However, the subtleties of peroxide-mediated signaling are not well understood, in part because the generation, degradation, and diffusion of H2O2 are highly volatile within different cellular compartments. Therefore, the direct measurement of H2O2 in living specimens is critically important. Fluorescent probes that can detect small changes in H2O2 levels within relevant cellular compartments are important tools to study the spatial dynamics of H2O2. To achieve temporal resolution, the probes must also be photostable enough to allow multiple readings over time without loss of signal. Traditional fluorescent redox sensitive probes that have been commonly used for the detection of H2O2 tend to react with a wide variety of reactive oxygen species (ROS) and often suffer from photostablilty issues. Recently, new classes of H2O2 probes have been designed to detect H2O2 with high selectivity. Advances in H2O2 measurement have enabled biomedical scientists to study H2O2 biology at a level of precision previously unachievable. In addition, new imaging techniques such as two-photon microscopy (TPM) have been employed for H2O2 detection, which permit real-time measurements of H2O2in vivo. This review focuses on recent advances in H2O2 probe development and optical imaging technologies that have been developed for biomedical applications.Mounting evidence supports the role of hydrogen peroxide (HThe online version of this article (doi:10.1186/2045-3701-4-64) contains supplementary material, which is available to authorized users. The aberrant production of H2O2 within cellular compartments is connected to serious pathological conditions such as cancer [The role of Hnew data –7. H2O2 otection , 5–10. Hs cancer , ageing s cancer –14, diabs cancer , and neus cancer –18.2O2 in complex biological environments is measuring the spatial and temporal dynamics of this reactive oxygen metabolite. Much of the data implicating H2O2 both pathological and physiological roles in cultured cells and in vivo has been acquired using treatment with exogenous H2O2[2O2 lysing/scavenging agents [2O2, a key to fully understanding the mechanistic bases of redox biology is measuring the amount of H2O2 generated in specific intracellular compartments.A substantial challenge in elucidating the diverse roles of Hnous H2O2, 20, oveg agents , 21, or g agents . Althoug2O2 in living systems suffer from several issues: 1) The probes are often nonspecific and react with other reactive oxygen species such as hydroxyl radicals and superoxide, as well as reactive nitrogen species. 2) H2O2 is generally produced at a low concentration and can have a short half-life due to the activity of enzymes that neutralize it. 3) H2O2 is often produced in specific cellular compartments and rapidly diffuses across the membranes. For example, dichlorofluorescein (DCF), which has served as the workhorse for the redox biology community, detects multiple types of reactive small molecules, such as superoxide (O2•-), hydroperoxy radical (HO2•), singlet oxygen (1O2), peroxy radical (RO2•). DCF is not an ideal tool to determine the localization of H2O2 production. In addition to this lack of specificity of DCF for H2O2 detection, it can directly result in the creation of further ROS and thiol oxidation when exposed to UV radiation, and interacts with cytochrome c, rather than ROS as a consequence of apoptosis [2O2 detection over related ROS, particularly superoxide, nitric oxide, and hydroxyl radical and 2) improving photostability and determining the localization of H2O2 production. Recently, specific and highly sensitive fluorescent H2O2 probes have been developed to circumvent these issues, including chemoselective fluorescent probes, fluorescent proteins, and nanoparticles [Traditional approaches to measuring Hpoptosis –25. To oarticles , 27.2O2 levels. However, due to scattering and tissue penetration, imaging H2O2 levels in whole organisms often requires deep tissue imaging techniques. For example, two-photon microscopy (TPM) [in vivo studies [Confocal microscopy coupled with chemoselective fluorescent reporters permits the imaging of localized intracellular Hpy (TPM) , 28 and  studies , 30.2O2 production in cells and whole organisms.In this review, we describe and compare various methodologies for detection and imaging of H2O2 are generally based on the oxidation–reduction processes between the H2O2 and reduced probe, which fluoresces upon oxidation. Several novel fluorescent probes capable of detecting H2O2 with high selectivity have been reported, and some of them have been used to monitor intracellular H2O2. A comprehensive list of fluorescent H2O2 probes is listed in Table Small-molecule fluorescence probes for H2O2-selective probes utilizes a boronate-deprotection mechanism [2O2-mediated transformation of arylboronates to phenols. Arylboronates are appended to profluorescent molecules, such that reaction with H2O2 generates a fluorescent product. The monoboronate-based family of probes  can detect physiological changes in endogenous H2O2 levels. Since a wide color palette of such probes has been developed, various combinations can be selected for multicolor imaging experiments. The addition of acetoxymethyl ester-protected pro-anionic groups gives rise to the dye peroxyfluor-6 acetoxymethyl ester (PF6-AM), which increases cellular retention and further increases sensitivity to H2O2[2O2 localization studies, quantitative analysis of H2O2 generation using fluorescent probes is still challenging. Specifically, the signal from the single-wavelength emitting probes can be affected by the concentration of the probe. To address this, a monoboronate-based probe was synthesized, Ratio Peroxyfluor 1 (RPF)-1 that provides a ratiometric change of two fluorescent signals upon reaction with H2O2, which can potentially permit normalization to probe concentration [One approach for the development of Hechanism , 42, 45.y to H2O2. Althougntration . Becausentration , it is c2O2 levels with spatial resolution. In particular, several mitochondria-targeted probes have been generated, including Mitochondrial Peroxy Yellow 1 (MitoPY1) and SHP-Mito [2O2 levels, which has been shown to function in vivo[Combining the boronate–based probe strategy with organelle-targetable functional groups has provided probes that can measure HSHP-Mito , 43, 51,SHP-Mito , 51. Nucn in vivo.2O2, meaning the fluorescent signal is based on the accumulated H2O2 generated. By contrast, Redoxfluor (RF)-1 uses a reversible disulfide-based redox sensing mechanism, allowing multiple reversible redox reactions in the cellular environment to be detected over time [All of the boronate-based probes react irreversibly with Hver time . Howeverin vivo studies. The visible light excitation affects probe photobleaching, tissue and organ penetration, and subsequent imaging detection. To tackle these issues, Peroxy Caged Luciferin (PCL)-1 and near-infrared (NIR) probe quinone Cy-7 (QCy7) were developed [in vivo H2O2 detection throughout whole mice [2O2 signaling upon injection of lipopolysaccharides (LPS) into mice [One limitation of ROS fluorescent probes is their application in eveloped , 47, 49.ole mice , 47, 48.Compared with small-molecule fluorescent probes, nanoparticles offer several advantages , 54: (1)2O2[in vivo imaging of global H2O2 in mouse model [2O2 simultaneously in the liver of living mice and in real time [2O2[A number of nanoprobes were listed in Table 2O2, 55–57. se model . Semiconeal time . This natime [2O2–61, whic2O2 imaging. Allowing a dynamic measurement for the reversible detection of H2O2, Belousov, et al. developed a group of genetically encoded fluorescent proteins HyPer and their mutants to enable transient live-cell imaging [2O2 in which cpYFP is inserted into the regulatory domain of an Escherichia coli peroxide sensor OxyR [2O2in vitro, to micromolar levels of H2O2 exogenously added to cells, or changes of intracellular H2O2 upon growth factor stimulation [2O2 production, which can further improve H2O2 imaging with high signal-to-background noise from tissue auto-fluorescence in biological systems. HyPer probes are pH sensitive. To address this issue pH-specific probes are used as controls. HyPer-C199S would be an ideal control as a pH-sensitive and H2O2-insensitive version [In the past, researchers have largely relied on the use of fluorescent dyes for ROS sensing due to their good sensitivity, high signal-to-noise ratio, cell permeability, and ease of measurement. The emerging genetic fluorescent proteins offer another option for high resolution selective H imaging , 70–75. sor OxyR . HyPer imulation . Due to  version .2O2 detection [To improve the dynamic range of half-oxidation and half-reduction responses, HyPer-2 and HyPer-3 were developed which shows an expanded dynamic range. HyPer-3 showed faster oxidation-reduction kinetics and a higher fluorescence ratio (F500/F420) than what was reported for HyPer, demonstrating its advantage for Hetection . Both Hy2O2-sensing signaling peroxidase Orp1 [2O2 sensor that has been used to detect and quantify physiological levels of H2O2in vivo. In roGFP2-Orp1, Orp1 relays a disulfide bridge to redox-sensitive GFP (roGFP). The redox equilibrium of the engineered cysteines is associated with measurable ratiometric fluorescent changes. Contrary to HyPer probes, this redox-dependent fluorescence is insensitive to pH changes in the physiological range. RoGFP2-Orp1 has been successfully used to measure physiologically relevant changes in H2O2 levels in Drosophila tissues and in living larvae by real-time imaging [Redox-sensitive GFP (roGFP) , 73 coupase Orp1  is a gen imaging –81.2O2 detection ex vivo. Most fluorescent probes were developed for confocal microscopy. However, confocal microscopy has limitations, such as optical scattering, photodamage, photobleaching, and limited imaging depth for use in real time in vivo studies. Furthermore, prolonged visible light exposure can result in artifactual ROS generation and signal amplification [Confocal microscopy uses a scanning laser beam that is focused on the sample for imaging, with a pinhole placed in front of the detector. Confocal microscopy increases optical resolution and contrast by using a pinhole that prevents the out-of-focus photons from reaching the detector. It is the most popular imaging technique for Hfication .in vivo confocal microscopy is based on microendoscopy. By utilizing a miniprobe for confocal microendoscopy, PY1 was demonstrated for in vivo H2O2 imaging in colorectal cancer [The recent advance of l cancer .TPM provides sub-micron resolution imaging with lower phototoxicity and deeper tissue penetration than confocal microscopy . In the 2O2 production in live cells and tissues [2O2 in rat primary astrocytes using the chemoslective fluorescence probe PF6-AM. Figure 2O2 imaging mechanism of trappable probe PF6-AM [2O2. As a comparison, Figure TPM was demonstrated for imaging intracellular H tissues , 50, 51.in vivo TPM, it has motivated new trends of technology development including long wavelength lasers [in vivo TPM for deep tissue H2O2 imaging in real time.For the deep tissue h lasers –88, fasth lasers , 90, andh lasers –93. Thes2O2 probes have been used for ratiometric imaging based on wide-field microscopy, confocal microscopy and TPM [Ratiometric imaging is the division of one fluorescence channel by another one to derive the ratiometric channel. Ratiometric imaging has been widely used to detect intracellular ion concentrations, protein distributions, voltage or pH changes . Compare and TPM , 94–96.2O2 production. This imaging technique provides a solution for deep tissues H2O2 quantitative analysis.Figure in situ  or in vitro under conditions closely resembling intracellular environments. Figure 2O2 production in zebra fish larvae using representative FLIM of HyPer-3 [2O2 with higher concentrations of the oxidant at the wounding site. This imaging technique provides another solution for H2O2 quantitative analysis.FLIM is an optical imaging technique based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample , 98. Bec HyPer-3 . The patIn vivo chemi-/bioluminescence imaging is a popular method to monitor enzymatic light emission by a living organism [2O2 in organs. By using cooled charge-coupled device (CCD) cameras, this optical imaging modality gets high sensitive. The field of view is up to scores of centimeter covering a whole small animal, but the resolution is at the millimeter level. The recent advances of bioluminescence and chemiluminescence probes enable whole animal studies of H2O2 production [organism –101. Theoduction , 62, 1022O2 production in a mouse model using Peroxalate nanoparticles [2O2 reacts with the peroxalate ester of (1) Peroxalate nanoparticles to produce a high-energy dioxetanedione intermediate within the nanoparticles (2), which then chemically excites the encapsulated dye, leading to light emission from the nanoparticles (3). Peroxalate nanoparticles were mixed with various concentrations of H2O2 and injected, intramuscularly, into the legs in Figure 2O2, (II) 1 μM H2O2, (III) peroxalate nanoparticles only, and (IV) negative control. The chemi-/bioluminescence imaging is the only technique for whole animal global H2O2 monitor in real time.Figure articles . H2O2 re2O2 production in real time for live cells and in vivo studies. Among these fluorescent probes, chemoselective probes offer an attractive approach to H2O2 detection due to their general compatibility with an array of biological systems without external activating enzymes and genetic manipulation. Nanoprobes were recently developed for in vivo imaging of H2O2. A benefit arising from the multifunctional nanotechnology, probe is that it is designed for both chemiluminescence imaging and ratiometric imaging. Furthermore, genetic fluorescent probes that target redox sensitive proteins to specific cellular locations provide high sensitive targeted imaging technology for real time H2O2 imaging.It is imperative to develop fluorescent probes that are able to monitor spatio-temporal intracellular H2O2 detection using: 1) high resolution fluorescence imaging such as confocal microscopy and TPM; 2) large field of view global imaging involving in vivo chemi-/bioluminescence imaging; and 3) ratiometric imaging or FLIM for the quantification of cellular H2O2 levels.These emerging probes enable H


In nine cases, the microorganisms isolated from the preservation fluid concurred with those grown from the donor blood cultures, and in one case, the isolate matched with the one obtained from bile culture. No liver transplant recipient developed an infection due to the transmission of an organism isolated from the preservation fluid. Our findings indicate that contamination of the preservation fluid is frequent in liver transplantation, and it is mainly caused by saprophytic skin flora. Transmission of infection is low, particularly among those recipients given targeted antimicrobial treatment for organisms isolated in the preservation fluid.The role of contaminated preservation fluid in the development of infection after liver transplantation has not been fully elucidated. To assess the incidence and etiology of contaminated preservation fluid and determine its impact on the subsequent development of infection after liver transplantation, we prospectively studied 50 consecutive liver transplants, and cultured the following samples in each instance: preservation fluid , blood, and bile from the donor, and ascitic fluid from the recipient. When any culture was positive, blood cultures were obtained and targeted antimicrobial therapy was started. We found that the incidence of contaminated preservation fluid was 92% (46 of 50 cases of liver transplantation per year), but only 28% (14/50) were contaminated by recognized pathogens. Blood and bile cultures from the donor were positive in 28% and 6% respectively, whereas ascitic fluid was positive in 22%. The most frequently isolated microorganisms were  Liver transplantation has become the definitive treatment of several end-stage liver diseases –3. In reContamination of preservation fluid has been identified as a potential source of post-transplant infection ,11, althTo detect allograft contamination and improve the early diagnosis and approach of infection-related complications, some transplant centers now routinely take intra-operative cultures of organ preservation fluid. However, evidence supporting this practice is scarce, and little is known about the most appropriate management strategy for liver recipients with confirmed preservation fluid contamination. In this study, we aimed to clarify the incidence and etiology of preservation fluid contamination in liver transplantation to determine the role of this contamination in the subsequent development of infection.We conducted a prospective observational study at a tertiary university referral hospital for adults in Barcelona, Spain, where some 50 liver transplants are performed annually. For this study, we included all liver transplants from September 2012 to August 2013, collecting demographic and medical information from the donors and the recipients  as well as the operative data. All data were carefully recorded in a database. Differences in donor, baseline, and surgical characteristics of those undergoing liver transplantation were evaluated.None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. Written informed consent was considered not necessary for the study, as it was an analysis of our usual everyday work. The data of the patients were anonymized for the purposes of this analysis. The confidential information of the patients was protected according national normative. This manuscript was approved for its publication by the Clinical Research Ethics Committee of Bellvitge University Hospital.The microbiology reports for all the positive cultures (blood and preservation fluid) were reviewed daily by the infectious disease team, and patients were then followed up by an infectious disease physician. When pathogens were isolated from donor or preservation fluids, blood cultures were taken from the recipient and targeted antimicrobial therapy was prescribed. However, treatment was stopped if no clinical signs of infection developed or if cultures from the recipient were negative.Grafts were routinely preserved in Celsior solution at 4°C. The liver was placed in a sterile plastic double-bag and transported in a third non-sterile bag. Back-table procedures were performed in a separate dedicated room near to where the organ retrieval was performed.In order to detect the exact time when the PF is contaminated, and to understand which is the main mechanism involved in the contamination, three samples of preservation fluid were routinely obtained under sterile conditions . Under sterile conditions, we also systematically obtained blood and bile samples from donors and ascites samples from recipients.Preservation fluid contamination during the back-table procedure was defined as isolation of any organism from the sample obtained after the back-table procedure, but not in the sample recovered before the back-table procedure. Contamination of the preservation fluid during liver transplantation was defined as isolation of any organism from the sample obtained just before the graft implantation, but not recovered from the sample obtained before the back-table procedure.Infections were defined according to the CDC/NHSN guidelines . In casePrior transplantation referred to re-transplantation of the same organ. Empirical antibiotic therapy was considered inadequate if the treatment regimen did not include at least one antibiotic with in vitro activity against the infecting microorganism. Shock was defined as a systolic blood pressure of less than 90 mmHg, which was unresponsive to fluid treatment or required vasoactive drug therapy . Acute aPneumocystis jiroveci infection was with trimethoprim–sulfamethoxazole given as a single dose, three times a week, for the first 6 months after transplantation. Prophylaxis against cytomegalovirus (CMV) was given according to the current guidelines [All patients received perioperative antibacterial prophylaxis with teicoplanin plus aztreonam for up to 24 hours after transplantation. Antifungal prophylaxis was given to high-risk recipients according to current recommendations . Prophylidelines .Samples were processed by the BACTEC 9240 method . The inoculated bottles were incubated for five days at 35°C before being discharged. Microbial identification was performed using commercially available panels , by standard biochemical and/or enzymatic test, or by matrix-assisted laser desorption ionization . The Clinical and Laboratory Standards (CLSI) criteria were used to define susceptibility or resistance to antimicrobial agents. Antibiotic susceptibility was tested using the micro-dilution method, following CLSI guidelines .The microbiologic results of the preservation fluid culture were compared to the results of samples from the donor and the recipient. The criteria for microorganism matching were based on species identification and susceptibility profiles.U test. All statistical tests were two-tailed and the threshold of statistical significance was <0.05.The statistical analysis was performed using PASW Statistics for Windows, Version 18.0 . Categorical variables were characterized by percentages and compared with chi-square tests, while continuous variables were expressed as means and standard deviations, or as medians and interquartile ranges (IQR), according to their distribution. Comparisons were made using the Mann- Whitney We recorded the details of 50 consecutive liver transplantation among 47 patients during the study period. coagulase-negative staphylococci in seven cases, and streptococci viridans and candida spp. in one case each. In another case, the preservation fluid isolate (Enterobacter spp.) matched that obtained from the bile culture. The microorganisms isolated are detailed in full in During the study period, only six recipients suffered infections during the first month after liver transplantation, although none of these was due to possible pathogen transmission from the donor to the recipient. Three recipients required re-transplantation for ischemic cholangitis, acute cellular graft rejection, and hepatic artery thrombosis. One recipient died because of an acute hepatic artery thrombosis.Finally, we compared various donor and surgical features as well as the outcomes between liver recipients with and without the growth of pathogens in the preservation fluid culture collected just before implantation. In this prospective study of 50 consecutive liver transplants, the incidence of preservation fluid contamination was 92% just before implantation. True pathogenic contamination accounted for 28%. Despite this high incidence, however, we did not identify any cases of infectious transmission.Previous studies have reported a wide variability in the incidence of preservation fluid contamination ,12–14, wGiven that the prevalence of preservation fluid contamination was high, it is reasonable to speculate that the lack of infection transmission is related to the use of early, targeted antimicrobial treatment. However, we identified matches between isolates in the preservation fluid and isolates in the blood and bile cultures of donors, suggesting that this is a plausible route of infection transmission. It should be noted, however, that we did not perform a molecular epidemiology analysis, which would be necessary to confirm that these were truly the same microorganisms.Few reports in the literature have shown the transmission of infection from donor to recipient through preservation fluid contamination ,13,23,24We collected preservation fluid samples at different times during the transplant procedure in an attempt to detect when preservation fluid was contaminated. In this regard, our findings concurred with those found by previous researchers who suggested that preservation fluid is most frequently contaminated during harvesting and manipulation in the back-table procedure . Indeed,Among the strengths of this study are the prospective design and that we included a large number of different samples collected at different points during the transplant process, finding that contamination varies according to the time of samples. However, it should be acknowledged that our study has some limitations. For example, this was a single center study; in addition, due to the small size of our series, it was not possible to compare the differences between positive and negative cultures to identify risk factors for preservation fluid contamination.Preservation fluid contamination is frequent during liver transplantation, and it is mainly caused by superficial saprophytic flora. Transmission of infection is low among those recipients given targeted antimicrobial treatment for organisms isolated in the preservation fluid. However, prospective multicenter studies, which should include molecular epidemiological analyses, are required to determine the optimal strategies for the prevention and management of subsequent infection.S1 File(SAV)Click here for additional data file.


IntroductionThis study aimed to explore the potential for computed tomography (CT) perfusion and 18-Fluorodeoxyglucose positron emission tomography (FDG-PET) in predicting sites of future progressive tumour on a voxel-by-voxel basis after radiotherapy and chemotherapy.MethodsTen patients underwent pre-radiotherapy magnetic resonance (MR), FDG-PET and CT perfusion near the end of radiotherapy and repeated post-radiotherapy follow-up MR scans. The relationships between these images and tumour progression were assessed using logistic regression. Cross-validation with receiver operating characteristic (ROC) analysis was used to assess the value of these images in predicting sites of tumour progression.ResultsPre-radiotherapy MR-defined gross tumour; near-end-of-radiotherapy CT-defined enhancing lesion; CT perfusion blood flow (BF), blood volume (BV) and permeability-surface area (PS) product; FDG-PET standard uptake value (SUV); and SUV:BF showed significant associations with tumour progression on follow-up MR imaging (P < 0.0001). The mean sensitivity (±standard deviation), specificity and area under the ROC curve (AUC) of PS were 0.64 ± 0.15, 0.74 ± 0.07 and 0.72 ± 0.12 respectively. This mean AUC was higher than that of the pre-radiotherapy MR-defined gross tumour and near-end-of-radiotherapy CT-defined enhancing lesion . The multivariate model using BF, BV, PS and SUV had a mean AUC of 0.8 ± 0.1, but this was not significantly higher than the PS only model.ConclusionPS is the single best predictor of tumour progression when compared to other parameters, but voxel-based prediction based on logistic regression had modest sensitivity and specificity. In malignant glioma, over 80% of tumour progression occurs within 2 cm of the original tumour site after radiotherapy and concurrent and adjuvant temozolomide chemotherapy.Functional imaging is gaining popularity as it has demonstrated utility in differentiating tumour grade, predicting treatment response and survival and in identifying active tumour after treatment prior to progression.CT perfusion can simultaneously measure BF, BV and permeability-surface area product (PS) in brain tumours,No functional imaging studies have been reported near the completion of radiotherapy as an attempt to detect residual malignant gliomas. This study investigated the associations between the pre-radiotherapy gross tumour as defined on contrast-enhanced MR, near-end-of-radiotherapy enhancing lesion as defined on contrast-enhanced CT, near-end-of-radiotherapy CT perfusion and FDG-PET parameters with post-treatment MR-defined tumour progression. We then assessed the value of using these imaging parameters for predicting post-treatment MR-defined tumour progression. Therefore, the goal of this study was to assess whether functional imaging has the potential in predicting sites of future progressive tumour on a voxel-by-voxel basis after radiotherapy and chemotherapy. This can help identify biologically significant volumes of disease present at the end of conventional radiotherapy that could serve as a target for treatment intensification.All study procedures were approved by Institutional Research Ethics Board. Patients with newly diagnosed malignant glioma were prospectively recruited to the study with informed consent prior to the last week of radiotherapy. The inclusion criteria were: (1) at least 18 years of age, (2) histologically confirmed malignant glioma, (3) Karnofsky Performance Status ≥60, (4) no previous cranial radiotherapy and (5) pre-radiotherapy MR were performed within 12 weeks of radiotherapy treatment planning. Recurrent gliomas, multiple intracranial lesions, or patients that were not suitable for radical radiotherapy (40–60 Gy in 15–30 fractions) were excluded.After surgery but before radiotherapy, each patient underwent a standard MR imaging comprised of at least one series of T1- and T2-weighted images using a 1.5 T MR scanner . This MR scan was performed within 12 weeks of the treatment planning CT. Contrast-enhanced T1-weighted images were acquired using a fast spoiled gradient echo sequence  after an intravenous injection of gadopentetate dimeglumine .A FDG-PET and CT perfusion scan was performed during the final week of radiotherapy using a hybrid PET/CT scanner . After an intravenous injection of FDG (385 MBq), positron emission data were collected in list mode for 60 min and binned into 5 min frames in the last 40 min. Each PET data set consisted of 47 axial PET images with a 3.26 mm slice thickness and a 26 cm axial field of view (FOV).The FDG-PET CT attenuation correction maps in the FDG-PET study were used to select eight 5 mm sections to maximally cover the tumour for a 150 sec two-phase CT perfusion scan. A non-ionic contrast bolus  was injected at a rate of 2–4 mL/sec at 3–5 sec before the start of the scan. While the selected brain sections were scanned continuously for 45 sec and the images reconstructed at 1-sec intervals during the first phase, the same sections were scanned once every 15 sec for seven times during the second phase. The scanning parameters for both phases were 80 kVp, 190 mA, 1 sec per rotation and a 25-cm FOV.Post-radiotherapy follow-ups were both clinical and radiological. Routine MR images were obtained at 4–6 weeks post-radiotherapy and every 3 months thereafter, and were reviewed by a neuro-oncologist or radiation oncologist. Tumour progression was defined as a continual enlargement of new enhancing lesion (without subsequent resolution) on serial MR imaging along with deterioration of clinical signs and symptoms.2CT perfusion images were analysed using the prototype version of CT Perfusion 4D . Arterial and venous time-attenuation curves were measured from an anterior cerebral artery and the posterior superior sagittal sinus respectively. BF, BV and PS maps were calculated by deconvolving the arterial time-attenuation curve with the tissue time-attenuation curve from 2 × 2 voxel blocks of CT images.Software provided with the PET/CT scanner was used to correct the PET emission data for random and scatter coincidences, dead time and attenuation. PET images from the last 5 min bin were rigidly co-registered with the average CT using the 3D Slicer software.Gadolinium-enhanced T1-weighted MR images from pre-radiotherapy and at progression were rigidly co-registered with the near-end-of-radiotherapy average CT. All images (MR and average CT) and functional maps (CT perfusion and PET SUV) were then resampled to 170 × 170 voxels to minimise registration error. From here on, the contrast-enhancing lesion in the pre-radiotherapy T1-weighted MR lesion is the gross tumour, the contrast-enhancing lesion on the average CT acquired at the last week of radiotherapy is the near-end-of-radiotherapy enhancing lesion, and the contrast-enhancing lesion on follow-up T1-weighted MR at the time of progression is the progressive tumour. Contours around the pre-radiotherapy gross tumour, near-end-of-radiotherapy enhancing lesion and progressive tumour were delineated by a radiation oncologist (G. B.). Table A custom MATLAB  program was developed for this analysis. A 2-cm bounding box around the union of the pre-radiotherapy gross tumour and the progressive tumour was created to reduce the amount of information for analysis for each patient. Major blood vessels, surgical cavities, ventricles and voxels outside the bounding box were excluded. Each voxel inside the bounding box was assigned three binary statuses: pre-radiotherapy gross tumour (yes/no), near-end-of-radiotherapy enhancing lesion (yes/no) and progressive tumour (yes/no). Logistic regression was used to assess the association between tumour progression and the following sets of independent imaging variables: (1) pre-radiotherapy gross tumour, (2) near-end-of-radiotherapy enhancing lesion, (3) BF, BV, PS and SUV and (4) BV, PS, SUV:BF.Leave-one-out cross-validation was used to assess the value of using different imaging parameters to predict tumour progression.16P-values <0.05 were considered significant.Pre-radiotherapy gross tumour, near-end-of-radiotherapy enhancing lesion and progressive tumour volumes within the 4 cm CT perfusion scan length were compared using the Wilcoxon signed-rank test. AUCs of the different logistic models were compared using the Friedman test followed by the Wilcoxon signed-rank test. Models with AUCs that are significantly higher than the AUCs of the pre-radiotherapy gross tumour and end-of radiotherapy enhancing lesion were reported. All statistical tests were done using SPSS version 19.0 , and Ten patients with newly diagnosed malignant glioma were prospectively recruited to the study between the years 2008 and 2011. After surgery, all patients underwent radiotherapy (60 Gy in 30 fractions) with concurrent and adjuvant temozolomide chemotherapy. Patient characteristics are listed in Table 3 respectively. The differences in volumes were not significant (P > 0.05). Figure The mean volumes ±standard deviation (SD) of pre-radiotherapy gross tumour, near-end-of-radiotherapy enhancing lesion and the progressive tumour within the CT perfusion scan volume were 10.8 ± 11.1, 14.1 ± 20.8 and 16.1 ± 18.0 cmP < 0.0001)  and 43.3 , respectively, for tumour recurrence. These odds can be expressed as recurrence probabilities of 54.4% and 69.8% respectively. Multivariate logistic regression showed that the odds of tumour progression increased with lower BF, BV and SUV; and with higher PS and SUV:BF , which were 0.64 ± 0.14 and 0.65 ± 0.14 respectively. Although the AUCs of the two multivariate models were higher than that of PS, the differences were not statistically significant (P > 0.05). Table The AUCs for the three models (1) PS alone, 2) BV, PS, SUV:BF and 3) BF, BV, PS and SUV were 0.72 ± 0.12, 0.74 ± 0.13 and 0.77 ± 0.11 respectively. These AUCs were significantly higher than the AUCs of the pre-radiotherapy gross tumour and near-end-of-radiotherapy enhancing lesion .20The observation that both lower BF and BV showed significant associations with tumour progression is noteworthy. A lower BF and BV may suggest resistance to therapy due to the presence of hypoxia. In support of this, dynamic susceptibility contrast MR studies at pre- and mid-radiotherapy showed that a drop in relative BF and BV were predictors of poor survival.We found that a lower SUV, when considering the entire tumour, is associated with an increased risk of progression. This is contrary to studies that showed an increased FDG uptake is associated with poor survival in patients at various stages of treatmentSome limitations of this study must be considered. Although the AUCs of the multivariate models were higher than those of the univariate PS model, we were unable to demonstrate the statistical significance of multivariate models superiority. A larger sample size is required to determine whether (1) BF, BV, PS and SUV and (2) BV, PS and SUV:BF can provide more information than PS alone for identifying tumour regions that are likely to progress. Other radiotracers with a higher tumour-to-background ratio in the brain can improve the detection of residual tumour at the end of radiotherapy. For example, both 3′-Deoxy-3′-18F-fluorothymidine (FLT)We developed a technique for voxel-wise analysis of CT perfusion and FDG-PET images acquired at the end of radiotherapy for patients treated for malignant glioma. On the basis of our analysis, near-end-of-radiotherapy PS was the best predictor of tumour progression on a voxel-by-voxel basis when compared to other CT perfusion and FDG-PET parameters. However, voxel-based analysis had only modest sensitivity and specificity. Exploration of these findings among a larger patient cohort would help confirm the value of PS imaging in predicting recurrence and in confirming the trend towards improved prediction with multivariate imaging data.


Malignant neuroendocrine tumour of the appendix is a rare finding in the paediatric population. Metastases to the loco-regional lymph nodes at time of diagnosis are very rare. The literature reviewing the treatment and follow up of such cases is not definitive regarding best practice and management guidelines vary due to the complexity and rarity of these tumours.A twelve year old girl presented acutely to the emergency department, with a two day history of right iliac fossa pain associated with nausea and vomiting. No fevers or rigors were reported. The patient denied urinary symptoms and was pre-menstrual. She had a background of recurrent presentations to the emergency department with non specific abdominal pain in the preceding months. This girl had also been investigated by the paediatric service regarding episodes of dizziness, headaches and recurrent epistaxis. All previous investigations were normal. On this occasion, she had tenderness, localised guarding and rebound in the right iliac fossa. Her inflammatory markers were elevated, white cell count (WCC) 14.2 and a C reactive protein (CRP) of 23.4. Intravenous antibiotics were commenced and a plan for surgical intervention was made.The patient underwent an emergency uncomplicated laparoscopic appendicectomy. At the time of surgery, it was noted that the apex of the appendix was distended. Figure  There we.  demonstrated a rare case of infiltrating neuroendocrine tumour of the appendix. The tumour penetrated the mucosa, submucosa and muscularis propria with extraluminal extenstion into the mesoappendix. This tumour was well differentiated measuring 25 mm in maximal diameter. The tumour stained positive for chromogranin, CD 56 and synaptophysin. The Ki67 proliferative index measured 15%, therefore consistent with a Grade 2 (G2) neuroendocrine tumour. A microscopic lymph node of the mesoappendix was also analysed which stained positive for chromogranin A, confirming a small metastatic deposit in the centre of the lymph node. Figure  Urinary After discussion at the gastrointestinal multidisciplinary meeting, the decision to perform a laparoscopic right hemicolectomy was made. The pathological examination of the subsequent right hemicolectomy specimen showed no evidence of residual tumour grossly or microscopically. Forty seven loco-regional lymph nodes were retrieved. Two of these lymph nodes were positive for metastatic neuroendocrine tumour. These were located at 1 cm and 5 cm from the appendicectomy site. All peri-ileal lymph nodes were free of disease. The immunohistochemistry profile again was positive for chromogranin, synaptophysin and CD 56. Figure .Figure 4The patient is currently doing well and has been enrolled on a clinical and magnetic resonance imaging (MRI) surveillance programme.Laparoscopic appendicectomy is a common procedure in the paediatric age group. Rarely has an unexpected diagnosis of a malignant neuroendocrine tumour of the appendix been noted. Interestingly benign neuroendocrine tumours are the most common tumour of the gastrointestinal tract in children, however malignant neuroendocrine tumours are rare . Table 1Up to 90% are diagnosed incidentally after laparoscopic appendicectomy . UsuallyThe presenting features of both benign and malignant neuroendocrine tumours usually follow that of acute appendicitis as highlighted in our case. The well described carcinoid syndrome of flushing, diarrhoea and cardiac disease is rarely reported within the paediatric population as this is associated with liver or retroperitoneal metastases. It is in such cases that the 5 hydroxyindoleacetic acid (5HIAA) testing is positive ,3,5,6.et. al studied neuroendocrine tumours in children over a five year period. They noted that mean age of presentation was 13 yrs (4.5-19.5), the majority of those presented with signs of acute appendicitis and the diagnosis was made histologically. The location of the tumour similar to our case primarily was at the apex of the appendix (70%) with extension into the mesoappendix in 63%. Extension into the mesoappendix was more likely if the size of the primary tumour was over 15 mm.Boxberger It has been confirmed across the literature that site, size and grade are significant in predicting aggressive behaviour of tumours ,7. PrognDecision on further operative treatment after histological confirmation of malignant neuroendocrine tumours is based on the size of the tumour. If the tumour is less than 2 cm appendicectomy alone is the operation of choice. A low proliferative index, an apical location of the tumour, and lack of angiolymphatic or mesoappendiceal invasion are other factors that influence surgery. A right hemicolectomy is the operation of choice if the tumour is greater than 2 cm, or if there is histological evidence of mesoappendiceal extension or location of the tumour at the base with caecal extension. However it must be noted that only 20% of resected specimens will show any residual disease.et. al investigated Ki67 as a predictive index of tumour spread. This important study reported that a Ki67 index of 2.8% or greater gave a specificity of 86.8% of having metastases or recurrence [The World Health Organisation revised the classification system for neuroendocrine tumours in 2010 and places considerable emphasis on the Ki67 proliferative index. The Ki67 index is used to subdivide the neuroendocrine tumours into G1 or G2 neoplasms. If the Ki67 index is less than 3%, these are classified as G1. A Ki67 index between 3-20% classifies the tumours as G2. G3 is represented by a Ki67 greater than 20%. Ki67% has been studied as a factor for predicting metastases or recurrence. In 2013 Yamaguchi currence . When ascurrence .et. al confirmed that tumours greater than 2 cm and mesoappendiceal invasion are associated with metastatic disease [et. al suggested that a right hemicolectomy for a child with a neuroendocrine tumour of the appendix is a radical procedure as the prognosis is quite good. 5 year survival is reported between 90-100%. There is a trend towards limited resection as an alternative option to the classic right hemicolectomy, with perhaps an ileocaecal resection deemed appropriate [The presence of lymph node involvement as in our case, is rare and has been reported sporadically in 4-5% of paediatric cases ,6. A rev disease . D’Aleo No definitive follow up has been quoted in the literature. Despite the incidence of recurrent disease being low, follow up is recommended. In general terms clinical follow up, including chromogranin A (CgA) and 5 HIAA testing is recommended. No studies have assessed the sensitivity of these biologic markers to detect metastases or local recurrence ,15,16. TConsent was obtained from the patient for publication.


Currently, there are no consistently effective chemotherapies for recurrent and inoperable meningiomas. Recently, cucurbitacin I (JSI-124), a naturally occurring tetracyclic triterpenoid compound used as folk medicines has been found to have cytoxic and anti-proliferative properties in several malignancies thru inhibition of activator of transcription (STAT3) activation. Previously, we have found STAT3 to be activated in meningiomas, particularly higher grade tumors.Primary leptomeningeal cultures were established from 17, 20 and 22 week human fetuses and meningioma cell cultures were established from 6 World Health Organization (WHO) grade I or II meningiomas. Cells were treated with cerebrospinal fluid from patients without neurologic disease. The effects of cucurbitacin I on cerebrospinal fluid stimulation of meningioma cell DNA synthesis phosphorylation/activation of JAK1, STAT3, pMEK1/2, p44/42MAPK, Akt, mTOR, Rb and caspase 3 activation were analyzed in human leptomeningeal and meningioma cells.Cerebrospinal fluid significantly stimulated DNA synthesis in leptomeningeal cells. Co-administration of cucurbitacin I (250 nM) produces a significant blockade of this effect. Cucurbitacin I alone also produced a significant reduction in basal DNA synthesis. In grade I and II meningiomas, cerebrospinal fluid also significantly stimulated DNA synthesis. Co-administration of cucurbitacin I (250 nM) blocked this effect.In the leptomeningeal cultures, cerebrospinal fluid stimulated STAT3 phosphorylation but not p44/42MAPK, Akt or mTOR. Cucurbitacin I had no effect on basal STAT3 phosphorylation but co-administration with cerebrospinal fluid blocked cerebrospinal fluid stimulation of STAT3 phosphorylation in each. In the grade I meningiomas, cerebrospinal fluid stimulated phosphorylation of STAT3 and decreased MEK1/2 and cucurbitacin I had no effect on basal STAT3, p44/42MAPK, Akt, JAK1, mTOR, or Rb phosphorylation. In the grade II meningiomas, cerebrospinal fluid stimulated STAT3 phosphorylation in all and reduced phosphorylation of MEK1/2 in all and p44/42MAPK in one. Cucurbitacin I had no effect on basal phosphorylation of STAT3 but reduced phorphorylated p44/42 MAPK in 2 grade II meningioma cells lines.These studies raise the possibility that cucurbitacin I might have value as an adjunct chemotherapy. Additional studies are warranted to evaluate the effects of cucurbitacin I on meningiomas in vivo. Appro3.Meningioma growth is regulated by complex interactions between multiple mitogenic/anti-apoptotic signaling pathways . PreviouCucurbitacins are a family of naturally occurring tetracyclic triterpenoid compounds that have been used as folk medicines in China, India, Peru and Brazil for centuries -29 Figu. Six forPreviously we have found that cerebrospinal fluid stimulation of leptomeningeal and meningioma cell proliferation is associated, in part, with activation of STAT3. In search of a STAT3 inhibitor, we tested cucrbitacin I on STAT3 activation in one leptomeningeal and meningioma culture . In the Primary leptomeningeal cultures were established from de-identified postmortem leptomeninges from 17, 20 and 22 week human fetuses with University of Rochester Institutional Review Board approval and postmotrm exception but prior autopsy/surgical maternal consent for use of the tissue. Remnant, excess, discarded tissue/meningioma cells were also collected  with University of Rochester Institutional Review Board approval and surgical consent for use of tissue. M1-M3 were WHO grade I meningothelial meningiomas from the clivus, sphenoid wing or frontal lobe and all had detectable Merlin protein. M4-M6 included a WHO grade II meningothelial, transitional and microcystic meningioma from the frontal lobe and the 2 analyzed also had detectable Merlin protein. These cells were maintained in DMEM with 10% fetal bovine serum (FBS) which is insufficient for normal glia and endothelial cell survival and proliferation. The cells used were screened for features of leptomeningeal and meningioma cells following procedures described previously including detection of a leptomeningeal marker, epithelial membrane antigen (EMA) . For expRemnant, discarded lumbar cerebrospinal fluid was retrieved from samples collected at the University of Rochester Medical Center  and sent to the Dept. of Pathology for analysis between March, 2009 and September, 2012. As discarded remnant tissue, it was exempted by the University of Rochester Institutional review board from patient consent forms beyond standard patient consent forms for tissue use. Samples from patients found to be free of neurological disease that had cytopathology analysis showing no erythrocytes or increased lymphocytes and normal indices were classified as “normal”. In patients who had cerebrospinal fluid collected as part of staging protocol for a peripheral lymphoma, only cerebrospinal fluid from those with no cells by cytology and no neurologic disease were used. Due to limited volume, in some cases multiple samples were combined as pools to achieve some uniformity and enough volume to treat large numbers of cells in T75 flasks. Samples were initially frozen at -17°C then stored at -80°C.Confluent cells from human 17, 20 and 22 week fetal leptomeninges (L1-L3), 3 WHO grade I (M1 -M3) and 3 WHO grade II (M4- M6) were plated in 96 well plates, serum deprived overnight then treated with serum free DMEM, undiluted cerebrospinal fluid, DMEM and cucurbitacin I (250 nM) or cerebrospinal fluid with cucurbitacin  for 72 hrs. This dose was chosen based on the dose response curves and previous literature using doses as high as 10 uM  or lowerFor analysis of effects of various doses of cucurbitacin on cerebrospinal fluid stimulation of a grade I (M1) meningioma were also plated in 96 well plates, serum deprived overnight then treated with serum free DMEM, DMEM with cerebrospinal fluid with or without cucurbitacin I 25, 75 or 250 nM for 72 hrs. Cells were subsequently analyzed in 6 replicates by CyQUANT Cell Proliferation assay . Differences between treatment groups were analyzed by unpaired, two-tailed T-tests .For analysis of effects on PDGF-BB, cells of 20wk fetal leptomeninges, (L2) were also plated in 96 well plates, serum deprived overnight then treated with serum free DMEM, DMEM with recombinant human PDGF-BB  with or without cucurbitacin I 50, 100 or 200 nM for 72 hrs. Cells were subsequently analyzed as above.Confluent cells from 17, 20 and 22 week primary fetal leptomeningeal cell cultures (L1-3), 2 WHO grade I (M2 -M 3) and 2 WHO grade II primary meningioma cultures (M 4 and M5) were serum deprived overnight then treated with serum free DMEM, cerebrospinal fluid without or with 250 nM cucurbitacin I. Lysates of the cells were then analyzed by Western blots.For Western blots, meningioma cells were scrapped in RIPA Lysis Buffer (Upstate Biotechnology) with 1:100 Protease Inhibitor Cocktail (Sigma) then vortexed vigorously and frozen at –85°C. Protein concentrations were quantified using a Bradford assay (BioRad Protein Assay reagent), then 10–35 ug protein from each was loaded on 7.5% acrylamide gel then transferred to 0.45 um nitrocellulose membrane. The membrane was blocked 1 hour in 5% milk in Tris-Cl buffer with Tween 20 then reacted with an affinity purified primary antibody overnight at 4°C. This was followed by horseradish peroxidase conjugated secondary antibody treatment. Detection was achieved with Western Lightening (Perkin Elmer) on Xomat film (Kodak).Western blots were analyzed with monoclonal antibodies to STAT3, phospho-STAT3 phosphorylated at tyrosine 705, JAK, phospho-JAK1 phosphorylated at tyrosine 1022/1023, phosphso-MEK1/2 phosphorylated at serine 221, MAPK 44/42, phospho-MAPK 44/42 phosphorylated at threonine 180/tyrosine 182, Akt, phospho-Akt phosphorylated at threonine 308, phospho-mTOR phosphorylated at serine 2448, Rb and p-Rb phosphorylated at serine 608 . Loading was assessed with an antibody to actin.To screen for Caspase 3 activation by ARP cleavage, near confluent cells from cultures of 2 leptomeninges (L1 and L3) and a WHO grade I (M1) primary meningioma culture were treated with serum free DMEM with and without cucurbitacin I (250 nM) or cerebrospinal fluid with or without cucurbitacin (250 nM). Cucurbitacin I’s effects on apoptosis was measured by Caspase 3 activation. Poly (ADP ribose) polymerase 1 (PARP) fragmentation is an indirect, semiquatitative measurement of caspase 3 activation and apoptosis. Cleavage of the 117-kDa PARP into the 85 kDa product was assessed by Western blot with antibody to PARP -1  and blotting procedures detailed above.In the leptomeningeal cells, cerebrospinal fluid significantly stimulates DNA synthesis (p < 0.005). Co-administration of cucurbitacin I 250 nm) produces a significant blockade of this effect (p <0.0005). Cucurbitacin I alone also produced a significant reduction in basal DNA synthesis in L2 (p < 0.05)  and l grade 2 (M6). Cucurbitacin I (25 nM) had no effect on cerebrospinal fluid stimulation of DNA synthesis while 75 nM and 250 nM significantly reduced this stimulation Figure b.In L2, cucurbitacin I produced a dose dependent reduction in PDGF-BB stimulated DNA synthesis at 50 nM (p = 0.005) 100 nM (p = 0.01) and 200 nM (p = 0.01) doses Figure a.Cerebrospinal fluid had no effect on JAK1 phosphorylation in any of the meningioma cells studies (M2-M6) but stimulated STAT3 phosphorylation in all 3 leptomeningeal cultures and meningioma cells M1, M3–M6. Cucurbitacin I decreased STAT3 phosphorylation in L1-L3. Co-administration of cerebrospinal fluid and cucurbitacin I decreased phosphorylation in M5. In M4 an increase was seen with co-administration. Cerebrospinal fluid reduced MEK1/2 phosphorylation in M2, M4-M6. Cerebrospinal fluid with and without cucurbitacin I was also associated with a reduction of MEK1/2 phosphorylation in M2, M4-M6. Cerebrospinal fluid with or without cucurbitacin I also decreased p44/42MAPK phosphorylation in L2, M4-M6 .In the present study, curcubitacin I blocked cerebrospinal fluid stimulation of leptomeningeal, WHO grade I and II cell proliferation in each of the primary cell cultures. These findings suggest that cucurbitacin may be useful in treatment of select meningiomas. Cucurbitacin I appears to be a potent inhibitor in meningioma cells and effective at concentrations similar to that effective on nasopharyngeal carcinoma cells  and loweIn previous studies cucurbitacin selectively inhibited phosphorylation of STAT3 but not ERK 1 or 2 (P44/42 MAPK), JNK-1 or AKT in NIH 3 T3 cells and adenocarcinoma cell lines . NonethePreviously we have found that STAT3 phosphorylation/activation was associated with cerebrospinal fluid stimulation of irradiated leptomeningeal cell  and meniSTAT3 is one of a family of proteins with a phosphotyrosine and DNA binding domain that acts as a transcription factor -47. LateCucurbitacin I effects on meningioma cell apoptosis are uncertain. Curcubitacin I had no detectable effect on cleavage of PARP, a marker of caspase 3 activity. Nonetheless, due to the limitations inherent to using primary cultures and limited amounts of cerebrospinal fluid from patients without neurological disease, our analysis was limited in scope and time points analyzed. In another study, in combination with gemcitabine, cucurbitacin increased PARP cleavage in pancreactic carcinoma cells .in vivo.Cerebrospinal fluid from multiple different patients stimulated leptomeningeal and meningioma cell proliferation. These findings extend our previous observations that cerebrospinal fluid from a numerous different adults of various ages and both genders has the potential to stimulate leptomeningeal or meningioma cell proliferation under some circumstances in vivo and may participate in the pathogenesis of meningiomas . For exaCucurbitacin I is a potent inhibitor of meningeal and meningioma cell proliferation that warrants further study as a nonsurgical alternative therapy for meningiomas. While its mechanism of action may be primarily thru inhibition of STAT3 phosphorylation/activation, other mechanisms may contribute to its growth inhibitory effects.The authors declare that they have no conflicts of interest in performance or reporting of this work.MJ contributed to the design analyzed the data and wrote the paper. MO contributed to the design, performed the assays and edited the manuscript. KW contributed the tissue, design and editing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/303/prepub


The monitoring of infectious diseases like the porcine reproductive and respiratory syndrome (PRRS) using pen-wise oral fluid samples becomes more and more established. The collection of individual oral fluid, which would be useful in the monitoring of PRRSV negative boar studs, is rather difficult. The aim of the study was to test two methods for individual oral fluid collection from pigs and to evaluate the specificity of a commercial ELISA for detection of PRRSV antibodies in these sample matrices. For this reason, 334 serum samples from PRRSV negative pigs (group 1) and 71 serum samples from PRRSV positive pigs (group 2) were tested for PRRSV antibodies with a commercial ELISA. Individual oral fluid was collected with a cotton gauze swab from 311 pigs from group 1 and 39 pigs from group 2. Furthermore, 312 oral fluid samples from group 1 and 67 oral fluid samples from group 2 were taken with a self-drying foam swab (GenoTube). The recollected oral fluid was then analysed twice with a commercial ELISA for detection of PRRSV antibodies in oral fluid.All serum samples from group 1 tested negative for PRRSV antibodies. The collection of oral fluid was sufficient in all samples. Sampling with GenoTubes was less time consuming than sampling with cotton gauze swabs. False positive results were obtained in 7 (measure 1) respectively 9 (measure 2) oral fluid samples recollected from cotton gauze swabs and in 9 and 8 samples from GenoTubes. The specificity of the oral fluid ELISA was 97.4% for cotton gauze swabs and 97.3% for GenoTubes. 70 out of 71 serum samples and all oral fluid samples from group 2 tested positive for PRRSV antibodies. The sensitivity of the oral fluid ELISA was 100%. According to the kappa coefficient, the results showed an almost perfect agreement between serum and oral fluid collected in both ways (kappa > 0.8).Both methods used for individual oral fluid collection proved to be practical and efficient and can be used for PRRSV antibody detection. It has to be considered, however, that false positive results may occur more often than in serum samples. In recent years, the applicability of oral fluid samples for diagnostics of infectious disease like porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV) has seen increased discussion in scientific literature. Several methods of detecting PRRSV RNA and PRRSV antibodies (Ab), using both different molecular diagnostic methods and serological techniques, were developed -3. SamplFor the collected oral fluid samples, test systems with a high specificity and sensitivity are needed, as they are continuously developed and improved for serum samples ,11. A reThe objective of the study was to test the efficacy and practicability of oral fluid collection from individual pigs via cotton gauze swabs and a dry foam swab (GenoTube) as well as the re-collection of oral fluid from these materials. Furthermore, the specificity of the IDEXX PRRSV OF ELISA for the detection of PRRSV Ab in oral fluid collected with these methods was evaluated in comparison to the IDEXX PRRS X3 in serum samples. To ensure the sensitivity of the oral fluid ELISA, a number of PRRSV positive pigs were tested as well.A total of 395 pigs (405 samples) were included in the study. The pigs consisted of 2 groups. Group 1 (n = 334) included 152 boars from four German boar studs, 67 boars from one Austrian boar stud, 35 fatteners from one German pig breeding farm and 57 sows and gilts as well as 23 nursery piglets from two Austrian pig breeding farms. All farms were classified as PRRSV negative . Group Oral fluid was collected from the above mentioned pigs in two different ways while they were fixated for blood sampling or, in case of the boars, during semen collection:1. Individual oral fluid samples were collected via cotton gauze swab. For this purpose, the swab was held into the mouth of the respective pig with a serrefine and the oral fluid was allowed to soak into the swab  only cotton gauze swabs were used and in another 23 pigs (nursery piglets from Austria) only GenoTubes were utilised. Group 2: Oral fluid samples were collected from 35 pigs both via cotton gauze swab and via GenoTube. In another 4 pigs only cotton gauze swabs and in 22 pigs (32 samples) only GenoTubes were collected.Oral fluid collection was done on the same day that the blood samples were taken from the respective pigs.All serum samples were analysed with the IDEXX PRRS X3 Ab test for the presence of antibodies against PRRSV.All oral fluid samples were analysed with the IDEXX PRRS OF ELISA, designed for detection of antibodies against PRRRSV in oral fluid. To test the reproducibility of results, samples of group 1 were tested in two different measures. The capacity of the foam swab of the GenoTube was measured experimentally. For this reason, 10 GenoTubes were dived into oral fluid for some seconds and the amount of fluid soaked into the swab was measured with weighing. The average was at approximate 200 μl with no considerable deviation. To reconstitute the dried oral fluid, the foam swab of the GenoTube was re-suspended in a 1.5 ml microcentrifuge tube with 400 μl of the dilution buffer of the ELISA kit  and interpreted according to Landis and Koch .From all cotton gauze swabs, a sufficient amount of oral fluid (between 0.5 and 5.0 ml) could be collected. The collection from each pig took between 30 seconds and three minutes. The limiting factor for the collection of oral fluid samples via cotton gauze swabs was the dryness of the mouth of the respective pig. This was more often the case in smaller pigs while the mouths of sows and especially breeding boars contained more oral fluid. The collection of oral fluid via cotton gauze swabs from boars during semen collection is possible without any fixation.The collection of oral fluid with GenoTubes took only a few seconds and went therefore much faster than with cotton gauze swabs. The usage of GenoTubes in fatteners and adult pigs is mostly possible without fixation of the pig.The serum samples of all group 1 pigs tested negative for PRRSV antibodies. The calculated specificity of the serum ELISA was therefore 100%.The results of the oral fluid ELISA from cotton gauze swabs and GenoTubes compared to the serum ELISA are shown in Table The S/P values of the PRRSV antibody ELISAs in group 2 can be seen in Figure Descriptive test parameters and measurements of agreement for all three samplings are shown in Table In this study, two different ways of individual oral fluid sampling were evaluated for their effectiveness and practicability. Using these oral fluid samples, the descriptive test parameters of the IDEXX PRRSV OF ELISA for detection of PRRSV antibodies in oral fluid were calculated in comparison to the IDEXX PRRSV X3 ELISA in serum samples.Brachyspira DNA in rectal swabs of pigs, respectively classical and African swine fever virus DNA in wild boars were detected by PCR. One study is published that describes the usage of GenoTubes for sampling and detection of antibodies against African swine fever virus [Until now, besides the usage of GenoTubes Livestock for DNA analysis of the tested individuals, only a few studies described the use of GenoTubes for detection of infectious agents in animals ,15. In ter virus . HoweverBoth of the sampling techniques used, cotton gauze swabs as well as GenoTubes, proved to be efficient for oral fluid collection from individual pigs. The collection by both sampling methods was successful in all pigs. Individual oral fluid collection by cotton or polyester ropes is not always that successful even in trained pigs (success between 37.5 and 87.5% of the cases) . The colAccording to the kappa coefficient, almost perfect agreement κ > 0.80)  was foun0  was foThis study shows that oral fluid samples can be used for the PRRSV antibody detection on individual pig level. The use of GenoTubes proved to be an especially practical method both for oral fluid collection and for storage and transport of the samples. The ELISA detecting PRRSV Ab in oral fluid collected by cotton gauze swabs and GenoTubes proved to be highly sensitive. It has to be considered, however, that false positive samples needing re-testing may occur. This can cause irritations, especially in PRRSV negative farms like boar studs.


Spermophilus lateralis and Spermophilus tridecemlineatus. The coefficient of variance (CV) of each probe was calculated and there were 178 probes common between the lowest 10% CV of both datasets (n = 1258). All 3 lists were analysed by NormFinder. Our results suggest that the most invariant probe for S. tridecemlineatus was 02n12, while that for S. lateralis was 24j21. However, our results showed that Probes 02n12 and 24j21 are ranked 8644 and 926 in terms of invariancy for S. lateralis and S. tridecemlineatus respectively. This suggests the lack of common liver-specific reference probes for both S. lateralis and S. tridecemlineatus. Given that S. lateralis and S. tridecemlineatus are closely related species and the datasets are comparable, our results do not support the presence of genus-specific reference genes.The expressions of reference genes used in gene expression studies are assumed to be stable under most circumstances. However, studies had demonstrated that genes assumed to be stably expressed in a species are not necessarily stably expressed in other organisms. This study aims to evaluate the likelihood of genus-specific reference genes for liver using comparable microarray datasets from  Gene expression analysis is examining the variations in gene expression by measuring DNA expression levels over time. These variations may be a result of many factors, such as environmental, developmental, and metabolic changes, or treatments. Quantitative real-time polymerase chain reaction (qRT-PCR) is one such used technique to quantify and analyse gene expressions , 2. HoweCandidate reference genes, which are commonly assumed to be invariant, can be identified using statistically based algorithms, such as geNorm , NormFinGAPDH  [UBQ (ubiquinone) [GAPDH [UBQ [Polr32 has been suggested to be stably expressed in mouse heart [Polr32 is not stably expressed in mouse oocytes and embryos. GAPDH and PPIA are suitable reference genes for the human heart [However, several studies had refuted the possibility of universal reference genes –14 that ogenase)  or UBQ (quinone) . However) [GAPDH  and UBQ PDH [UBQ  vary in se heart , but Mamse heart  had showan heart , but thean heart  for the an heart . Using ean heart . In bactS. lateralis and S. tridecemlineatus to draw conclusions as to whether there are reference genes that are both genus specific and liver specific. The liver transcriptomes of Spermophilus lateralis, a ground-roaming golden-mantle ground squirrel, and Spermophilus tridecemlineatus, a tree-habiting 13-lined ground squirrel, under different states of feeding and hibernation had been studied using the same microarray platform . This aS. lateralis consisting of 35 samples (GSE2024) and another was from S. tridecemlineatus consisting of 26 samples (GSE2021). Briefly, these gene expression datasets represent the liver transcriptome for animals sampled during summer, interbout arousal, and late torpor [Two datasets were obtained from publicly available microarray database, Gene Expression Omnibus, National Centre for Biotechnology Information, of which one of them was from Z-score transformation based on the method previously described [Z-scoreProbe =  − µAssumed)/SDDataset, where ProbeInitial is the original probe log-ratio intensity value in the dataset, µInitialProbe is the mean of the initial probe log-ratio values for a particular sample, µAssumed is mean of both datasets consisting of 61 samples, and SDDataset is the standard deviation for each initial dataset. The Z-scores for each probe across different original datasets will then be comparable. The absolute values for Z-scores were used for further analyses.The intensity of each probe was calculated as the log-ratio of liver reference against sample. After which, each original probe log-ratio value in the original dataset was normalized using escribed , 27. BriZ-test for correlation coefficient [The coefficient of variation (CV) of every probe was calculated as the quotient of standard deviation and arithmetic mean. From 12575 probes, the probes were ranked in ascending CV for each dataset, and the rank difference was calculated. Spearman's correlation was used to determine the correlation between the CV values between GSE2021 and GSE2024. From 12575 probes, the data was separated into groups of 10% each. The top 10% of probes in GSE2021 and GSE2024 were identified. These were called CV10-2021 and CV10-2024, respectively . A thirdfficient , 28.Z-test was performed on the test statistic using the mean and standard deviation of the bootstrap distribution.The significance of overlap, CV10-common, was determined using bootstrapping method  where thn = 12565) and denoted as Random-10. The rest of the 12,555 probes that were not in NF-10 or Random-10 were denoted as Others-2021. Pair-wise Pearson's correlation is performed between each of the probes in NF-10 and each of the probes in Others-2021 to give a sample of the expression correlation of invariant probes and other probes . The random correlations were estimated by pair-wise correlation between each probe in Random-10 and Others-2021. To prevent biasness, the process of randomly selecting 10 probes that were not in NF-10 and pair-wise correlation calculation was repeated 5 times.The expression correlations of the invariant probes with other probes in GSE2021 were found. This tests the hypothesis that the expressions of invariant probes were less correlated to the transcriptome compared to randomly selected probes. Ten least variant probes  were identified from NF-2021 and denoted as NF-10. Ten probes which were not in NF-10 were randomly selected from GSE2021 , suggesting correlation between both datasets.The two datasets used in this study, GSE2021 and GSE2024, were of the same microarray platform (GPL1706), examining differential gene expressions in the liver of 2 species of squirrels in similar states of hibernation and activity. This allows for a comparative study to evaluate potential genus-specific and organ-specific reference genes. The microarray consists of 12575 probes. Spearman's correlation coefficient of the coefficient of variation (CV) of the probes in GSE2021 and GSE2024 after n = 1258), 178 probes (14.2%) were common in both datasets, which is statistically significant from a bootstrap mean of 43.11% with a bootstrap standard deviation of 1.33%  as resampled from the lowest 10% of CV in both datasets. However, Spearmans correlation for the NormFinder ranks of these 178 common probes between GSE2021 and GSE2024 was 0.322, and Z-test suggests that the ranks are correlated . Among the lowest 10% of CV in both datasets . NFC-2021 is the result of NormFinder analysis of CV10-common, using data from GSE2021. Coversely, NFC-2024 is the result of NormFinder analysis of CV10-common, using data from GSE2024. When ranked by NormFinder stability indices, the ranks of Probe ID 07I20 are 926 and 127 in NF-2024 and NFC-2024 , respectively.CV10-2021, CV10-2024, and CV10-common were analysed by NormFinder. There is one common probe  are 8644, 84, 321, 225, 654, and 650, respectively. Similarly, the NormFinder stability ranks of Probe IDs 24j21, 12c07, 24n06, 12g19, 15b23, and 12j13 in NFC-2021 (S. tridecemlineatus) are 127, 4, 53, 19, 101, and 89, respectively.However, there are 6 common probes in the 20 most invariant probes of NF-2024 and NFC-2024  between 10 of the most invariant probes of GSE2021 (NF-10) and Others-2021 (those probes not found in NF-10 or Random-10) was 0.055 with standard deviation of 0.136. The average coefficient of determination (r2) of the 5 replicates between (Random-10) and Others-2021 ranged from 0.115 to 0.147 with standard deviation ranged from 0.133 to 0.167 . By calculating Pearson's correlations of the expression of a probe with itself, the floating-point error in calculation can be estimated to be zero with a standard deviation of 2.79e − 12. Hence, we reject the null hypothesis that the correlations between invariant probes and random variant probes are equal and accept the alternate hypothesis that they are significantly lower.The average coefficient of determination  for the entire dataset (n = 12575) based on CV ranks showed correlation between GSE 2021 and GSE 2024, suggesting transcriptional similarities between the livers of S. lateralis and S. tridecemlineatus. This supports Williams et al. [P  value = 1.10 × 10−5). This is also expected, due to the overall correlation of the whole dataset. In addition, Natale et al. [Spearman's correlation , showing that there is little correlation between the invariance of a gene in S. lateralis and its invariance in S. tridecemlineatus, which suggests that genus-specific reference genes are unlikely. Moreover, our results demonstrated a lack of commonality between the top 20 probes in NF-2021 and NF-2024. This implies that the suitable liver reference probes identified by NormFinder for S. tridecemlineatus (GSE2021) are not suitable for S. lateralis. The ratio of overlap between the 10% lowest CV of both datasets (CV10-common) is 14.2%, which is much lesser than the expected 43.1% from bootstrapping. This result is significant (S. tridecemlineatus) with 84 the best rank. This suggests that there are 83 more suitable reference probes for S. tridecemlineatus if Probe 12c07 is to be used as a reference probe across both species. In addition, our results showed that the most stable probe for S. lateralis (Probe 24j21) is ranked 8644 in S. tridecemlineatus. Similarly, the common probe for NF-2021 and NFC-2021 (S. tridecemlineatus), Probe 07l20, is ranked lowly for S. lateralis (ranked 926 and 127 respectively). This is supported by our findings that there are no common probes in the top 20 NormFinder identified reference probes of NF-2021 (S. tridecemlineatus) and NF-2024 . A suitable reference probe for S. lateralis and S. tridecemlineatus, by extension, a genus-liver-specific reference probe for S. lateralis and S. tridecemlineatus, should have low ranks in both datasets. Taken together, these results show that the reference probes suitable for one species may not be suitable for other closely related species, which also suggests that genus-specific reference genes are unlikely.However, there are 6 probes in the top 20 of NF2024 that also fall in NFC-2024 (NF-common of GSE2024), but these probes are ranked poorly in NF2021   and Lingr2 = 0.055), whereas the coefficient of determination (r2) between the randomly selected probes (Random-10) and probes from Others-2021 ranges from 0.115–0.147. This result is significant , rejecting the null hypothesis that correlation between invariant probes (NF-probes) is equal to correlation between variant (random) probes. This supports the use of correlation as a method to identify reference probes/genes, which had been previously reported [Our results demonstrate that the correlation between the NF-10 probes (invariant) and the Others-2021 (variant) is low (reported . The data used in this study is from these two closely related species under the same experimental conditions and from the same organ. However, our results suggests that reference probes found in one species may not be suitable in other as we are not able to conclusively determine suitable genus-specific reference genes even in such closely related species and for the same organ. Despite having similar organ transcriptomes , our resIn conclusion, our results do not suggest the presence of a liver-specific reference gene/probe by analysing transcriptome profiles of 2 closely related species under similar conditions despite evidence supporting previous studies on the similarity of the transcriptomes. Hence, this study does not support the presence of genus-organ-specific reference gene. Therefore, reference genes should be identified for each organism and for each organ individually.