derived damage to the hepatic endothelium may be, at least in part, due to KLF2 down-regulation. Indeed, simvastatin pre-treatment maintains KLF2 vasoprotective pathways and efficiently protects the hepatic microcirculation from WIR and prevents subsequent liver injury. Our data strongly suggest that simvastatin administration may represent an effective and simple approach to prevent or reduce liver damage in clinical situations associated with warm ischemia-reperfusion liver injury.Male Wistar rats from Charles River Laboratories  weighting 300–350 g were used. Rats were treated with simvastatin  or its vehicle (DMSO 0.1%) 30 minutes before ischemia. Animals were kept in environmentally controlled animal facilities at the Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with European Community guidelines for the protection of animals used for experimental or other scientific purposes (EEC Directive 86/609).−1) was calculated as: portal pressure/. Blood pressures and flows were registered on a multichannel computer-based recorder . The external zero reference point was placed at the midportion of the animal. Hemodynamic data were collected after a 20-min stabilization period.Under anesthesia with intraperitoneal ketamine  and midazolam  partial warm ischemia affecting 70% of liver volume was induced by clamping the portal triad irrigating the medial and left lateral lobes with an atraumatic clamp for 1 hin vivo hemodynamic study, liver vascular responses were assessed in the isolated, in situ liver perfusion system, as described−7 to 10−5 M) after pre-constriction with methoxamine (10−4 M).Immediately after the Aliquots of the perfusate were sampled for the measurement of transaminases and lactate dehydrogenase (LDH) using standard methods at the Hospital Clinic of Barcelona’s CORE laboratory. At the end of the study, liver samples from lobules that suffered WIR were stored for molecular analysis as described below.Hepatocytes, Kupffer cells and liver sinusoidal endothelial cells (LSEC) were isolated from control rat livers by collagenase digestion followed by percoll gradientTM System followed by 24 h reoxygenation.Hepatocytes and Kupffer cells were treated with simvastatin (5 μM) or its vehicle for 24 h. LSEC were treated with simvastatin (1 μM) or its vehicle 1 h before undergoing 1 h anoxia using a BD GaspakLiver samples were fixed in 10% formalin, embedded in paraffin and sectioned. After antigen retrieval procedure and endogenous peroxidase activity inhibition, sections were incubated with anti-von Willebrand Factor  or anti-P-selectin  1 h at room temperature. HRP-Rabbit/Mouse (Dako) secondary antibody was added. Colour development was induced by incubation with a DAB kit (Dako) and the sections were counterstained with hematoxylin. Sections were dehydrated and mounted. The specific staining was visualized and images were acquired using a microscope equipped with a digital camera and the assistance of Axiovision software. vWF relative volume was determined by point-counting morphometry on immunoperoxidase-stained sections, using a point grid to obtain the number of intercepts over vWF positive cells over the tissue. Six fields were counted in each liver. All measurements were performed by two independent blinded observers. The relative volume was calculated by dividing the number of points positive in sinusoidal areas by the total number of points over liver tissue.Liver samples were processed and western blot performed as describedIn situ O2− levels were assessed in LSEC and liver tissue with the oxidative fluorescent dye dihydroethidium  as described44Levels of cGMP, a marker of NO bioavailability, were analyzed in liver homogenates using an enzyme immunoassay  as previously described3 and salicylaldehyde (200 mM). Afterwards, the aqueous solution was removed and sections were mounted in Fluoromount G medium . Negative and positive internal controls were included. Fluorescence images were obtained with a fluorescence microscope and quantitative analysis of at least six images per sample was performed with Image J 1.44 m software.Quantitative tyrosine nitration detection was performed as previously described45For the evaluation of neutrophil infiltration, paraffin embedded slides were stained for specific esterase activity using a commercial available kit  following manufacturer’s instructions. Six images per preparation were obtained and positive cells per field were counted.Liver cryosections of 6 μm thickness were fixed with acetone at −20 °C for 10 min, incubated with anti-VCAM-1  2 h at room temperature and incubated with secondary antibody Alexa Fluor 555  and 4′,6-diamino-2-fenilindol  and mounted in Fluoromount G medium.For CD68 detection paraffin embedded samples were used. After antigen retrieval procedure, sections were incubated with anti-CD68  1 h at room temperature and incubated with secondary antibody and mounted as described before.Ten images per sample were obtained with a fluorescence microscope and percentage of positive area (VCAM-1) or positive cells per field (CD68) were quantified.In Situ Cell Death Detection Kit  according to the manufacturer’s instructions.Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed in deparaffined liver sections using an t test when adequate. Differences were considered significant at p < 0.05.Statistical analyses were performed with the IBM SPSS Statistics 19 for Windows statistical package. All results are expressed as mean ± standard error of the mean. Comparisons between groups were performed with analysis of variance followed by LSD post-hoc test, or with Student’s How to cite this article: Hide, D. et al. Effects of warm ischemia and reperfusion on the liver microcirculatory phenotype of rats: underlying mechanisms and pharmacological therapy. Sci. Rep.6, 22107; doi: 10.1038/srep22107 (2016).


We describe the first six mecC MRSA isolates of human origin from Austria and report the application of a rapid PCR test. Candidate isolates (n = 295) received between 2009 and 2013 were investigated phenotypically by cefoxitin screening and streaking on ChromID MRSA plates. The presence of mecC was confirmed in six isolates from blood cultures, wound swabs and screening samples of four female and two male patients (age range 7–89 years) by an in-house PCR method and the new Genspeed MRSA test . The mecC MRSA were further characterized by whole genome sequencing, multilocus sequence and spa typing. Antimicrobial susceptibility testing was performed by Eucast disk-diffusion method and Vitek 2. The six mecC MRSA isolates were from two clonal lineages  and four different spa types . Analysis for virulence factor genes yielded lukED, eta, etd2 and edin-B (CC130 isolates) and tst, lukED, eta and sel (ST599 isolates). The Genspeed MRSA test identified mecC in all isolates whereas Vitek 2 failed to detect methicillin resistance in one isolate. The strains were susceptible to a wide range of non-β-lactam antibiotics. All patients were successfully treated or decolonized. mecC MRSA are present in Austria as colonizers but may also cause infections. Thus, laboratories must choose appropriate test methods such as cefoxitin screening and confirmation using molecular assays specifically targeting mecC.Reports of  Staphylococcus aureus (MRSA) isolates carrying the mecA homologue mecC have been reported from all over Europe mecA fail to recognize these strains due to nucleic acid divergences between mecA and mecC. Published clinical data concerning mecC MRSA in humans include reports about colonization as well as skin and soft tissue infections Methicillin-resistant Staphylococcus spp. many of which have been extensively studied and typed using molecular methods S. aureus carrying mecC using the conventional phenotypic approach followed by molecular confirmation with an in-house PCR method as well as one of the first commercially available systems also able to detect mecC, the Genspeed MRSA test . In addition, clinical and molecular typing data on four mecC-positive isolates detected as part of routine screening are presented, describing for the first time the presence of mecC MRSA in human samples from Austria.The National Reference Centre for Antimicrobial Resistance and Nosocomial Infections at the Elisabethinen Hospital Linz receives bacterial isolates of human origin for identification, confirmation and typing from Austrian laboratories. Its strain collection contains over 5000 isolates of S. aureus isolates (n = 295) that had tested negative for mecA and positive for femA using previously published primer sets between the years 2003 and 2012 were chosen from the strain collection mecC were included in this study. Strains were subcultured overnight on trypticase soy agar containing 5% sheep’s blood  at 36 ± 1°C in an aerobic atmosphere. Species identification of all isolates was done by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry using the IVD MALDI Biotyper  and the Vitek 2 system .Candidate mecC MRSA for a panel of substances .Susceptibility testing was performed according to the Eucast disk-diffusion method. All strains were screened phenotypically for methicillin susceptibility using cefoxitin 30 μg disks in quintuplicate. They were also inoculated onto ChromID MRSA agar plates (bioMérieux) that were read after 24 hours of incubation at 37°C. The broader antimicrobial susceptibility of bstances  was assebstances . The suset al.mecA, mecC and lukF-PV after extraction of bacterial DNA with InstaGene Matrix , with modified primer concentrations. Additionally, a commercially available rapid PCR system, the Genspeed MRSA  was evaluated. This system uses rapid bacterial lysis followed by conventional PCR and detection of mecA and mecC, including specific probes for S. aureus, via hybridization on a chip.All isolates showing a mean cefoxitin zone diameter <22 mm and/or growth on selective media underwent confirmatory PCR testing using a protocol published by Stegger mecC were further analyzed by pulsed-field gel electrophoresis of SmaI restriction fragments. They were also typed by amplification of the polymorphic X region of the protein A gene (spa) followed by sequencing and assignation of the spa type mecC-positive isolates was performed as previously described mecC gene status, to determine their multilocus sequence type (ST) and to identify virulence factor and antibiotic resistance genes. Nucleotide sequences have been deposited in the European short-read archive as ERR387183, ERR387184, ERR387185, ERR387090, ERR 490434 and ERR490433 for isolates 4402, 5127, 5590, 5625, 5675 and 5752, respectively.Isolates carrying mecC MRSA was obtained from medical records retrospectively. Consent of the local ethics committee was provided (C-60-13).Clinical information on patients with Staphylococcus aureus with MALDI-TOF scores of >2.0. Phenotypic screening of the 295 isolates from the strain collection revealed two cefoxitin screening–positive isolates (0.7%) with mean diameters of 14 mm (range 13–14 mm) and 15 mm , respectively. Fourteen isolates (4.7%), including the two that were cefoxitin screening positive, showed growth on ChromID MRSA agar. Cefoxitin diameters of the other 12 isolates were in the susceptible category (range 27–32 mm); thus, sensitivity of the ChromID MRSA agar for phenotypic methicillin resistance detection was 100% and specificity was 95.9%.All strains were confirmed as mecC and negative for mecA by conventional PCR as well as with the new Genspeed MRSA test. In accordance with their susceptibility to cefoxitin, the other 12 isolates carried neither mecA nor mecC. Additionally, the four strains that were received in our capacity as reference laboratory were analyzed. All four isolates had a positive cefoxitin screening test with diameters of 17, 18, 20 and 21 mm, respectively. Presence of mecC and absence of mecA was confirmed with both PCR assays, and lukF-PV was not detected in any of the strains.Further analysis by molecular testing was performed on these fourteen isolates. The two cefoxitin-resistant isolates were positive for spa sequencing data showed four different spa types among the six isolates: t5930 (n = 2), t1535 (n = 2), t843 (n = 1) and t3256 (n = 1). Whole genome sequencing of all isolates confirmed that each isolate encoded mecC within an SCCmec type XI. Sequence types derived from the genome sequences included three ST130, a single-locus variant of ST130 and two ST599. All six isolates were positive for the genes encoding leukocidin ED (lukED) (GenBank accession no. Y13225) and exfoliative toxin A (eta) (GenBank accession no. CAQ49592). The four isolates belonging to CC130 additionally carried the epidermal cell differentiation inhibitor B gene (edin-B) (GenBank accession no. AHC54577) and the exfoliative toxin D gene (etd2) (GenBank accession no. HF563069). The two ST599 isolates were also positive for the toxic shock syndrome toxin-1 (tst) (GenBank accession no. Q9F0L4) and the enterotoxin-L (sel) gene (GenBank accession no. CAI80052)  . Other tSmaI pulsed-field gel electrophoresis patterns showed the strains to be unrelated, with the exception of isolates 5590/2012 and 5625/2012, which were isolated in the same area of Austria within 8 weeks of each other. The Dice coefficient for this pair was 85.9%, indicating possible relatedness.Analysis of mecC MRSA were subjected to antimicrobial susceptibility testing and yielded susceptible results for all non-β-lactam antibiotics  was identified as methicillin-susceptible Staphylococcus aureus (MSSA) with a negative cefoxitin screen also upon repetition of the test. The remaining susceptibility data of the AST GP card matched the results of manual testing.Subsequently, the six ibiotics . Two isomecC MRSA strains as well as the year of detection are indicated in Clinical data are summarized in mecC MRSA in humans in Austria. We can confirm the presence of such strains back to the year 2009 resulting from the investigation of our strain collection. The only other data on mecC MRSA in Austria covers livestock- and wildlife-origin strains To our knowledge, this is the first report concerning the detection of mecC-positive isolates in this study were phenotypically recognizable as MRSA using cefoxitin in the Eucast disk-diffusion test, which has been shown to reliably detect these strains mecC MRSA isolates mecC MRSA as described by Cartwright et al.mecC MRSA from other European countries All mecC MRSA. In contrast, in our small sample also containing ST130 strains, all isolates grew well on ChromID agar. However, the cefoxitin MICs of our isolates were considerably higher than those reported by Cuny et al.Previous reports have shown reduced efficiency of growth on commercially available selective chromogenic media, also including ChromID agar mecA gene. However, the newly discovered mecC element shares only 69% identity of its DNA sequence with mecA, leading to negative results using mecA-based PCR protocols and a need for additional primers to detect mecC. At the moment, not all commercially available assays cover this requirement. The Genspeed MRSA test that we used in this study can be completed in 75 minutes from culture; it can also be performed directly from swabs. It may be used as screening tool for colonization with S. aureus (MSSA) as well as mecA or mecC MRSA.Up to now, PCR methods for MRSA detection have successfully covered all known SCCmec types as a result of the high homology of the mecC MRSA in our study belonged to CC130, which agrees with previously published data from other countries that also show CC130 to be predominant tst and staphylococcal enterotoxin genes. However they were all positive for exfoliative toxin genes (eta and edinB). The occurrence of mecC in MRSA isolates belonging to ST599 is relatively uncommon, but the two isolates in our study were positive for the PTSAg genes tst and sel, which matches the toxin gene profile of previously published ST599 mecC MRSA isolates spa types were recovered in this study, all of which have previously been reported among mecC MRSA. t843 was the dominant spa type of mecC MRSA in a range of reports spa types t1535 and t3256 have also been described Four of the six mecC MRSA to cause clinical disease in a wide range of patients has been established by several reports. In a Spanish case series, most patients were elderly men who were colonized, but a patient with lethal sepsis was also described et al.mecC MRSA isolates from Denmark, with equal sex distribution and a mean patient age of 51 years. Interestingly, the majority of the isolates were from infections, mostly skin and soft tissue infection, and not from colonization screening. In a recent report on the prevalence of human mecC MRSA in England, more than half of the isolates were identified from screening samples mecC MRSA mecC MRSA were identified via routine screening, but in the remaining four patients, it was clearly associated with their clinical presentation.The capability of mecC MRSA do not seem to be highly prevalent in Austria at the moment, which reflects the situation in most other European countries mecC MRSA has increased significantly to a proportion of 2% of the total annual human MRSA cases in Denmark mecC is carried on a mobile genetic element (SCCmec), it has the potential to spread into different lineages mecC MRSA with appropriate phenotypic and molecular screening and confirmation methods is needed.Overall, None declared.