Acute myocardial infarction (AMI) launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC) transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI).Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI) were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection.Twenty-six patients  from the previously reported FINCELL study (n = 80) were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups . At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group .BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI. The heart has limited anaerobic metabolism, and myocytes are dependent on oxygen and a constant flow of nutrients. In acute myocardial infarction (AMI), the ischemic event launches cellular trauma, an intense inflammatory response, and a remodelling process, all of which contribute to changes in ventricular geometry and stiffness . The cytMyocardial repair is orchestrated by the controlled release of proinflammatory cytokines, which enhance the inflammation process ,3. The iIn our previous double-blinded clinical study, we observed increased EF in ST segment elevation myocardial infarction (STEMI) patients treated with bone marrow mononuclear cell (BMMNC) transplantation after thrombolysis and percutaneous coronary intervention [PCI] . In addiThe data was collected in Finland, at the University Hospital of Oulu or the University Hospital of Turku, between October 2001 and February 2007. A total of 522 consecutive patients with STEMI treated with intravenous thrombolytic therapy were screened for the original study . InclusiThis was a double-blinded study in which the patients were randomly assigned in a 1:1 ratio to have either BMMNC or placebo treatment  13]. Bo. Bo13]. Under local anaesthesia, 80 mL of bone marrow was aspirated from the posterior iliac crest into a heparin-treated syringe to harvest mononuclear cells from the bone marrow on the morning of the PCI day. The bone marrow aspirate was subjected to density-gradient centrifugation on Ficoll-Hypaque to exclude granulocytes and erythrocytes. After the mononuclear cells were collected from the interphase, they were washed twice with heparinized physiological saline. Mononuclear cells were suspended in 10 mL of medium containing 5 mL of the patient’s own serum and heparinized physiological saline. The BMMNC suspension was filtered and quality-control procedures  were performed. After the BMMNC separation procedure, the cells were administered by intracoronary injection. The placebo medium contained physiological saline. The validity of the cell preparation system was assessed as described previously .The culprit coronary lesion supplying the infarct area was recognized, and PCI was performed using the standard techniques with implantation of paclitaxel drug-eluting stents for all patients. After stenting, medium containing BMMNCs or placebo medium was administered by intracoronary injection over the wire balloon using intermittent balloon inflation in the stent.To evaluate cardiac function, the left heart ventricular angiography was performed at baseline with catheterization with PCI, and was repeated identically 6 months later . The lefBlood sampling was performed 0–10 days after the onset of symptoms. The mean time to PCI and injection  was 56 hours after STEMI, and the final sample was taken at the control visit 6 months after the index event. The serum was stored at -20°C until cytokine analysis. The plasma samples were collected in ethylenediamine tetra-acidic acid tubes on ice and immediately spun down, and the plasma was stored at -70°C until analysis. An Innotrac Aio! analyser  was used to determine the concentration of troponin-I (TnI) from plasma samples .2) of the 5-parameter logistic regression standard curves was between 0.998 and 1.000 in all assays. For each analyte, the assay working range was determined using the Milliplex software on the basis of intra-assay precision (%CV) and standard curve recovery.The serum samples from baseline, 2 days post-PCI, and 4 days post-PCI were used in the cytokine measurements . The cytThe balance between anti-inflammatory and proinflammatory cytokines was measured to evaluate the effect of BMMNC transplantation on the inflammation process after AMI. The sum of the concentrations of the proinflammatory cytokines  and anti-inflammatory cytokines  was calculated at baseline, 2 days post-PCI, and 4 days post-PCI. The percentage change from baseline to 2 days post-PCI and from baseline to 4 days post-PCI was measured, and Kendall’s tau was used to evaluate the correlation between the changes in anti-inflammatory cytokines and proinflammatory cytokines.t-test or the Mann-Whitney U test was used to assess the distribution of variables between the study groups. P-values less than 0.05 were considered statistically significant. Due to skew distribution and rather small sample size, Kendall’s tau was used to measure correlation between two rates; a value of 0.6 was considered a significant finding.Statistical analysis was performed using SPSS SmartViewer version 22.0 . The Shapiro-Wilk test was used to test normality. Student’s This is a sub-study of the previously reported FINCELL study, in which 522 patients were assessed for eligibility and 80 patients were included the trial . For thiThe TnI concentration in the plasma samples was determined at 2 and 4 days after the onset of symptoms . At 2 daLeft ventricular angiograms were available for the 11 control group patients and 13 BMMNC group patients at baseline and at 6 months . The meaTo test our preliminary hypothesis, cytokine concentrations were measured at baseline, as well as 2 and 4 days after PCI and injection . At baseSimilarly high variability was observed regarding anti-inflammatory cytokine levels . IL-10 aWe also measured the correlation of percentage changes in anti-inflammatory cytokines  and in pro-inflammatory cytokines  from baseline to 2 days after PCI and injection and from baseline to 4 days after PCI and injection . We obseOur initial aim was to evaluate the changes in cytokine levels as a possible mechanism leading to improved EF in patients with thrombolysis and PCI-treated STEMI after BMMNC transplantation. The study groups were relatively equal with respect to demographic and clinical parameters, diseases, and medication at discharge and at 6 months after STEMI. Increases in the troponin level did not differ significantly, indicating that the groups had similar infarction damage. Indeed, the ejection fraction was improved in the BMMNC group, although the difference was not statistically significant after the exclusion of patients from whom we did not obtain reliable serum samples at all time points. To examine our initial hypothesis, levels of a wide variety of cytokines were measured to analyze the acute inflammatory reaction.In our earlier study, we did not detect any significant association between IL-6 and changes in EF in BMMNC-treated or placebo patients . We thouThe proinflammatory phase following AMI is necessary and plays a crucial role in ventricular remodeling and proper healing . A proloIn the present study, we performed BMMNC transplantation via an intracoronary route. Due to difficulty of sampling, the cytokine concentrations were measured in blood samples obtained from systemic vein. It should also be kept in mind that few cells administered by intracoronary transplant remain in the myocardium, and the transplanted cells affect other organs in addition to the myocardium ,23. Our Inflammatory M1 macrophages dominate at the early phase of infarction following abundance of M2 reparative macrophages on day 7 after injury. Ben-Mardechai et al. showed that MSC injection favours M2 polarization. These mOne limitation of this study is the relatively small number of patients. As a result, the increase in EF in the BMMNC-treated patients was not statistically significant, even though our previous study (which included more patients) demonstrated a marked increase. Another limitation is the variation in time delay from onset of symptoms to PCI, which determines the baseline time point and additional cytokine samples; this might explain the different initial cytokine levels and other unknown variables, such as infectious status. Baseline sample collection times varied from 40 to 77 hours from the onset of symptoms, and cytokine measurements were performed at 2 days and at 4 days after baseline  . These sOur aim was to study the effect of BMMNC transplantation on proinflammatory and anti-inflammatory cytokines in STEMI patients. We measured the concentrations of IL-4, IL-10, and IL-13 to explore the anti-inflammation process after AMI. The rationale for choosing these anti-inflammatory cytokines was that all selected cytokines have been shown to reduce the inflammatory process in many models and conditions –28. The Our object was to study mechanism leading for improvement in cardiac function in STEMI patients treated with BMMNC transplantation. We conclude that BMMNCs have a balancing effect on inflammation process that has a crucial role in remodeling and cardiac repair after myocardial infarction. This study demonstrates a correlation between the anti-inflammatory and proinflammatory cytokines in BMMNC-treated STEMI patients at 4 days. Stem cell transplantation has a smaller effect than anticipated on individual cytokines, but is capable of restoring the balance in the inflammatory process. This might partly explain the favorable effect of bone marrow cell injection on cardiac repair.Intracoronary BMMNC transplantation during PCI for STEMI patients influences systemic cytokine levels by maintaining the balance between proinflammatory and anti-inflammatory cytokines.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.


P = 0.03752, P = 0.03078, P = 0.00512, P = 0.00512). PGA-PM-scaffolds demonstrate increased observed initial fixation strength in cadaver femoral head defects relative to PGA-scaffold, particularly when fibrin glue is used for fixation.This study investigates the adhesion capacity of a polyglycolic acid- (PGA-) hyaluronan scaffold with a structural modification based on a planar polymer (PM) surface in a cadaver cartilage defect model. Two cadaver specimens were used to serially test multiple chondral matrices. In a cadaver hip model, cell free polymer-based cartilage implants with a planar bioinspired PM surface (PGA-PM-scaffolds) were implanted arthroscopically on 10 mm × 15 mm full-thickness femoral hip cartilage lesions. Unprocessed cartilage implants without a bioinspired PM surface were used as control group. The cartilage implants were fixed without and with the use of fibrin glue on femoral hip cartilage defects. After 50 movement cycles and removal of the distraction, a rearthroscopy was performed to assess the outline attachment and integrity of the scaffold. The fixation techniques without and with fibrin fixation showed marginal differences for outline attachment, area coverage, scaffold integrity, and endpoint fixation after 50 cycles. The PGA-PM-scaffolds with fibrin fixation achieved a higher score in terms of the attachment, integrity, and endpoint fixation than the PGA-scaffold on the cartilage defect. Relating to the outline attachment, area coverage, scaffold integrity, and endpoint fixation, the fixation with PGA-PM-scaffolds accomplished significantly better results compared to the PGA-scaffolds ( In the regenerative cartilage surgery, many surgical techniques were developed for coating focal articular cartilage defects. Current available surgical cartilage treatments include lavage and debridement, subchondral penetration procedures , autogenous osteochondral transplantation (AOT), and autologous chondrocyte implantation (ACI) with or without using a scaffold –7.As a single-stage procedure, the most used treatment of focal cartilage defects represents the microfracture (Mfx) technique in which a penetration of the subchondral bone layer is performed with subsequent effluent of progenitor cells from the bone marrow into the articular cartilage lesion , 2, 8. TScaffold-assisted single-step techniques were developed to enhance cartilage tissue regeneration by the combination of the Mfx with a biomaterial. By combination of the Mfx with a scaffold, the mesenchymal stem cells (MSCs) are attracted into the biomaterial and the chondrogenic differentiation of the MSCs to cartilage repair tissue is better assured such as the sole application of the Mfx , 11. HowThe importance of the primary fixation of the scaffolds is common, but there is no study available which examines the influence of a structural modification of the scaffold surface in order to achieve a higher adhesion ability .Here we compare and quantify the primary fixation stability of a commonly used scaffold for cartilage repair attached by a planar bioinspired polymer (PM) surface in a cadaver hip model. The aim of our experimental study was that the arthroscopic implantation of a cell free polymer-based scaffold with a structure derived from tree-frog footpads was more stabile compared to an unprocessed scaffold.The study was approved by the local ethical committee of the Ruhr-University Bochum (registration number 4611-13). The surgery was performed by two orthopaedic surgeons experienced in hip arthroscopy . The fluoroscope was placed diagonally across the table . In this study, two right human cadaver hips were arranged for hip arthroscopy in a supine position demonstrated in The resorbable PGA-hyaluronan scaffold  of 10 mm × 15 mm × 1.1 mm was impregnated with an embossing stamp. The embossing stamp consisted of a thermoplastic polymer (PM) surface composed of acrylnitrile butadiene styrene (ABS). The PM was designed of a moulding form with computer-aided design (CAD) software from a 3D printer . The PGA-hyaluronan scaffold was loaded with the PM embossing stamp with a defined 4 kilo force for about 20 minutes in a laboratory-type drying cabinet at 80°C. The PGA-PM-scaffold was produced only for experimental use. For the fixation, the scaffold was placed directly onto the defect of the subchondral bone without additional material.After evacuating the saline solution, the scaffold was implanted by the use of a tissue grasper and simply released above the full-thickness cartilage defect. By using a tissue elevator, the scaffolds were shaped into the defect to reach a 100% filling of the defect. Before fixation of the scaffold with fibrin glue, we used an application device with a 35 cm tip (Evicel) through the instrumentation portal. After removing the distraction, 50 cycles of flexion (90°) and extension (10°) were performed by moving the leg manually. The cycles were conducted by an independent third-party-assistant that was blinded to the assessment of the measurements. Then, the hip joint was refilled with saline solution and the defect area with the implanted scaffold was inspected Figures  and 5. ATo classify the differences concerning adhesion and integrity between the tested scaffolds, we modified the scoring system developed by Bekkers et al. . Outline U test with level of significance defined at *P < 0.05.For each individual fixation technique, the average scores and standard deviations per scoring item were calculated after 50 motion cycles. The arithmetic mean value and the SD were calculated for the variables above and measured with Microsoft Excel . The values were recorded in IBM SPSS Statistics 22 . Mean values of each set were performed using Mann-WhitneyP = 0.03752, P = 0.03078, P = 0.00512, P = 0.00512). Concerning the outline attachment and endpoint fixation, lower statistically significant scores were found for the unfixed PGA-PM-scaffold compared to the PGA-scaffold . With regard to the area coverage and scaffold integrity, no statistical difference was observed between the unfixed PGA-scaffold and the unfixed PGA-PM-scaffold after 50 cycles (P = 0.17384   P = 0.06576). Closer examination showed an incomplete detachment of the PGA-scaffold without fibrin glue to a complete attachment of the PGA-PM-scaffold with adhesive fixation. However, no rupture of the scaffold fiber was observed during arthroscopic implantation. Relating to the scaffold integrity, the PGA-PM-scaffold with fibrin glue achieved the best result after the motion cycles. The fibrin glue fixation with PGA-PM-scaffolds provided the best scaffold integrity as compared to scaffolds without adhesive fixation after 50 cycles. The endpoint fixation strength of the scaffold was highest in the PGA-PM-scaffold fixed with fibrin glue. All the fixation techniques were feasible through the initial incision. None of the specimens showed macroscopic damage at the opposing articular cartilage surface. We compared the unfixed PGA-scaffold versus the unfixed PGA-PM-scaffold plus the PGA-scaffold with fibrin glue versus the PGA-PM-scaffold with fibrin glue. The results of the scaffold fixation are shown in In the present study, we assessed the primary arthroscopic fixation stability of scaffold-based tissue-engineered implants of full-thickness cartilage defects in cadaver hips to simulate the adhesion properties. Regarding the fixation, we showed that the adherence on the cartilage defect was higher with the PGA-PM-scaffold than with the unmodified PGA-scaffold. These results were collected in experimental settings with high similarity to the intended application. They confirm the initial assessment from the development process. in situ cadaver legs; thus the transfer of the findings to a clinical setting is difficult. However, we did not accomplish intra-articular pressure measuring, but it can be assumed that the anatomical precondition of the hip joint with additional burden during motion generated a shearing force on the scaffold, which is similar to the in vivo conditions. This shear force will partially be conveyed directly by friction with the opposing cartilage and partially by the intra-articular remaining saline solution. Further biomechanical studies are needed to prove whether a slightly submerged implant could protect it from the friction in vivo, although in one rabbit model trial, it was demonstrated that submerged metal grafts in focal cartilage lesion deranged the integrity of the articulating cartilage surfaces [This study was limited by the reduced number of implanted scaffolds and the lack of power analysis. Another limitation of our study was that we did not apply static uniaxial tensile tests onsurfaces .Bekkers et al. demonstrated the need for a compromise between fixation stability and scaffold integrity . TherefoThe present study examines for the first time different arthroscopic fixation techniques of cartilage implants of the hip joint in an experimental setup. It demonstrates that bioinspired structural alterations of scaffolds may support arthroscopic application significantly. Since the role of hip arthroscopy and consequently the number of surgeons who apply this technique have significantly increased in the recent years our study presents an important impulse towards easier procedures and subsequently broader clinical application .Furthermore, a stickier scaffold may allow cartilage repair as a simple one step procedure when extended fixation techniques, for example, transosseous fixation or biodegradable pin fixation, are impossible due to tight anatomical conditions or operational circumstances like the hip joint.The presented technique is a single-step procedure. In a clinical study, Fontana et al. showed that the arthroscopic autologous chondrocyte transplantation (ACT) is superior to arthroscopic debridement of hip chondral defects . HoweverIn an arthroscopic hip study, Mancini and Fontana compared the clinical outcome of the arthroscopic matrix-induced autologous chondrocyte implant (MACI) with the autologous matrix-induced chondrogenesis (AMIC) techniques which is matchable to our technique . The autThis study focuses on the arthroscopic application of enhanced cartilage implants on femoral hip cartilage lesions. Although this essential aspect is difficult to objectify, the authors of this paper underline the improved implantation conditions by using the PGA-PM-scaffold and eagerly anticipate the experiences of other arthroscopic hip surgeons. PGA-PM-scaffolds demonstrate increased observed initial fixation strength in cadaver femoral head defects relative to PGA-scaffold, particularly when fibrin glue is used for fixation. Further investigations including biomechanical adhesion analysis and studies with a greater number of cases are necessary.


MicroRNAs are small non-coding RNAs that can regulate expressions of their target genes at the post-transcriptional level. In this study, we propose a tri-component strategy that combines the conservation of microRNAs, homology of mRNA coding regions, and conserved microRNA binding sites in the 3′ untranslated regions to discover conserved microRNA-mRNA interactions. To validate the performance of our conservation strategy, we collected the experimentally validated microRNA-mRNA interactions from three databases as the golden standard. We found that the proposed strategy can improve the performance of existing target prediction algorithms by approximately 2–4 fold. In addition, we demonstrated that the proposed strategy could efficiently retain highly confident interactions from the intersection results of the existing algorithms and filter out the possible false positive predictions in the union one. Furthermore, this strategy can facilitate our ability to trace the homologues in different species that are targeted by the same miRNA family because it combines these three features to identify the conserved miRNA-mRNA interactions during evolution. Through an extensive application of the proposed conservation strategy to a study of the miR-1/206 regulatory network, we demonstrate that the target mRNA recruiting process could be associated with expansion of miRNA family during its evolution. We also uncovered the functional evolution of the miR-1/206 regulatory network. In this network, the early targeted genes tend to participate in more general and development-related functions. In summary, the conservation strategy is capable of helping to highlight the highly confident miRNA-mRNA interactions and can be further applied to reveal the evolutionary features of miRNA regulatory network and functions. MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules that are ∼22 nucleotides in length and are involved in numerous biological processes, such as development, differentiation, and growth Recently, the evolution of miRNAs has been studied extensively The mature miRNA sequences from eight species, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Ornithorhynchus anatinus, Bos taurus, Mus musculus, and Homo sapiens, were obtained from miRBase Release 19 In this study, three algorithms, TargetScan To assess the performance of the proposed conservation strategy, we compiled an experimentally validated miRNA-mRNA interaction dataset from the union of three databases, TarBase V5.0 n species, there would be at least n member miRNAs. This step groups evolutionarily conserved miRNAs into families. In one species, one target mRNA set can be predicted for and assigned to a mature miRNA by an existing algorithm. Therefore, for one miRNA family conserved across n species, up to n number of target sets in n species can be predicted by one algorithm. Then, one target mRNA of the miRNA family i and its orthologues, which were predicted as target mRNAs of the miRNA family i in other species, were considered to be conserved target mRNAs of the miRNA family i. This step selects target mRNAs with conserved coding regions. Accordingly, our strategy required the conserved miRNA binding sites located in the homologue genes’ 3′ UTR and targeted by the members of one miRNA family. Consequently, the conserved miRNA-mRNA interactions with conserved miRNAs, target mRNA coding regions, and miRNA binding sites in the 3′ UTR can be identified by the tri-component conservation strategy.In this study, we proposed a tri-component conservation strategy to discover conserved miRNA-mRNA interactions, to improve the performance of existing miRNA target prediction algorithms, and to investigate the evolution of miRNA regulatory networks . This stn species, a target mRNA that meets the criteria of the strategy in k species would be assigned a conservation level of k. To have further restriction, we required the conserved miRNA-mRNA interactions to be detected in both the oldest and youngest species; thus, k is from 2 to n.Additionally, we further defined the conservation level of one conserved miRNA-mRNA interaction by the number of species in which this miRNA-mRNA interaction could be detected. For example, for a miRNA family with In this study, we developed a tri-component conservation strategy that combined the conservations of miRNAs, mRNA coding region, and miRNA binding sites in the 3′ UTR to predict highly conserved and confident miRNA-mRNA interactions . This stAfter applying our conservation strategy, the precision and F-measure values substantially increased by 2–4 fold compared to the original algorithms and combination data sets, i.e. intersection, union, and IntSec(hsa) . F-measuThe conservation level of miRNA-mRNA interactions also affects the precision . The conIn contrast to the other target prediction algorithms, our proposed conservation strategy combined three major components (1) conservation of miRNAs (2) orthologues of target genes, and (3) conserved miRNA binding sites in the 3′ UTR. We combined these three features to identify the conserved miRNA-mRNA interactions during evolution. This strategy facilitated our ability to trace the homologues in different species that are targeted by the same miRNA family. Due to this intrinsic advantage, the conservation strategy can be further applied to study the evolution of miRNA regulatory networks. In this study, we used the miR-1/206 family to demonstrate this application. The combination putative target gene dataset of IntSec(hsa) was used to perform this analysis.D. melanogaster to D. rerio  et al. In this study, we proposed a tri-component conservation strategy to identify the conserved miRNA-mRNA interactions and demonstrated its ability to improve the performance of existing target prediction algorithms. The improved performance of the proposed conservation strategy implies that conserved miRNA-mRNA interactions might be highly confident Figure S1The work-flow of the tri-component conservation strategy. First, we obtained the mature miRNA sequences from miRBase 19 and 3′ UTR sequences from Ensembl BioMart for eight studied species. With the above two datasets, we run three existing target prediction algorithms (TIF)Click here for additional data file.Figure S2The mature sequences of miR-1/206 family. This figure shows the mature sequences of miR-1/206 family. The background colors represented the different types of nucleotides. The RNAs in seed regions were colored in white.(TIF)Click here for additional data file.Figure S3The size variety of miR-1/206 regulatory network during evolution. The human target gene sizes in the most distant species were shown at y-axis. There is a dramatic increasing of target gene size in between D. melanogaster and D. rerio.(TIF)Click here for additional data file.Figure S4The correlation between the evolutionary distance and GO level. The correlation that older target genes tend to be enriched in lower level GO functions was further confirmed by other three criteria, top 20, 30, and 10%. (Mya: Million Years Ago).(TIF)Click here for additional data file.Figure S5The correlation between the evolutionary distance and the number of literatures. The correlation that older target genes don’t tend to be studied more was further confirmed. (Mya: Million Years Ago).(TIF)Click here for additional data file.Table S1The top 10 enriched functions in the most distant species.(PDF)Click here for additional data file.


Herpes simplex esophagitis (HSE) is commonly identified in immunosuppressed patients. It is rare among immunocompetent patients and almost all of the reported cases are due to HSV-1 infection. HSV-2 esophagitis is extremely rare. We report the case of a young immunocompetent male who presented with dysphagia, odynophagia, and epigastric pain. Endoscopy showed multitudes of white nummular lesions in the distal esophagus initially suspected to be candida esophagitis. However, classic histopathological findings of multinucleated giant cells with eosinophilic intranuclear inclusions and positive HSV-2 IgM confirmed the diagnosis of HSV-2 esophagitis. The patient rapidly responded to acyclovir treatment. Although HSV-2 is predominantly associated with genital herpes, it can cause infections in other parts of the body previously attributed to only HSV-1 infection. Herpes simplex virus (HSV) has been recognized with increased frequency as an opportunistic invader of the esophagus in immunosuppressed or severely ill patients as a result of primary infection or more commonly viral reactivation . PatientWe present a case of a 28-year-old gentleman who presented to the hospital with four days of progressive dysphagia, odynophagia, and epigastric pain and two days of subjective fever. He was unable to tolerate oral intake and had lost approximately 8 pounds over the last several days. He had been in a sexual relationship with one partner for six months and was unaware of any potential exposure to HSV. His past medical history is significant for asthma controlled without steroids. At admission he was febrile with a temperature of 102.8 and tachycardia of 110/min. There were no oral or pharyngeal lesions. He had tenderness in the epigastric area, but no palpable masses or hepatosplenomegaly. Laboratory workup was significant only for atypical lymphocytes noted on smear. Esophagogastroduodenoscopy (EGD) revealed small ulcers with overlying exudate with normal intervening mucosa with some areas of confluence in the lower and middle third of the esophageal mucosa . The conHSE is common in immunosuppressed patients. HSE is rare, but distinct entity in immunocompetent patients. , 3. The HSV-1 is most commonly associated with HSE, although HSV-2 infections were reported rarely [This case also presented with a unique challenge for endoscopic diagnosis of HSE. Initially, due to the white-plaque-like raised appearance of the lesions, candida esophagitis was suspected. There were more characteristic discrete nummular lesions hidden around the large plaque-like lesions. HSV-2 was ultimately diagnosed by immunohistochemistry for HSV and IgM antibodies for HSV-2. IgM/IgG antibodies were not detected for HSV-1. Although tissue immunohistochemistry and PCR for HSV-2 would have been alternate methods to confirm the diagnosis, due to the technical difficulties, we were not able to perform them. However, we believe that the evidence is compelling in this case to confirm the diagnosis of HSV-2 esophagitis.


Amplitude changes in the P-wave of intracavitary electrocardiography have been used to assess the tip placement of central venous catheters. The research assessed the sensitivity and specificity of this sign in comparison with standard radiographic techniques for tip location, focusing on factors influencing its clinical utility. Both intracavitary electrocardiography guided tip location and X-ray positioning were used to verify catheter tip locations in patients undergoing central venous catheter insertion. Intracavitary electrocardiograms from 1119 patients  showed specific amplitude changes in the P-wave. As the results show, compared with X-ray positioning, the sensitivity of electrocardiography-guided tip location was 97.3%, with false negative rate of 2.7%; the specificity was 1, with false positive rate of zero. Univariate analyses indicated that features including age, gender, height, body weight, and heart rate have no statistically significant influence on P-wave amplitude changes (P>0.05). Multivariate logistic regression revealed that catheter insertion routes  and basal P-wave amplitude  have statistically significant impacts on P-wave amplitude changes. As a reliable indicator of tip location, amplitude change in the P-wave has proved of good sensitivity and excellent specificity, and the minor, zero, false positive rate supports the clinical utility of this technique in early recognition of malpositioned tips. A better sensitivity was achieved in placement of centrally inserted central catheters (CICCs) than that of peripherally inserted central catheters (PICCs). In clinical practice, a combination of intracavitary electrocardiography, ultrasonic inspection and the anthropometric measurement method would further improve the accuracy. Central venous access, including centrally inserted central catheters (CICCs) and peripherally inserted central catheters (PICCs) placement, is widely used in the clinical management of cancer patients, and is frequently a necessity for the delivery of chemotherapy, for surgical operations and for critical care. Correct catheter tip location is crucial for the safe use of the central venous catheters (CVC) and for the prevention of complications. The Infusion Nurses Society (INS) recommends a post-insertion chest X-ray to check the CVC tip position. This technique, however, cannot be used to identify abnormal tip positions during the process of catheter insertion, and it is not always feasible, for patients who are in the immediate post-operative state, or in critical conditions. In addition, the X-ray inspection increases the exposure of patients to radiation.Intracavitary electrocardiography(ECG) guided tip positioning has been introduced since 1980’s. Studies indicated that the CVC entering superior vena cava induced changes in the P-wave, typically including increased amplitude, notching of the P-wave , or a dual-phase P-wave. The changes in the P-wave are typically seen when the catheter tip reaches the superior vena cava at the right atrial junction . These cDespite this, some studies have found that the specific P-wave changes are not always observed in all patients during CVC insertion. The success rate of tip positioning during intracavitary electrocardiography-guided CVC insertion ranged from 76% to 97% ,4. In onThe study objects were cancer patients undergoing central venous catheter placement in the CVC department of Sichuan Cancer Hospital from September 2013 to April 2014. Eligibility required age ≥16 years, confirmation of sinus rhythm by the doctor in charge of central venous access; and planned access via PICC, or CICC using the internal jugular vein. The study has been approved by the Ethics Committee of Sichuan Cancer Hospital. Informed written consent was obtained from all subjects. Excluded were patients who had one or more of the following conditions: 1) patients not to undergo PICC or CICC placement for a change in access plan; 2) patients with a cardiac pacemaker; 3) patients with dynamic electrocardiography equipment; and 4) patients whose testing imaging showed unstable basal ECG or intracavitary ECG; unidentifiable p-wave and/or QRS-wave.A total of 1200 patients consented to participate in the study, age ranging from 18 to 91 years old. The final number of subjects achieving standards of enrollment were 1160, as 9 subjects withdrew from CVC insertion before the procedure, while 31 subjects were excluded for conditions list above.The ultrasonic instrument used to guide the puncture was the GE portable ultrasonic apparatus. The intracavitary (IC) electrocardiography monitor used was the Mindary iMEC8 monitor with electrocardiac signal amplifier and lead wires from Braun, Germany. The venous access devices were classified as follows: short term central venous catheters  and peripherally inserted central catheters . For the procedure, 2% lidocaine hydrochloride and 10 U/ml heparin sodium saline were used.A doctor and a nurse who had been trained for in CVC insertion prior the study were responsible for catheter insertion. One assistant nurse took charge of the connection and recording of intracavitary electrocardiography.The catheters were inserted according to the internal jugular vein placement protocol and PICC insertion protocol. The electrocardiography IC connection was set up when catheter was expected to enter the superior vena cava. Posteroanterior chest X-ray was performed after catheter placement. Once there was suspicious or unsatisfactory X-ray result, alternative means were taken to confirm that the tip location, e.g. a lateral view. The tip positions for all subjects was commented on by the same radiologist, with catheter direction and tip location of each subject reviewed and recorded by nurses trained in specific.The intracavitary electrocardiography lead connection was set up by connecting one end of the lead wire to electrocardiography monitor via an adaptor; the other end connected to the external end of the supporting guide wire via a metal clip.The amplification ratio of the ECG wave amplitude was set to 1times, and lead II was selected to monitor the electrocardiac signal. Prior to CVC insertion, the patient was in the supine position for recording of a baseline surface ECG and a desired placement length. The desired length of catheter is predicted by measuring the distance from the puncture point to the sternoclavicular joints plus the distance from the sternoclavicular joints to the 3rd intercostal space on the right parasternal line. After the expected length was reached, CVC advancement took a pause and the lead connections were secured; Intracavitary electrocardiogram was then printed. When the catheter tip reach the expected length but no P wave amplitude change or just some disturbed waves were observed, the connection should be adjusted and re-established, and the catheter should be drawn-back and re-attempt for no more than three times.The criteria for proper identification of tip location on chest X-ray include: radiological landmark of the lower 1/3 of SVC, under the carina but within the first distal 3 cm. The change in amplitude of the P-wave on intracavitary electrocardiogram was used as the indicator for tip location at the target position. The correlation between tip location by intracavitary electrocardiography and by X-ray check was analyzed to explore factors that might influence changes in the amplitude of the P-wave.The amplitude was defined as the distance between the P-wave peak position and ECG baseline, with the unit of millivolt (mV).The change in P-wave amplitude was defined as ≥1 mV changes of the absolute value of the P-wave amplitude on intracavitary electrocardiogram  compared to baseline P-wave amplitude ,5.A SPSS 13.0 software package was used for statistical analysis. The t-test and chi-squared test were used for univariate analysis, and logistic regression was used for multivariate analysis.50 of 3 mV.As shown in When the chest X-ray was used as the standard method for CVC tip location, the sensitivity of electrocardiography guided tip positioning accounted 97.3%; the specificity 1, with a false negative rate of 2.7% and a false positive rate of zero. The crude agreement rate was 97.3%  as a most accurate method for tip location, and quite a few authors have evaluated the accuracy of the IC-ECG method in comparison with X-ray ,2,4–6. OConsidering that the ideal position for the tip of the catheter is restricted in the lower 1/3 of the superior vena cava or at the cavo-atrial junction , accuratIn this study we compared the tip location results from intracavitary electrocardiography-supported placement with the results of X-ray examination, and found there are 1129 cases out of 1160 cases obtained consistent results from both examine approaches. Taking X-ray results as standard, intracavitary electrocardiograph has a sensitivity rate of 97.3%, a specificity rate of 100%, and a false positive rate of zero. Furthermore, all the suspected cases judged as malpositioned were first detected by intracavitary electrocardiography, then confirmed and corrected by ultrasonic assessment. No false positive case was observed. In this sense, tip location by intracavitary electrocardiography has significant advantages in clinic use, especially for early detection of errors in CICC and PICC tip location.In the current study, 31 cases with proper placement showed no specific changes in P-wave amplitude. The sensitivity was lower than that as reported by Pitti et al . The reaIn the current study, based on 1160 cases performed intracavitary electrocardiography-guided tip location, we explored the factors that might have impact on P-wave amplitude change. The results showed that age, gender, height, body weight and heart rate had no statistically significant association with P-wave amplitude changes (P>0.05), indicating that intracavitary electrocardiography guided tip location has good applicability to varied patient-specific conditions. Kremser et al.  suggesteAs to the effects of basal P-wave amplitude, Moureau et al.  excludedResults in the present study showed that clinically useful P-wave amplitude changes were more readily found in CICC placement than in PICC placement. We suggest that the variation might be related to the differences in the catheters. Shorter, larger catheter provides more stable lead connection/signal transductions in CICC placement compared with PICC placement. The CICC guide wire used in this study consists of multiple metal strands and the diameter of CICC measures 0.8 mm, in contrast to the ≤0.5mm diameter of the PICC. Better sensitivity was then achieved in CICC placement in intracavitary electrocardiography-guided tip location. We believe that by applying specially designed ECG devices with better lead connection and improved signal transduction, the applicability of this technique will be further enhanced in clinical practice.


Gliomas represent the most common and devastating primary CNS tumor. While surgical resection is the mainstay of treatment, treating physicians are seeking a safer and more effective treatment modality that is non-invasive, deep-penetrating, and less prone to infection and damage to surrounding tissues. Photodynamic therapy, utilizing a photosensitizing agent with laser light, while effective in a variety of solid tissue tumors, has not proven to be especially advantageous in the treatment of gliomas, due to the poor penetration of the light. Sonodynamic therapy has been explored as an alternative to PDT. Sensitizers such as 5-aminolevulinic acid (5-ALA) and indocyanine green (IcG) have been shown to be preferentially taken up by glioma cells. Low-intensity ultrasound waves can provide enough energy to activate sensitizing agents and induce apoptosis, without the collateral damage to surrounding tissues seen with laser light in PDT. The aim of this initial study is to determine whether the sonosensitizers 5-ALA and IcG can be effectively delivered to, and detected in, the subcutaneous C6 glioma rat model.in vivo imaging system to visualize the fluorescence intensity of the tumor as compared with the adjacent normal tissues.A total of 12 rats were inoculated with the C6 rat glioma cell line for use in this initial study. The rats were then split into three groups: 4 rats were treated with 100 mg/kg of intravenous 5-ALA, 4 rats were treated with 150 mg/mL of intravenous IcG, and 4 rats were used as a control. Rats given 5-ALA were euthanized 2 to 4 hours later. Tumors were then harvested for fluorescent spectroscopy study. In rats which were given IcG, we used in vivo imaging. Future steps will include another proof-of-concept study to assess the ability of low-intensity ultrasound to cause hyperthermia-induced apoptosis in C6 gliomas via activation of 5-ALA and IcG. A larger study would then be needed compare the effectiveness of these sonosensitizers, and further optimize ultrasound parameters. Ultimately we would like to move this work towards a clinical trial.Tumor assays for all 8 of the experimental rats demonstrated uptake of the sensitizing agents. None of the assays for the control rats were positive. Our results confirm that 5-ALA and IcG were enriched in the subcutaneous C6 glioma in rats via the fluorescence spectroscopy or


Articular cartilage defect can lead to degradation of subchondral bone and osteoarthritis (OA).  To determine the healing effect of transplantation of adipose-derived mesenchymal stem cells (Ad-MSCs) in full-thickness femoral articular cartilage defects in rabbit. 6 cells of third passage suspended in 1 mL of DMEM was injected into articular defect. The control group just received 1 mL of DMEM. Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and 2 mM L-glutamine were used for cell culture. To induce cartilage defect, 4 mm articular cartilage full-thickness defect was created in the knee. For histological evaluation in each group , 3 rabbits were sacrificed 4 weeks and 3 animals, 8 weeks after cell transplantation. 12 rabbits were equally divided into cell-treated and control groups. In cell-treated group, 2×10In cell therapy group post-transplantation, no abnormal gross findings were noticed. Neo-formed tissues in cell-treated groups were translucent with a smooth and intact surface and less irregularity. In cell-treated group after 8 weeks post-transplantation, the overall healing score of experimental knees were superior when compared to other groups. We showed that Ad-MSCs, as an available and non-invasive produced source of cells, could be safely administered in knee osteochondral defects.  So the More therapeutic alternatives for full thickness cartilage defects were previously described such as resurfacing procedures with perichondrium, periosteum, and osteochondral bone plugs/allografts , 7. CellHowever, the procedure has limitations, which include the sacrifice of undamaged cartilage within the same joint and the lack of availability of cell numbers especially in elderly patients. In addition, hyaline cartilage is not always seen in the repair tissue after ACI . In treatment of severe cartilage injuries or chondral defects, osteochondral allograft transplantation has been used with good outcomes in clinical practices. However, allograft chondrocyte death due to apoptosis may happen during storage or as a result of implantation stress. Chondrocytes can undergo apoptosis in allo-transplantation. This apoptosis involves the caspase-3 cascade and indicates that chondrocytes may induce acute rejection .eg, bone marrow, adipose tissue, cord blood, etc. In contrast to articular chondrocytes, the expansion of MSCs does not have higher risk for replicative aging or unlimited growth of these cells making them a candidate for cell-based therapies and an attractive option in regenerative tissue repair .,17.16,17® for Windows® ver 11.5  was used for statistical analysis. Mann-Whitney U test was used to compare the histotological criteria between groups. A p value <0.05 was considered statistically significant.SPSSFibroblastic adherent cells were seen in the culture flask and in all subsequent subcultures . It was When specimens were evaluated histologically, in transplanted cell group the knees were studied for structural characteristics, the nature of the predominant tissue, being free from inflammation, inflammatory cells and degenerative changes in adjacent cartilage in comparison to the control knees . After 8Among animals after 4 and 8 weeks post-transplantation, experimental knees were smooth and had intact surface with hyaline articular cartilage and good column alignment of chondrocytes without cell clustering identical to the morphology of native cartilage  and 4. IAfter 4 weeks of cell transplantation in the experimental knees, articular contours were restored, but the histological repair process appeared slower, more irregular and more immature than in groups after 8 weeks of the transplantation. The control knees showed a poor healing process with hypocellularity and cell number showed a decreasing trend in the defect area  and 4. IIn histological scorings, the repaired tissue in the experimental knee was overall superior to that in the control knee, which showed the adding value of transplanted cells . This ovIn this study, safety, feasibility and regenerative potential of Ad-MSCs in knee articular cartilage full-thickness defects in rabbit were assessed. No adverse effects were seen in Ad-MSCs transplantation group. Tissue repair was visible after 4 weeks based on histological findings and after 8 weeks for knee articular cartilage full-thickness defects. The results were much more prominent when compared to the control group. This difference may be due to less time for implanted cells in 4 weeks group when compared to the 8 weeks group . Cell-based therapy in the form of autologous chondrocyte implantation (ACI) was first described in 1994 . This teThe safety and efficacy of cartilage repair in the human knee using arthroscopic microfracture and injections of MSCs and hyaluronic acid were previously reported . In cellSome authors reported augmenting the scaffolds with either autologous articular chondrocytes cultured from biopsies of non-weight-bearing local cartilage or MSCs that lead to better regeneration of the cartilage and subchondral bone. The MSCs may themselves differentiate into the desired phenotype and can recruit and activate local MSCs toward regeneration, or may reactivate local differentiated cells for this purpose. So the early regenerative effect in cell-treated group may be because of paracrine or trophic effects of MSCs on residing cells rather than their innate differentiation potential. This paracrine effect of MSCs was reported by others to be more important than the differentiation potential of the cells  as it waet al [Wakitani, et al , compareet al . The cliet al . in vivo tracing of green fluorescent protein (GFP) labeled MSCs showed that these cells were localized at the site of a surgically created full-thickness chondral defect and resulted into formation of neo-cartilage [in vivo tracing of labeled cells confirmed the presence of injected MSCs in the neo-cartilage [Viable injected cells were recovered in a goat knee with induced arthritis . In porcartilage . In porcartilage . In goatartilage .In rat after injection of GFP-labeled MSCs into the knees, it was found that the injected cells were mobilized to the injured sites . Again iIn our study, we isolated and evaluated the role of implantation of Ad-MSCs in repair of rabbit cartilage defects as a novel cell source. After 4 and 8 weeks, it was shown that the injured knees had histologically and morphologically, a superior cartilage repair in comparison to the control group. As articular cartilage is a highly differentiated, avascular tissue with a low self-regeneration capacity, many researchers tried to increase the repair potential of damaged cartilage using cell-based therapies, such as stem cell implantation similar to our study , 33, 34.It was demonstrated that there might be possible interactions between human nucleus pulposus (NP) cells and MSCs mediated by secreted proteins . It was in vitro chondrogenic differentiation potential. Even its in vivo cartilage regeneration potential is still under question [There are similar studies on application of allogenic Ad-MSCs as an alternative option in cell therapy of osteochondral defects , 34. Othquestion , 40, 41.in vivo and with hypo-immunogenic properties denoting to the important role of these cells in cell transplantation purposes while our findings confirmed their effect in healing of knee chondral defects of rabbit model [Ad-MSCs were shown to be a safe and an available tissue source in surgical wards that could be collected in a non-invasive way with suitability for immediate transplantation it model . Anotherit model . We alsoit model .We showed that Ad-MSCs, as an available and non-invasive source in surgical wards, could be safely administered for transplantation purposes in knee osteochondral defects. A longer follow up of more than eight weeks is suggested to clarify the healing potential of these cells after this period.


We have recently shown higher parietal-occipital EEG gamma activity during sleep in long-term meditators compared to meditation-naive individuals. This gamma increase was specific for NREM sleep, was present throughout the entire night and correlated with meditation expertise, thus suggesting underlying long-lasting neuroplastic changes induced through prolonged training. The aim of this study was to explore the neuroplastic changes acutely induced by 2 intensive days of different meditation practices in the same group of practitioners. We also repeated baseline recordings in a meditation-naive cohort to account for time effects on sleep EEG activity.High-density EEG recordings of human brain activity were acquired over the course of whole sleep nights following intervention.Sound-attenuated sleep research room.Twenty-four long-term meditators and twenty-four meditation-naïve controls.Two 8-h sessions of either a mindfulness-based meditation or a form of meditation designed to cultivate compassion and loving kindness, hereafter referred to as compassion meditation.We found an increase in EEG low-frequency oscillatory activities  over prefrontal and left parietal electrodes across whole night NREM cycles. This power increase peaked early in the night and extended during the third cycle to high-frequencies up to the gamma range (25–40 Hz). There was no difference in sleep EEG activity between meditation styles in long-term meditators nor in the meditation naïve group across different time points. Furthermore, the prefrontal-parietal changes were dependent on meditation life experience.This low-frequency prefrontal-parietal activation likely reflects acute, meditation-related plastic changes occurring during wakefulness, and may underlie a top-down regulation from frontal and anterior parietal areas to the posterior parietal and occipital regions showing chronic, long-lasting plastic changes in long-term meditators. Increasing evidence indicates that meditation practices induce long-term neuronal plastic changes, as reflected by an increase in central and posterior EEG gamma power. Berkovich-Ohana and coworkers related this EEG feature to enhanced attentional skills and sensory awareness . Lutz anThe aim of the present study was to investigate the acute effects of meditation practice, as they may be revealed by measuring EEG activity during sleep. Our prediction was that an intensive meditation session would result in sleep increase in low frequency activity (1–12Hz) in long-term meditators. The main line of evidence supporting this hypothesis comes from studies assessing the specific effects on the brain of different forms of training. It has been demonstrated that motor and mental trainings are able to induce task-related neuroplastic changes, in waking as well as during subsequent sleep –8. ExperIn this study we asked the same population of experienced practitioners who underwent baseline sleep recordings to intensely meditate for 8 hours in two different sessions, dedicated to the Vipassana and the Metta style of practice respectively. Vipassana meditation is a style of mindfulness or open monitoring practice . It invoinsight meditation. It has been pointed out that training self-observation not only improves cognitive, but also enhances the emotional regulatory abilities, by decreasing emotional reactivity and by monitoring the emotional tone of the subjective experience [loving-kindness (LK) tradition. Metta meditation specifically trains loving-kindness, or the wish of happiness towards ourselves and others, as well as compassion, the wish for all beings to be free from suffering [A population of twenty-nine right-handed long-term meditators  was initially recruited based on a history of daily meditation practice of at least 3 years and the participation to a minimum of 3 one-week intensive retreats. Mean duration of meditation training was 15.6 years . All participants were experienced in attentional and emotional styles of meditation practices, as taught within the framework of Theravada and Tibetan Buddhist traditions. From the perspective of the attentional regulatory strategies involved, the complexity and heterogeneity of these meditation practices can be related back to two main styles: focused attention (FA) and open monitoring (OM) [We also enrolled a group of 38 age and gender matched control individuals, from which the 24 best matches for the long-term meditators were chosen . The meditation naïve individuals did not undergo 2 intensive days of practice, but they were utilized as a control group to assess for potential, nonspecific effects of time on sleep architecture and EEG activity.After an initial phone screening to collect the medical and psychiatric history, each study participant underwent a thorough in-person screening, which included several questionnaires (see below). Sleep-disordered breathing and sleep-related movement disorders were also established/excluded with in-laboratory polysomnography (see below). All participants provided written informed consent and were instructed to maintain regular sleep-wake schedules in the week preceding EEG recordings. This study was approved by the Institutional Review Board of the University of Wisconsin-Madison.The experimental design was structured in 3 sessions, a baseline session, and two days of practice of Vipassana and Metta meditations, respectively. The order of the days of meditation was randomized across participants. The 3 sessions were separated by a time interval of 160.8 ± 38.0 days. Participants arrived at the sleep laboratory 5 hours before their usual bedtime for EEG set-up and waking recordings. The waking session was comprised of an auditory Go/No-Go attentional task and an assessment of positive and negative affects which was performed before and after the EEG recordings by means of the PANAS-now questionnaire, a version of the PANAS scale that is sensitive to day by day fluctuations of the emotional state . AdditioThe two days of practice closely reproduced the structure of a meditation retreat. Six 45-min sessions of sitting meditation, three in the morning, three in the afternoon, were separated by four 30-min sessions of walking meditation. The first 45-min sitting meditation session began at 8:15 am with a short guided meditation. The morning and afternoon sessions were separated by a 1-h lunch break, followed by a 30-min dharma talk.The order of Vipassana and Metta meditation sessions was randomized across participants. Two subjects didn't complete the Vipassana meditation session, and their data were excluded from the analyses. For the control participants, no intervention was performed.Sleep EEG recordings with a 256 channel hdEEG system , were collected for each of those three visits.The socioeconomic status  was admiParticipants’ reports of their average hours of formal  meditation training per week allowed us to assess the total amount of lifetime hours of practice , as well as to derive the specific contribution of meditation styles to their training in terms of percentage over the total.® Sleepware . Sleep EEG recordings were scored by a registered polysomnographic technologist who was blind to the experimental conditions. PSG recordings during the baseline session were reviewed by a board certified sleep medicine physician. Individuals with sleep disorders, which included sleep-related movement disorders , and sleep-disordered breathing , were excluded from the analyses. All-night sleep hdEEG recordings were collected at a sampling frequency of 500 Hz with vertex-referencing, using a NetAmps 300 amplifier and NetStation software . After applying a first-order high-pass filter  to eliminate the DC shift, data were band-pass filtered (1–50 Hz), and down-sampled to 128 Hz. Average-reference was performed after removing EEG channels in which artifacts affected most of the recording. Computation of the power spectral density was conducted on six-second epochs by means of a Welch’s averaged modified periodogram with a Hamming window. The epochs were divided in 8 segments with 50% overlapping using the built-in MATLAB function pwelch. A threshold based on the mean power at either a low (1–4 Hz) or high (20–30 Hz) frequency band was used to detect and remove artefactual six-second epochs from NREM sleep data [Six mastoid-referenced channels, which included F3, F4, C3, C4, O1, and O2, together with a sub-mental electromyogram and an electrooculogram were utilized to perform sleep staging, in 30-second epochs according to standard criteria  using Aleep data ,26. Artieep data . Power seep data .Differences in self-report variables between meditators and controls were investigated with a univariate, repeated-measures ANOVA. As for the socioeconomic status, the within factor had 9 levels . For all tests, the p-value threshold was set at 0.05.condition factor had 3 levels . The sleep cycle factor had 3 levels . The sleep stage factor had 4 levels . Total sleep time (TST) and wake after sleep onset (WASO) were entered in a separate ANOVA that included the condition factor and the sleep parameter factor (TST and WASO). For both ANOVA designs, the between group factor had two levels, long-term meditators and meditation-naïve controls. Greenhouse-Geisser correction was applied to p-value and degrees of freedom in case of violation of the sphericity assumption.Differences in sleep architecture across conditions organized in a 3-way factorial design were investigated with a univariate, repeated-measures ANOVA. The Differences in topographical NREM and REM sleep hdEEG power between conditions were assessed with statistical non-parametric mapping (SnPM), a permutation approach that accounts for the multiple comparison problem inherent in the analysis of functional neuroimaging datasets . SpecifiIn order to control for the effect of time and adaptation to the lab environment, we compared with SnPM the whole night recordings of age and gender matched control participants acquired at 3 different time points, corresponding to the baseline and the two days of meditation practice. Moreover, since we expected a potential effect of adaptation to the lab environment to become evident following the first time point, we compared the average changes in long-term practitioners after days of practice relative to baseline to the respective changes across time in naïve participants.We used SnPM to investigate the effect of lifetime meditation experience on the changes in spontaneous brain activity during sleep after an intense day of meditation practice over scalp channels, frequency bins, and sleep cycles.To increase sensitivity, we reduced the frequency space by integrating the frequencies above 15 Hz across 5-Hz bins while keeping a 1-Hz resolution for traditional sleep rhythms . We alsogroup factor, F1,45 = 0.1209, p = 0.73; interaction group*questionnaire items, F2,96 = 1.457, p = 0.24) and mental health .The two groups did not differ as for their socioeconomic status  . Specifically, participants were highly proficient in OM , followed by FA  and LK .Whole night NREM sleep EEG absolute power values were compared between conditions in 1-Hz bins across a range spanning the interval just above 1 Hz to 40 Hz. Since we did not find any difference in sleep night scalp power following Vipassana and Metta meditation practices, we averaged the intervention conditions together for the purpose of comparing against baseline. This analysis revealed a topographically localized, frequency specific, and time modulated increase in EEG power after a day of meditation practice in Vipassana and Metta meditation as compared to baseline . SuprathNo session effect was found by comparing whole night recordings at the same time points in age and gender matched control subjects . The betWe next investigated whether meditation experience could predict the significant changes after intense meditation practice. We found that lifetime meditation practice in open monitoring correlated with the increase predominantly involving theta-alpha power over prefrontal and left parietal electrodes during the sleep night following the intense meditation sessions . The overlapping with the changes in low-frequency activity after intense meditation practice  was moreAfter computing the spatial distribution of power normalized to the mean power across all electrodes, we also performed topographic EEG analysis of REM sleep. As previous literature has suggested functional differences between tonic and phasic REM, we separated these two REM sleep patterns in our analysis –33. SNPMIn this study we found a significant increase in scalp EEG power predominantly localized in low frequency activities (1–12 Hz) across sleep cycles after an intense 8-hour session of mindfulness meditation (Vipassana) and compassion meditation (Metta) practice, which positively correlated with the lifetime meditation experience. This increase was localized over prefrontal and parietal electrodes for both styles of meditations and was specific for NREM sleep. This effect peaked at the transition between the alpha and theta bands (8 Hz) during the first sleep cycle, whereas during the second cycle it extended to slow wave activity (SWA) and spread across the scalp surface involving also posterior regions.A localized increase in low-frequency (≤ 11Hz) sleep EEG activity has been shown to be induced by specific forms of training –8. FurthThe increase in alpha-theta power was not related to changes in self-reported sleepiness and was accompanied by increase in delta power compared to the baseline, thus suggesting that these findings are not due to a decrease in sleep depth. While alpha EEG during sleep is most frequently considered as a sign of arousal  or coverA prefrontal alpha-theta increase has been associated during wakefulness with different styles of meditative practices, including mindfulness-related practices. In addition, Vipassana meditation, has also been linked to state-related increase in posterior gamma power ,39, and Consistent with our previous finding of a positive correlation between sleep parietal-occipital gamma activity and the length of the lifetime daily meditation practice , the acuAt this stage, we can only speculate about the functional significance of our findings. One possibility is that prefrontal-parietal alpha-theta EEG activity could represent a persisting activation of underlying neuronal circuits engaged by the intense meditation practice during the day . Those cIn conclusion, we were able to show the acute effects of meditation training during subsequent sleep. Future work is needed to elucidate the functional significance of these changes. Specifically, the low-frequency oscillatory activities increase observed over prefrontal and left-parietal electrodes following an intensive day of meditation practice could index alpha mediated top-down inputs  regulati


The demand for meta-analyses in basic biomedical research has been increasing because the phenotyping of genetically modified mice does not always produce consistent results. Melanin-concentrating hormone (MCH) has been reported to be involved in a variety of behaviors that include feeding, body-weight regulation, anxiety, sleep, and reward behavior. However, the reported behavioral and metabolic characteristics of MCH signaling-deficient mice, such as MCH-deficient mice and MCH receptor 1 (MCHR1)-deficient mice, are not consistent with each other. In the present study, we performed a meta-analysis of the published data related to MCH-deficient and MCHR1-deficient mice to obtain robust conclusions about the role of MCH signaling. Overall, the meta-analysis revealed that the deletion of MCH signaling enhanced wakefulness, locomotor activity, aggression, and male sexual behavior and that MCH signaling deficiency suppressed non-REM sleep, anxiety, responses to novelty, startle responses, and conditioned place preferences. In contrast to the acute orexigenic effect of MCH, MCH signaling deficiency significantly increased food intake. Overall, the meta-analysis also revealed that the deletion of MCH signaling suppressed the body weight, fat mass, and plasma leptin, while MCH signaling deficiency increased the body temperature, oxygen consumption, heart rate, and mean arterial pressure. The lean phenotype of the MCH signaling-deficient mice was also confirmed in separate meta-analyses that were specific to sex and background strain . MCH signaling deficiency caused a weak anxiolytic effect as assessed with the elevated plus maze and the open field test but also caused a weak anxiogenic effect as assessed with the emergence test. MCH signaling-deficient mice also exhibited increased plasma corticosterone under non-stressed conditions, which suggests enhanced activity of the hypothalamic-pituitary-adrenal axis. To the best of our knowledge, the present work is the first study to systematically compare the effects of MCH signaling on behavioral and metabolic phenotypes. Meta-analysis is a statistical method of combining the results of individual studies to obtain the most reliable conclusions Melanin-concentrating hormone (MCH) is a 17-amino-acid cyclic neuropeptide that was originally isolated from teleost fish; in these fish, MCH causes the aggregation of melanosomes in the scales, which results in color changes Consistent with the orexigenic effect of the injection of MCH into the cerebral ventricles In addition to metabolic parameters, MCH signaling appears to be involved in a variety of behavioral phenotypes. MCH- and MCHR1-deficient mice exhibit decreased non-REM sleep time We performed a meta-analysis of the previously reported data regarding MCH signaling-deficient mice, MCH-deficient mice, and MCHR1-deficient mice. Because genetic background and sex affect behavioral and metabolic characteristics, we also performed separate meta-analyses segregated by background strain and sex.We performed an extensive electronic search to identify studies that had examined the effects of the deletion of MCH signaling on the behavioral phenotypes of mice. We searched PubMed (1948-Jan 2014) using the free-text search terms “melanin-concentrating hormone” or “melanin-concentrating hormone receptor” plus “knockout” or “deficient” on Jan 1, 2014. The first report about MCH signaling-deficient mouse was published in 1998 for MCH-deficient mouse A two-step procedure was used to identify the articles that were eligible for inclusion. First, the articles were assessed by reviewing their titles and abstracts to determine whether the articles met the following inclusion criteria: written in the English language; reported as an empirical article; used MCH-deficient or MCHR1-deficient mice; and included behavioral or metabolic experiments. These inclusion criteria were applied by two independent assessors, and the title and abstract reviews were conducted by same assessors. In cases in which the abstract provided insufficient information to make a final decision, the study was selected for full-text review. The full-text review was also conducted by two assessors. Next, the methodologies of the selected articles that were related to study quality were critically examined in full-text reviews. Studies that did not use wild-type littermates or wild-type animals from the same colony as the control mice were excluded. The parameter we used to index food intake was not normalized by body weight. The confirmation of the methodology was conducted by two assessors. The final decisions regarding inclusion were made by all of the present authors. Ultimately, 405 experiments from 33 studies were retained for the analysis. We did not include energy expenditure data in this study due to the inconsistency of the reported data .The effects of the deletion of MCH signaling on behavioral and metabolic phenotypes were derived by coding the performances in behavioral tests or the values that resulted from physiological and biochemical assays . Regardip<0.05.To assess potential publication bias, the tendency that significant results are more likely to be published than negative results, funnel plots were first created for the overall meta-analysis and for each separate meta-analysis. Because larger study samples tend to provide better estimates of the true effect sizes, inverted funnel shapes should be observed when the effect sizes are plotted against the sample sizes. Further, the funnel plots were assessed for asymmetry using the Egger test r to index effect sizes. The value of the test statistics resulting from two-tailed t-tests, one-way analyses of variance (two groups), chi-squared tests (2×2), and Wilcoxon-Mann-Whitney tests were converted to the correlation coefficient rp-value and the sample size n or the dft-value from these values. The obtained r values indicate how well a particular parameter correlates with the deletion of MCH signaling; an r of ±1.0 indicates a perfect (positive/negative) correlation. Correlations are positive when the parameter is suppressed or decreased in the MCH signaling-deficient mice. To estimate the 95% confidence intervals, we transformed the r values into Fisher’s z values. Fisher’s z is normally distributed, and its variance is 1/(n-3). We then calculated the 95% confidence interval the Fisher’s z metric and converted the Fisher’s z back to r. Statistical analysis was performed using Microsoft Excel.We used the correlation coefficient z-transformed correlations and converted the results back to the r metric. Then, the sample sizes were summed in those parameters. In the overall meta-analysis, significance testing was performed using the 95% confidence intervals. If the confidence interval did not contain zero, we judged the effect of the deletion of MCH signaling on that parameter to be statistically significant. In the separate meta-analyses, we adopted the parameters which were balanced in terms of MCH/MCHR1 KO, background strain, and sex. Further, the mean effect sizes of each group that were judged to be statistically significant were compared with each other using z-tests. Differences were considered significant at p<0.05. In the present analysis, the correlations were considered strong when the lower limit of the 95% confidence interval was >0.3.The mean effect size of each parameter was calculated. In our analyses, the number of experiments that we used to calculate the mean of each parameter was small. Hence, there was not enough power for a test of heterogeneity. We assume that there is one true effect size which is shared by all the included experiments and conducted the analyses using the fixed-effect model p>0.05).To assess potential publication bias, the tendency that significant results are more likely to be published than negative results, we examined the distributions of the included parameters  by funnep>0.05), which suggests that the results of the separate meta-analyses  and that MCH signaling deficiency enhanced locomotor activity, wakefulness, alcohol preference, motor activation by psychostimulants, aggression, male sexual behavior, and social interaction (p<0.05).The effects of MCH signaling on behavioral and metabolic parameters are summarized in p<0.05), and MCH signaling deficiency enhanced food intake, body temperature, oxygen consumption, heart rate, mean arterial pressure, corticosterone level, and norepinephrine transporter level in the globus pallidus and nucleus accumbens (p<0.05). Additionally, MCH-signaling deficiency decreased D1 receptor levels in the nucleus accumbens and the ventral tegmental area, D2 receptor levels in the caudate-putamen and olfactory tubercle, NR1 subunit levels in the hippocampal formation, and 5-HT levels in the prefrontal cortex (p<0.05).Regarding the metabolic parameters, the deletion of MCH signaling suppressed body weight, fat mass, leptin level, liver triglyceride level, and total cholesterol level  were observed among the parameters related to body-weight regulation, including oxygen consumption, body temperature, fat mass, heart rate, arterial pressure, plasma leptin, locomotor activity, and serum total cholesterol. Other parameters that exhibited strong correlations  were non-REM sleep time, wake time, stress-induced hyperthermia, aggression, male sexual behavior, 5-HT levels in the prefrontal cortex, and norepinephrine transporter levels in the globus pallidus.We did not find any inconsistency in the effect size between the earlier literatures before 2002 and recent literatures published in 2005 and later. For example, glucose level of MCH-deficient mouse reported in 1998 p<0.05). MCHR1 deficiency produced significant effects on body weight, plasma insulin, and plasma leptin, and the MCH-deficient mice did not exhibit any significant effects in these parameters.The different effects of MCH deficiency and MCHR1 deficiency on the behavioral and metabolic parameters are summarized in p<0.05). Interestingly, significant increases in food intake and body weight in the MCH-deficient mice were found only in the 129 (p<0.05) and not in the B6 background. The deletion of MCH signaling increased body temperature in the B6 and mixed backgrounds (p<0.05) and suppressed stress-induced hyperthermia in the B6 and mixed backgrounds (p<0.05). Due to the small number of studies that used mixed-background mice, data regarding body weight, fat mass, oxygen consumption, locomotor activity, and food intake were not available.Behavioral studies of MCH and MCHR1 deficiency were performed using mice of the C57BL/6 (B6), 129/Sv (129), and 129-B6 mixed (mixed) backgrounds. Because each strain has different behavioral and metabolic characteristics due to genetic variations, the phenotypes of the gene-modified mice may vary among the different strains. Nevertheless, the phenotypes to which alterations in MCH signaling mainly contribute are thought to be recognizable in MCH signal-deficient mice of different strains. The results of the separate meta-analyses of the effects of the MCH and MCHR1 deficiencies on the behavioral and metabolic parameters are summarized in p<0.05). The lack of MCH signaling exhibited a suppressive effect on glucose levels under a high-fat diet only in the 129 mice (p<0.05), and no significant effect was observed in the B6 mice. Strain-specific effects were also observed in the following parameters: anxiolytic effects, as assessed with an open field test and elevated plus maze test, were observed in the MCH-deficient mice of both the B6 and mixed backgrounds (p<0.05), whereas no significant effects were observed in the 129 background mice; MCH-signaling deficiency in the B6 background produced a significant increase in wake time and a significant decrease in non-REM sleep time, and MCH-signaling deficiency in the 129 background did not produce significant changes in sleep/wakefulness behaviors.Under a high-fat diet, the deficiency in MCH signaling reduced the fat mass and increased oxygen consumption in both the 129 and B6 backgrounds (p<0.05). The deletion of MCH signaling in the male mice suppressed body weight, anxiety, and plasma T4 (p<0.05). The deletion of MCH signaling in the female mice suppressed depressive behavior and plasma insulin (p<0.05) and enhanced food intake (p<0.05).To examine the differential role of MCH signaling in males and females, we performed separate meta-analyses, which are summarized in Nearly two decades ago and long after the report of the expression of MCH in the mammalian lateral hypothalamus Surprisingly, the present meta-analysis revealed that MCH-signaling deficiency caused mild but significant increases in food intake when the mice were given normal or high-fat diets. However, the separate analyses of the MCH-deficient mice and the MCHR1-deficient mice did not show such an effect. The combined meta-analysis of the ligand- and receptor-deficient mice had sufficient statistical power to detect mild hyperphagia in the MCH signaling-deficient mice. The increased food intake of the MCH signaling-deficient mice does not contradict the acute orexigenic effects of MCH that have been reported by independent researchers based on pharmacological experiments Our meta-analysis confirmed that the body weights were reduced and that oxygen consumption increased in the MCH signaling-deficient mice on both normal chow and high-fat diets. Thus, the lean phenotype of the MCH signaling-deficient mice is likely due to increased energy output and not due to reduced energy input. The increased food intake of the MCH signaling-deficient mice may be caused by feedback regulation that maintains stable body weights. Consistent with this notion, our meta-analysis revealed that the MCH signaling-deficient mice exhibited decreased plasma leptin levels; leptin is a major anorexigenic hormone that is secreted from adipose tissues. Suppressive effects of MCH signaling deficiency on the weight gains of mice on high-fat diets were also found in the separate meta-analyses of the MCH-deficient, MCHR1-deficient, B6, male, and female mice. The lack of a suppressive effect on diet-induced obesity in the 129 background may have resulted from the fact that the wild-type 129 strain is resistant to diet-induced obesity, which, due to a floor effect, would make it difficult to observe a suppressive effect on body-weight gain The major source of energy output in rodents is the brown adipose tissue; the thermogenic activity of this tissue is regulated by the sympathetic nervous system MCH neurons are actively firing during non-REM sleep and are more active during REM sleep. Our meta-analysis confirmed that MCH signaling deficiency enhanced wakefulness, suppressed non-REM sleep, and did not alter REM sleep. Because MCH neurons project to the locus coeruleus The lack of a significant effect on REM sleep seems to be inconsistent with the fact that the highest activity levels of the MCH neurons occur during REM sleep There have been contradictory reports about the anxiety-related behavior of MCH signaling-deficient mice .MCHR1 is most strongly expressed in the nucleus accumbens r = −0.704 and r = −0.832, respectively). MCHR1 is expressed in the ventromedial nucleus of the hypothalamus The present meta-analysis revealed that MCH-signaling deficiency was strongly correlated with enhanced aggression and male sexual behavior . This result could not be predicted based on the suppression of REM sleep following the injection of MCHR1 antagonists or the normal REM sleep of the MCH signaling-deficient mice.The prominent difference in MCH signaling between humans and rodents is that humans express MCHR2. Although MCHR2 is expressed in the brain in a pattern similar to that of MCHR1, the downstream target of MCHR2 is different from that of MCHR1 because MCHR2 is a Gq-coupled receptor, whereas MCHR1 is a Gi-coupled receptor Studies that utilized mice in which the MCH neurons were ablated with ataxin-3 or diphtheria toxin were excluded from our meta-analysis because the lack of the GABAergic neurotransmission of the MCH neurons may result in behavioral alterations that are independent of the MCH-MCHR1 signal. Nevertheless, MCH neuron-ablated mice are lean and exhibit increased oxygen consumption, hyperlocomotion, and reduced responses to cocaine; these phenotypes are consistent with the results of the current meta-analysis of MCH signaling-deficient mice The present meta-analysis is not free of limitations. The sample sizes varied across the parameters: many of the included studies examined body weight and food intake, but few studies examined oxygen consumption, largely because a special experimental apparatus is required to measure and control oxygen and carbon dioxide levels to allow for indirect calorimetry. The numbers of studies that we used to calculate the means of each parameter were small. Hence, there was not enough power for a test of heterogeneity. Furthermore, the number of parameters used for separate meta-analysis was small because we adopted the parameters which were balanced in terms of genotypes, sex, and strains.To the best of our knowledge, the present work is the first study to systematically compare the effects of MCH signaling on behavioral and metabolic phenotypes. Our analyses revealed the robust contribution of MCH signaling to energy metabolism, reward behavior, and anxiety. Large numbers of researchers and international consortia are working on phenotyping genetically modified mice Figure S1Funnel plot of effect sizes against sample sizes in separate meta-analysis by ligand-receptor. Black circles indicate behavioral or metabolic parameters in MCH KO (left panel) or MCHR1 KO mouse (right panel).(TIF)Click here for additional data file.Figure S2Funnel plot of effect sizes against sample sizes in separate meta-analysis by background strain. Black circles indicate behavioral or metabolic parameters in C57BL/6 (left panel), 129 (middle panel), or 129×C57BL/6 mouse (right panel).(TIF)Click here for additional data file.Figure S3Funnel plot of effect sizes against sample sizes in separate meta-analysis by sex. Black circles indicate behavioral or metabolic parameters in male (left panel) or female mouse (right panel).(TIF)Click here for additional data file.Table S1A descriptive summary of the studies used in the meta-analysis.(XLSX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


HTLV-1 genes are encoded on both strands of the provirus, such as tax in the plus and HBZ in the minus strand. The HBZ gene is constitutively expressed from the negative strand of the integrated provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is expressed only intermittently in vivo, perhaps to escape from host immune surveillance. However, it remains unknown what regulates this pattern of proviral transcription in vivo. We have found that CTCF binds to the HTLV-1 provirus. CTCF is a DNA-binding protein that plays a fundamental role in controlling higher-order chromatin structure and gene expression in vertebrates. We identified several candidate regions for CTCF binding in the HTLV-1 genome. Chromatin immunoprecipitation assays showed that CTCF bound selectively to the pX region of HTLV-1. Furthermore, electromobility shift assays revealed that CTCF bound directly to the pX DNA sequence. Consistent with the CTCF binding, there was a sharp border of histone modification patterns at the pX region, consistent with CTCF's role as a chromatin insulator. Finally, the CTCF-binding region (1bp) showed enhancer-blocking activity. The CTCF binding and epigenetic border were detectable not only in HTLV-1 cell lines and ATL cell lines but also in fresh PBMCs of ATL patients. These observations suggest that CTCF plays a central role in the regulation of HTLV-1 transcription.#Poster award winnder - 1st place


Arabidopsis thaliana. Treatment of A. thaliana wild-type with AITC resulted in a time- and dose-dependent depletion of cellular GSH. AITC-exposure of mutant lines vtc1 and pad2-1 with elevated and reduced GSH-levels, displayed enhanced and decreased AITC-tolerance, respectively. AITC-exposure also led to increased ROS-levels in the roots and loss of chlorophyll which are symptoms of oxidative stress. Following exposure to AITC, we found that GSH rapidly recovered to the same level as in the control plant, suggesting an effective route for replenishment of GSH or a rapid detoxification of AITC. Transcriptional analysis of genes encoding GSTs showed an upregulation in response to AITC. These findings demonstrate cellular effects by AITC involving a reversible depletion of the GSH-pool, induced oxidative stress, and elevated expression of GST-encoding genes.Allyl isothiocyanate (AITC) is a phytochemical associated with plant defense in plants from the Brassicaceae family. AITC has long been recognized as a countermeasure against external threats, but recent reports suggest that AITC is also involved in the onset of defense-related mechanisms such as the regulation of stomatal aperture. However, the underlying cellular modes of action in plants remain scarcely investigated. Here we report evidence of an AITC-induced depletion of glutathione (GSH) and the effect on gene expression of the detoxification enzyme family glutathione S-transferases (GSTs) in  Arabidopsis thaliana . Moreover, AITC was able to induce a shift in the cell cycle distribution in A. thaliana . The metabolite GSH functions in metabolism, mediating redox signaling, and detoxification of xenobiotics  are biodegradation products found in several plants belonging to the Brassicaceae family, and are produced upon disruption of the tissue by herbivores or pathogens. ITCs are stored as their biologically inert precursors glucosinolates that is physically separated from the hydrolyzing enzyme myrosinase. Upon damage to the cells both substrate and enzyme is released and the subsequent hydrolysis of glucosinolate yields several products including ITCs , Spectrum Plant Total RNA Kit and monodansylcadaverine (MDC) were purchased from Sigma Aldrich (Norway). Primers for qPCR were synthesized by Eurofins MWG (Germany). QuantiTect Reverse Transcription Kit and RNase-Free DNase Set were purchased from QIAGEN (Norway). SYBRgreen was purchased from Roche Applied Science (Norway).AITC (95% purity), Murashige-Skoog (MS), kit for determination of total glutathione content, 2′,7′-dichlorodihydrofluorescein diacetate  on MS-agar without lid was placed in a 13 cm petri-dish together with a filter paper added 200 μl of an AITC-dilution. The latter mentioned plate was sealed and incubated in room temperature for the desired time of AITC-exposure before transferring 10 plants to a 9 cm petri-dish containing fresh MS-agar medium with a slot for each plant subsequently filled with agarose to cover roots and avoid drought stress. Plants were then incubated as above and growth was monitored by capturing pictures every day for 9 days after treatment. The software ImageJ was used to estimate the diameter of the plants.Roots from untreated or AITC-treated seedlings were removed before snap-freezing in liquid nitrogen followed by grounding of plant material using a TissueLyser. 5-sulfosalicylic acid  was added to the grounded tissue for extraction and deproteinization. The supernatant was purified by centrifugation before assayed for GSH-content. The level of total glutathione was measured using a glutathione assay kit according to the provided protocol.A. thaliana plants were grounded in liquid nitrogen followed by extraction using 80% acetone and centrifugation. The collected supernatants were analyzed spectrophotometrically at wavelengths 645 and 663 nm to determine chlorophyll a and b contents, respectively, , 5 mM KOH, 50 μM CaCl2, 10 mM MES-Tris and pH 6.15 for 30 min, followed by washing in the same buffer without H2DCFDA. Pictures were captured through a fluorescence microscope.Following treatment with AITC, wild-type seedling of Clathrin) and At4g34270 (TIP41-like) were used as housekeeping genes. Primer sequences are given in Supplementary Table Quantitative PCR was performed as previously described  and subsequently washed three times in PBS. Detection was visualized by fluorescence microscopy.Staining of autophagosomes with MDC followed the protocol as previsously published  and displayed some bleaching of the leaves. Moreover, pad2-1 seedlings showed increased mortality when exposed to AITC. Vapor of 1 M AITC for 1 h caused 95% lethality whereas only 5% of the wild-type seedlings died from the same treatment  . In addition, the movement of the mitochondria was stagnated following AITC-treatment, which coincides with the results from a parallel report in which the treatment of A. thaliana with AITC resulted in inhibition of actin-mediated intracellular transport . ITC conjugating with GSH affecting the mitochondria has also been reported in mammalian cells, usually resulting in damage and reduced functionality of mitochondria . This may be linked to the difficulties with these methods as previously described  . Little is known of the role of GSH in the plant cell cycle. Interestingly, nuclear recruitment of GSH during the G1- and S-phase of the cell cycle has been shown to be important for the redox state of the cytoplasm and redox-related genes  without any significant response (Øverby et al., A. thaliana (Hara et al., GSTU19 and showed tendencies to upregulation of GSTF6, GSTF7, and GSTZ1 (Hara et al., GSTU5, GSTU13, and GSTF8 that were upregulated by AITC in our studies, showed no response to PEITC (Hara et al., 2O2, hypoxia, chilling and bacterial- and fungal stress in a previous report (Sappl et al., GSH1 and GSH2, respectively. A previous study with depletion of GSH in A. thaliana by cadmium and copper resulted in an upregulation of GSH1 and GSH2 (Xiang and Oliver, A. thaliana did not result in an upregulation of GSH1, but upregulation of GSH2 after 1 h of exposure. Our studies show genes of several GSTs to be upregulated in response to AITC, although further studies should be undertaken to uncover the role of GSTs in AITC-induced defense mechanisms in A. thaliana.GSTs comprise a family of enzymes commonly associated with detoxification and upregulated activity in response to various types of stress. Transcriptional analysis of a set of GST-encoding genes was therefore undertaken in order to investigate the response to AITC-exposure. Among the 55 GSTs in A. thaliana seedlings. AITC was also shown to cause GSH depletion, followed by repletion and upregulation of GST encoding genes. Although more work is required to fully understand a presumable physiological role of AITC in plants, this study provides evidence of cellular effects induced by AITC in A. thaliana.In conclusion, the present study shows that AITC causes growth retardation, bleaching and induction of oxidative stress in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.